Synthesis and biological characterization of new aminophosphonates for mitochondrial pH determination by 31P NMR spectroscopy

Article abstract of DOI:10.1021/jm301866e

A series of mitochondria targeted α-aminophosphonates combining a diethoxyphosphoryl group and an alkyl chain-connected triphenylphosphonium bromide tail were designed and synthesized, and their pH-sensitive ³¹P NMR properties and biological activities in vitro and in vivo were evaluated. The results showed a number of these mito-aminophosphonates exhibiting pK_a values fitting the mitochondrial pH range, short relaxation, and chemical shift parameters compatible with sensitive ³¹P NMR detection, and low cytotoxicity on green algae and murine fibroblasts cell cultures. Of these, two selected compounds demonstrated to distribute at NMR detectable levels within the cytosolic and mitochondrial sites following their perfusion to isolated rat livers, with no detrimental effects on cell energetics and aerobic respiration. This study provided a new molecular scaffold for further development of in situ spectroscopic real-time monitoring of mitochondrion/cytosol pH gradients.

Full text of DOI:10.1021/jm301866e

Article
pubs.acs.org/jmc
Synthesis and Biological Characterization of New
31
Aminophosphonates for Mitochondrial pH Determination by P
NMR Spectroscopy
Marcel Culcasi,† Gilles Casano,† Celine Lucchesi,† Anne Mercier,† Jean-Louis Clement,† Valerie Pique,†
́
́
́
Laure Michelet,‡ Anja Krieger-Liszkay,‡ Maxime Robin,† and Sylvia Pietri*,†
†Aix-Marseille Universite,
Radicalaire, Marseille, France
́ ́
CNRS UMR 7273, Equipe Sondes Moleculaires en Biologie et Stress Oxydant, Institut de Chimie
‡CNRS UMR 8221, Institut de Biologie et de Technologie de Saclay (iBiTec-S), CEA Saclay, Gif-sur-Yvette, France
ABSTRACT: A series of mitochondria targeted α-amino-
phosphonates combining a diethoxyphosphoryl group and an
alkyl chain-connected triphenylphosphonium bromide tail
were designed and synthesized, and their pH-sensitive 31P
NMR properties and biological activities in vitro and in vivo
were evaluated. The results showed a number of these mito-
aminophosphonates exhibiting pKa values fitting the mitochon-
drial pH range, short relaxation, and chemical shift parameters
compatible with sensitive 31P NMR detection, and low
cytotoxicity on green algae and murine fibroblasts cell cultures.
Of these, two selected compounds demonstrated to distribute at NMR detectable levels within the cytosolic and mitochondrial
sites following their perfusion to isolated rat livers, with no detrimental effects on cell energetics and aerobic respiration. This
study provided a new molecular scaffold for further development of in situ spectroscopic real-time monitoring of mitochondrion/
cytosol pH gradients.
pumps protons from the inner matrix and generates a proton-
motive force driving ATP synthesis in parallel with metabolite
and ions uptake.6,8,9
INTRODUCTION
■
Intracellular pH (pHi) is an essential marker of a variety of
physiological processes and the cellular response to external
stimuli. In normal cells, pHi homeostasis is controlled within a
narrow range of less than 0.2 units by the high buffering
capacity of cytosol and through the activity of transport systems
located into the plasma membrane, such as the Na+/H+
The need for a noninvasive, dynamic pH monitoring at the
subcellular level has led to extensive research into the
development of biocompatible weak acids or bases displaying
pH-dependent spectroscopic properties mainly relying on
fluorescence or nuclear magnetic resonance (NMR).10 From
the known pH-dependency of the 31P NMR chemical shift (δ)
of cytosolic inorganic phosphate (Pi) and metabolites, such as
ATP or glucose 6-phosphate, many synthetic alkyl- and
aminoalkylphosphonic acids, and alkylphosphonates have
been proposed as pH markers. However most of the pH-
sensitive compounds used in the pioneering studies (see ref 11
and references cited therein) showed limitations because of (i)
their relatively low NMR spread Δδab (defined as the mean
difference between the chemical shifts of their protonated (δa)
versus unprotonated (δb) forms), which can be considered as
an index of sensitivity,12 (ii) their poor cytosolic penetration,
and (iii) their lack or slow internalization or distribution in cell
organelles.10,13
exchangers and the Na+/HCO3 cotransporter.1 Their
−
regulatory role strongly depends on cell type and function
and varies in response to pathological situations.2,3 Although
the rapid acidosis occurring when cells are exposed to apoptotic
stimuli, during tumor size progression, ischemic episodes,
oxidative stress, or starvation has been largely documented, the
mechanisms of pHi decrease and the cellular targets of released
protons are still not fully understood.1−6
Beside the central role of plasma membrane regulating
systems in pHi control, local specific pHs are maintained within
several cellular organelles that are crucial in processes such as
the transport of various metabolites and ions over membranes,
enzyme structure and activity or the cellular redox state. Thus
acidic pHs < 5 exist in many cytoplasmic organelles, including
endocytic and secretory vesicles, lysosomes, or portions of the
endoplasmic reticulum or trans-Golgi complex, in which the
proton gradients are mediated by a ATP-dependent H+
pump.7,8 Conversely, pHs up to 8 were found in the
mitochondrial matrix of microbes and bacteria, as well as in
mammalian cells, as a result of the respiratory activity which
Previously, we have reported about nontoxic, linear, and
cyclic α- and β-aminophosphonates as pH markers having a
three to 4-fold larger 31P NMR sensitivity as compared to
Received: December 18, 2012
Published: February 25, 2013
© 2013 American Chemical Society
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Figure 1. General structures of linear (a) and cyclic (b) α- or β-aminophosphonates, and (c, d) new mitochondria targeted α-aminophosphonates
designed as sensitive 31P NMR pH indicators for biological studies. In previous studies,13,14 substituents in (a, b) were R = H or Me; R1 and R2 =
alkyl; R3 = alkyl or phosphonyl group; spacer = (CH2)n with n = 0, 1. In the target structures of mito-aminophosphonates substituents are (c) R1 = H
or alkyl, R2 = alkyl, spacer = (CH2)n with n = 6, 8, or 12 and (d) R1 = alkyl, R2 = H or alkyl, spacer = (CH2)3. Inset: Structures of DPP and
DEPMPH.
phosphonic acid derivatives, such as methyl-, phenyl-, or 2-
aminoethyl-phosphonate.11,14,15 The basic concept behind the
design of these compounds was that the 31P NMR shielding/
deshielding in the protonated ammonium (corresponding to
δA) and unprotonated amino (corresponding to δB) forms, and
the pKa would be strongly dependent upon the chain length,
and electronic and steric properties of substituents linked to P
and N atoms. This was explored and modeled on a series of
linear or cyclic secondary or tertiary amines having one α-
carbon linked to a phosphonyl group either directly or through
an aliphatic spacer (Figure 1a and b).11,14 This screening led to
find that diethyl(2-methylpyrrolidin-2-yl)phosphonate
(DEPMPH; pKa = 7.01) or diethyl(2-propylaminoprop-2-
yl)phosphonate (DPP; pKa = 6.99) are accurate probes within
the pH 5−8 window (Figure 1 insets), DEPMPH being the first
reported exogenous pH marker yielding non overlapping 31P
NMR signals in the cytosolic, extracellular, and acidic
compartments of the ischemic/reperfused isolated rat liver.13
To modulate the pKa of pH markers a general synthetic strategy
was to compensate the lowering effect of electron withdrawing
groups (such as phenyl, amino or carboxyl)11 by increasing the
spacer length and/or substituting the α-carbon with donors
such as alkyls (Figure 1a and b). We thus obtained compounds
able to probe the pH in the 3−5 window of liver acidic
vesicles13 and found some of them could specifically enter the
acidic compartments of Dictyostelium discoideum amoeba cells.15
In addition to their low toxicity, another advantage of
aminophosphonates is that they undergo fast cellular uptake
or washout by passive permeation contrary to phosphonic acids
probes which demonstrate a very low rate of intracellular
diffusion and distribution.13,15,16
On the other hand, the simultaneous assessment of cytosolic
and mitochondrial pH is a key feature to better understand a
variety of mechanisms including cell growth,6 trans-Golgi
network pH,8 mitochondrial depolarization in neurons,9 or
insulin secretion.17 Current measurement of mitochondrial pH
in cells is mainly based on fluorescent probes.6,8,9,17 However
31P NMR remains a privileged technique to obtain real-time
information on phosphorylated metabolites and pHi in whole
organs. In an earlier 31P NMR study on liver grafts
preservation,18 changes in the mitochondrial or cytosolic
resonances of Pi or certain endogenous molecules, such as
phosphorylcholine and phosphoenolpyruvate, were exploited to
study the mitochondrial versus cytosolic pH gradients at 4 °C.
However these endogenous mitochondrial pH probes become
irrelevant under normothermic conditions because of the
superimposition of the cytosolic and mitochondrial Pi
resonances. The Pi NMR signal was detected in isolated
mitochondria from rat heart19 or liver20 but its intensity was too
dependent on the ATP-ADP turnover to allow accurate pH
determination. Consequently, there is still a need for
developing novel pH reporters that can specifically accumulate
within the mitochondrial site.
Successful strategies for targeting the mitochondria have
included the preparation of lipophilic cations, such as
triphenylphosphonium, which actively enter the mitochondria
driven by the large membrane potential generated during
oxidative phosphorylation.21,22 This property has allowed the
design of vectorized antioxidants, cardioprotective or anticancer
drugs, and analytical tools for mitochondrial energetics
measurements.21−28
In this study, we have combined the concepts of
mitochondria targeting and enhanced 31P NMR pH probing
to synthesize and biologically evaluate a new class of α-
aminophosphonates bearing a triphenylphosphonium cation.
Their design should cope with (i) the known mitochondrial pH
of 7−8, (ii) a final pKa range of 6.5−7.0 with Δδab ≈ 10, and
(iii) the balance between the expected pKa decreasing effect of
the electron withdrawing Ph3P+ and the potential cytotoxicity
that may increase with the length of a lipophilic spacer
introduced to counterbalance this former effect. On the basis of
these criteria, we report on eleven mito-aminophosphonates
having linear structures (Figure 1c and d) derived from DPP. In
addition, we evaluated their toxicity in two cellular models and
assessed their cytosolic and mitochondrial permeation,
innocuity and resistance to biodegradation on the normother-
mic isolated perfused rat liver.
RESULTS AND DISCUSSION
■
Chemistry. Most of the mitochondria targeted pH markers
prepared in this study were built around linear secondary
amines bearing an α-diethoxyphosphoryl group as the pH-
sensitive 31P NMR probe, and a triphenylphosphonium salt
linked to the amino function by a hydrocarbon chain of
different length as the mitochondria vector. In most of the
syntheses, the key step was a Kabachnik−Fields29 one-pot
aminophosphorylation with ammonia and diethylphosphite
using a protocol previously optimized to prepare linear α-
aminophosphonates pH markers.14 To obtain a uniform
bromide counterion all phosphonium salts were ultimately
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submitted to an exchange using saturated aqueous sodium
bromide as previously described.26
In a first approach to obtain compounds bearing an alkyl
spacer consisting of n = 6 carbons, the aminophosphorylation
occurred after the triphenylphosphonium moiety was intro-
duced (Scheme 1). Hence, the amine functionality of
Scheme 2. Synthesis of Mito-aminophosphonates 7a−b and
a
8a−b
a
Scheme 1. Synthesis of Mito-aminophosphonates 4a and c
a
Reagents and conditions: (a) dry NH3 gas, room temp., 1 h; (b)
HP(O)(OEt)2, 65 °C, 4 h; (c) K2CO3, MeCN, 1,8-dibromooctane,
reflux, 24 h; (d) Ph3P, MeCN, reflux, 2 days, satd. NaBr; (e) 4-
iodobutyltriphenylphosphonium iodide, K2CO3, MeCN, reflux, 3 days,
satd. NaBr.
a
Reagents and conditions: (a) Boc2O, MeOH, room temp., 6 h, 99%;
(b) MsCl, Et3N, CH2Cl2; (c) NaI, MeCN, room temp., 2 days, 77%;
(d) Ph3P, dioxane, 80 °C, 12 h, 93%; (e) 4 M HCl, CH2Cl2, room
temp., 3 h; (f) Et3N, MgSO4, CH2Cl2, R1C(O)R2, room temp., 12 h;
(g) HP(O)(OEt)2, CH2Cl2, reflux, 2 days, satd. NaBr.
Scheme 3. Synthesis of Mito-aminophosphonates 4b and 18a
and b
a
commercially available 6-aminohexan-1-ol was protected with
Boc2O in methanol to yield compound 1 quantitatively. Then,
conversion of the hydroxyl group of 1 to the corresponding
mesylate followed by treatment with sodium iodide afforded
the iodide 2,30 which reacted with triphenylphosphine in
dioxane to give the triphenylphosphonium iodide 3.31 After
deprotection of the Boc group as described in ref 31, the
unprotected amine was subjected to aminophosphorylation14 in
the presence of either isobutyraldehyde or pivaldehyde to give
rise to mito-aminophosphonates 4a or 4c, respectively. Use of
acetone instead of an aldehyde failed to give the expected 4b
possibly because of the lack of reactivity of the imine
intermediate caused by steric effects.
a
Reagents and conditions: (a) aq. HBr, toluene, reflux, 2 days; (b)
Ph3P, MeCN, reflux 2 days under argon and in the dark; (c) 5a or 5b,
To modulate the length of the alkyl spacer still using
commercially available starting material, we further employed a
chemical pathway in which Kabachnik−Fields aminophosphor-
ylation was the initial step (Scheme 2). Thus α-amino-
phosphonates 5a and 5b were first prepared from the
appropriate carbonyl compounds and these compounds were
directly converted into the corresponding mito-aminophosph-
onates having a n = 4 spacer 8a or 8b by treatment with 4-
iodobutyltriphenylphosphonium iodide (IBTP).32 To increase
the length of the spacer up to n = 8, 5a and 5b were first
condensed with 1,8-dibromooctane to afford α-aminophosph-
onate bromides 6a and 6b and the phosphonium functionality
was introduced in the last step by treatment with
triphenylphosphine to give the corresponding mito-amino-
phosphonates 7a and 7b.
In a third approch to prepare mito-aminophosphonates
having a n = 12 hydrocarbon chain spacer, intermediates
bearing separately the triphenylphosphonium and the α-
aminophosphonate functionalities were prepared and coupled.
In the synthetic strategy (Scheme 3), which also allowed to
obtain compound 4b (n = 6), monobromo alcohols 11 and 12
were first prepared by selective bromination of commercially
available diols 9 and 10 as described33 and were then converted
K2CO3, MeCN, reflux, 5 days, satd. NaBr.
into the corresponding hydroxyalkylphosphonium salts 13 and
14 by reaction with triphenylphosphine in acetonitrile,
according to earlier procedures.34,35 These compounds were
then reacted with HBr in toluene under reflux as described in35
to afford the corresponding bromoalkyltriphenylphosphonium
salts 15 and 16, and the aminophosphonate functionality was
finally introduced by condensation with 5a or 5b to yield the
desired mito-aminophosphonates 4b (n = 6) or 18a and b (n =
12).
The series of mito-aminophosphonates was finally completed
by preparing two compounds having the shortest hydrocarbon
chain spacer (n = 3). The synthetic pathway (Scheme 4)
involved two steps starting from the iodoketone 19, which was
prepared from commercially available 5-iodopentan-2-one as
reported.36 Compound 19 was then converted into its
triphenylphosphonium iodide 20, which was then subjected
to an aminophosphorylation with ammonia or isobutylamine in
the presence of diethylphosphite to afford the desired
aminophosphonates 21 or 22, respectively, with rather good
yields. It is noteworthy that 21 is the only mito-amino-
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a
represent a drawback to maintain pKa values of mito-
aminophosphonates near neutrality. We therefore prepared a
first group of nine mito-aminophosphonates inspired by the
structure of the neutral DPP, and having a n = 4−12 spacer to
progressively move the Ph3P+ group away from the amine
function (structure A in Table 1, entries 3−11). As could be
predicted from our former semiempirical model,11 the
experimental pKa increased (i) with alkyl substitution at α-C
for a given n value (see Table 1, entries 3 and 4 versus 7 and 8,
respectively), and (ii) with increasing n for the same (R1, R2)
set (see Table 1, entries 3−6 and 7−10). The sensitivity Δδab of
pH probing of all structure A compounds was very high and
similar to that of DPP and for this parameter n = 8 was the
optimal value for the spacer chain length (see Table 1, entries 5
and 9 versus 6 and 10, respectively).
For the future purpose of developing vectorized mito-
aminophosphonates, we prepared two compounds where both
the phosphonyl and phosphonium groups are branched to the
same α-C, leaving one amino substituent ready for suitable
substitution. Because of the short n = 3 spacer present in these
structure B compounds, pKa values were expectedly the lowest
measured while Δδab values were still within an acceptable
range (Table 1, entries 12 and 13).
Scheme 4. Synthesis of Mito-aminophosphonates 21 and 22
a
Reagents and conditions: (a) Ph3P, toluene, reflux, 12 h (70%); (b)
dry NH3 gas, room temp., 1 h; (c) HP(O)(OEt)2, room temp., 1 h
then 65 °C, 4 h, satd. NaBr; (d) 2-methylpropan-1-amine, room temp.,
1 h, satd. NaBr.
phosphonate of the series having a primary amine as the
titrating function.
31P NMR pH-Titrations. 31P NMR titrations were
performed at 25 °C in a Krebs−Henseleit (KH) buffer and
the chemical shifts were measured with respect to external
phosphoric acid. Of the two 31P NMR peaks exhibited by mito-
aminophosphonates, the chemical shift corresponding to the
Ph3P+ group remained quite constant at ∼24 ppm, while that of
(EtO)2P(O)-group was strongly pH-dependent in the 1.5−12
pH range (Figure 2). For this latter resonance, all compounds
Finally, regarding their NMR spectroscopic properties all
prepared mito-aminophosphonates had very short spin−lattice
relaxation times T1, thus allowing easier quantitative determi-
nations than when using Pi (Table 1). Altogether from our
initial requirements for optimized mitochondrial 31P NMR pH
probing, compounds 4b, 7b (Figure 2), and 18b (almost
neutral) or the slightly more sensitive 7a and 18a (rather
acidic) appeared as the best candidates to probe the alcaline
mitochondrial pH domain.
Cytotoxicity Studies. Since millimolar intracellular levels
of phosphorylated molecules are at least needed to achieve a
satisfactory 31P NMR sensitivity, the toxicity of the new mito-
aminophosphonates was first assessed in vivo against algae
(Chlamydomonas reinhardtii) and in vitro in mammalian (3T3
murine fibroblasts) cells by determining viabilities in a large
concentration range and the data were compared to that of
DEPMPH, DPP, the spin trap α-phenyl-N-tert-butylnitrone
(PBN) and its mitochondria targeted derivative mitoPBN.26
The results are summarized in Table 2 as lethal doses (LD) and
lactate dehydrogenase (LDH) leakage values, following a 1
(algae) or 3 h (fibroblasts) incubation, and are examined
together with predicted lipophilicities (clogD).
Of particular pertinence to the study in C. reinhardtii is the
fact that in plants or algae there is a high interest in determining
the pH inside chloroplasts and mitochondria. pHi (i.e., inside
the lumen and the stroma of the chloroplasts) varies largely
during the light period (i.e., the photosynthetic active period)
as compared to night (respiratory period). In addition, the
cytosolic pH can change drastically depending on the
physiological conditions, like for example when cells are kept
anaerobic.37−40 If in those studies using 31P NMR, the
intracellular Pi signal efficiently probed the chloroplastic and
cytosolic compartments (pH 6.9−7.4) or the acidic vacuoles
(pH ∼ 5.7), there was no information gained on the
mitochondria.38,39
Figure 2. Titration curves of the two 31P NMR resonances of mito-
aminophosphonates 4b and 7b in Krebs−Henseleit buffer at 25 °C.
afforded monophasic titration curves, which could be fitted to
the equation of a single experimental curve suggesting that
protonation always occurred at the nitrogen atom as found
earlier for DPP and its analogues.11 The fits allowed the
calculation of the pKa, δa, δb, and Δδab values which are
reported in Table 1 together with data for Pi and DPP, which
both were within the range reported in our earlier study.11
Previously, we found the basicity of linear α-amino-
phosphonates having the structure shown in Figure 1a to be
mostly affected by inductive effects of substituents R1−R3. Thus
pKa increased upon substitution by electron-donating groups,
such as alkyls, and decreased by withdrawing substituents, such
as dialkoxyphosphonyl, phenyl, or benzyl, this latter effect being
weakened by intercalation of one methylene spacer around the
amine protonation site.11,14,15 On this basis it was obvious that
the pKa-decreasing, electron-withdrawing effect of Ph3P+ may
Here compounds, such as DEPMPH and PBN, which are
moderately lipophilic and have no mitochondria targeting
properties, were not very active on photosynthetic and
respiratory activities compared to mitoPBN (Table 2, entries
2−4). This background cytotoxicity of compounds containing
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a
b
Table 1. 31P NMR Titration and Relaxation Parameters of Mito-aminophosphonates in Comparison with Inorganic
Phosphate (Pi) and DPP
c
a
δa and δb, limiting 31P NMR chemical shift in acidic and basic medium, respectively; Δδab = δa−δb; data were determined at 25 °C in Krebs−
b
Henseleit buffer and are given as means SEM from three repeated experiments. T1 = spin−lattice relaxation time determined at pH ≈ pKa.
c
d
−
2−
Refers to the second acidity of the H2PO4 /HPO4 couple. nd: not determined.
the Ph3P+ group, which has been documented in mitoPBN and
other mitochondria targeted antioxidants derived from vitamin
E or coenzyme Q, is generally not considered as a drawback
since applying very low nontoxic concentrations (e.g., micro-
molar or nanomolar) leads to local millimolar mitochondrial
levels still enabling NMR detection.21,22,27
In structure A, mito-aminophosphonates either increasing
the length of the spacer from n = 4 to 12, or replacing a methyl
by an isopropyl group in the diethoxyphosphonyl arm for a
given n value expectedly resulted both in an increase of clogD
and a decrease of LD50 values (see Table 2, entries 5−8 versus
9−12). Overall, structure A compounds 8a, 4a, 8b, 4b, and 7b
together with Structure B compound 22 having a short n = 3
spacer demonstrated the better innocuity in C. reinhardtii and
thus may have the best potential as mitochondrial pH reporters
in plant cells. Since these molecules (excepting 22) cover a pH
range ∼5.0−8.0 with a maximal 31P NMR sensitivity (Table 1),
they could be interesting alternatives to other pH measurement
techniques to follow the hypoxia-induced fluctuations of the
external and mitochondrial matrix pHs.41
Regarding toxicity 3T3 fibroblasts were more resistant than
green algae and it was found that their viability and LDH
release values still paralleled lipophilicity in either structure A
compounds having the same R1 substituent (Table 2, entries
5−8 versus 9−12) or when carbon substitution of the R1 group
increased for the same n = 6 spacer length (Table 2, entries 8, 4,
and 11). Of interest was the observation of a significant cell
necrosis when mitoPBN (but not PBN itself) or only 3 of the
10 novel mito-aminophosphonates tested were added to the
culture medium at a concentration that preserved 90% cell
viability. This allowed to select the more biocompatible 4b and
7b in the next perfusion experiments, which are best able to
preserve cellular function and integrity, and showed pKa values
compatible with the more accurate pH determination.
Previously, similar LD50 values (up to 50 μM) were obtained
in cells for a series Ph3P+-containing antioxidants, and it was
postulated that this toxicity is mainly determined by the
lipophilic cation.21,22,24−27 Here we found this cytotoxicity to
be lowered up to a factor of 80 in fibroblasts for mito-
aminophosphonates having a n = 4−8 alkyl spacer (Table 2).
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Table 2. Toxicity and Distribution Coefficients of Mito-aminophosphonates and Reference Compounds in Chlamydomonas
a
b
reinhardtii and 3T3 Murine Fibroblasts
C. reinhardtii
d
3T3 cells
c
d
e
e
f
entry
compd
DPP
clogD
LD50 phot. (μM)
LD50 respectively (μM)
LD10 (mM)
LD50 (mM)
LDH release (UI/mg prot.)
reference compounds
g
1
2
3
4
0.86
0.43
1.32
4.61
nd
nd
76
100
102
7.2
11.9 1.9
13.1 1.4
14.2 1.9
49.7 2.8*
DEPMPH
PBN
>1000
>500
100
>1000
78
>500
4.1
0.05
mitoPBN
50
0.2
mito-aminophosphonates
5
8a
2.42
3.21
4.18
5.71
2.15
3.12
3.85
5.45
3.71
2.92
250
130
25
150
130
25
0.3
0.6
0.5
0.3
0.02
0.7
0.6
0.3
0.1
0.3
0.7
10.7 1.8
15.9 1.9
49.4 1.4*
52.7 3.8*
10.2 1.2
14.9 2.1
17.7 1.9
44.7 3.1*
16.7 1.1
12.9 2.2
6
4a
0.2
7
7a
0.05
0.01
0.4
8
18a
8b
4b
7b
18b
4c
<10
500
250
100
<25
nd
<10
400
100
75
9
10
11
12
13
14
0.2
0.15
0.05
0.2
<25
nd
22
300
300
0.5
a
b
*
Data are means of at least three independent experiments. Data are means SEM of 3−6 independent experiments. P < 0.05 vs untreated cells,
c
d
by one-way ANOVA followed by Duncant test. Calculated at pH 7.4. Concentration causing 50% loss of photosynthetic or respiratory activity after
1 h exposure at 25 °C, as compared to control. LD10 and LD50 = compound concentrations at which 90% or 50% of the cells were viable,
respectively, after 3 h incubation at 37 °C. Measured after 1 h exposure at the concentration corresponding to LD10. In untreated cells the baseline
value was of 9.7 2.5 UI/mg prot. and the total LDH activity was 680 12.5 UI/mg prot. (n = 6). nd: Not determined.
e
f
g
Table 3. Intracellular Distribution, Effect on Tissue ATP, and Mitochondrial Function of Spin Traps and pH Markers (0.1 mM)
a
after 1 h Normothermic Perfusion in the Isolated Perfused Rat Liver
b
b
b
c
c,d
compd
none
cytosol (μmol/g)
mitochondria (μmol/g)
tissue ATP (μmol ATP/g)
membrane potential (mV)
respiratory control ratio
2.38 0.21
2.11 0.22
1.95 0.11*
2.33 0.12
2.29 0.29
2.08 0.30
180.9 3.4
178.9 3.2
170.7 3.2
181.3 1.9
174.2 2.9
171.9 3.0
6.2 1.3
5.5 0.5
4.7 1.1*
5.9 0.6
5.7 0.7
5.2 0.7
e
PBN
mitoPBN
DPP
4b
0.88 0.11
0.12 0.04
0.56 0.12
0.93 0.15
0.64 0.13
<0.05
1.95 0.17
e
<0.05
1.55 0.28
1.32 0.23
7b
a
*
Data are means SD of 3−9 independent experiments. P < 0.05 vs Krebs-Henseleit perfused livers, by one-way ANOVA followed by Duncant
b
c
d
test. Expressed per gram of fresh liver tissue. From isolated mitochondria prepared at the end of perfusion. Defined as the ratio of the oxidation
rate under phosphorylating vs nonphosphorylating conditions. Below the detection limit.
e
This could be in part related to the protective effect of α-
aminophosphonates seen in ischemic-reperfused isolated rat
hearts and livers we previously assigned to an intrinsic effect of
the diethoxyphosphoryl group.13,15
DPP seen here could be a consequence of a release into the KH
buffer used during washout by passive membrane diffusion, a
mechanism that was evidenced for uncharged aminophospho-
nates.15
Experiments on the Isolated Perfused Rat Liver and
Mitochondrial Function. To check for their intracellular
uptake and mitochondria targeting properties in a highly active
metabolizing environment, compounds 4b and 7b were
compared to mitoPBN using isolated rat livers perfused for 1
h with a KH buffer containing 0.1 mM of test compound
followed by a 10 min washout with plain cold KH. As
compared to mitoPBN, the more hydrophilic compounds 4b
and 7b exhibited (i) a good accumulation within the
mitochondrial fraction and (ii) a significantly better recovery
in the cytosolic fraction (i.e., the cytosolic distribution
percentage was 7%, 33%, and 33% for mitoPBN, 7b and 4b,
respectively; see Table 3). As expected, the mitochondrial
content of the parent DPP and PBN was neglectible (Table 3),
this latter result being in line with a study showing that
penetration of PBN within the nuclear and mitochondrial
fractions of isolated perfused rat hearts is very low.42 Compared
to the more lipophilic PBN, the relatively low cytosolic levels of
To illustrate the different patterns of cytosolic versus
mitochondrial distribution of Table 3, homogenates recovered
in each fraction from the same liver perfused with 7b or
mitoPBN were extracted with dichloromethane, evaporated to
dryness and redissolved in 1 mL CDCl3. Individual 31P NMR
spectra were then recorded (Figure 3) showing for both
compounds no new NMR peaks indicating significant
metabolic degradation, and the unique property of 7b to
indicate simultaneously mitochondrial (Figure 3a) and
cytosolic (Figure 3b) pH values unlike the nitrone, which
only accumulates within the mitochondria, giving a pH-
insensitive signal (see Figure 3c and arrow in Figure 3d).
The easy 31P NMR detection of 4b and 7b in a solvent volume
representative of liver cytosol and mitochondria spaces shows
that these compounds have accumulated to a large extent in
liver cells despite their low concentrations in the perfusate. This
is in line with previous studies on Ph3P+-containing compounds
reporting a 5−10-fold accumulation in the cytosol relative to
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Figure 3. 31P NMR signals in CDCl3 recorded from 1 mL samples of mitochondrial (a, c) and cytosolic (b, d) extracts from the same rat liver that
underwent a 60 min normothermic perfusion with Krebs−Henseleit buffer containing 0.1 mM of 7b (a, b) or mitoPBN (c, d). Inset a: Typical
spectrum of the 7b-supplemented perfusion fluid. Chemical shifts (in ppm relative to 85% H3PO4 in D2O): Pi, 2.44; Ph3P+-, 24.10 in buffer and
24.50 (7b) or 24.68 (mitoPBN) in CDCl3, and (EtO)2P(O)-, 31.18 in perfusion buffer (corresponding to pH 7.35) and 31.40 in CDCl3. The arrow
indicates the hardly visible cytosolic mitoPBN peak. The signal-to-noise ratios measured for Ph3P+ signal peaks in a, b, c, and d are 36.7, 14.0, 262.9,
and 2.8, respectively.
extracellular concentration followed by a 100−6000-fold
accumulation in mitochondria (versus cytosol), giving milli-
molar levels into the mitochondrial matrix.25,27
7b which demonstrated a unique potential to finely monitor pH
gradients at the subcellular level in plant and animal studies.
Table 3 shows that none of the tested compounds
significantly altered control tissue ATP levels at the end of
perfusion period, with the notable exception of mitoPBN.
Current opinion associates excess uptake of lipophilic cations
such as Ph3P+ in the mitochondria to disruption of ATP
synthesis as a result of their adsorption on the surface of the
inner membrane.25,27 Accordingly, mitochondria isolated from
livers after the end of perfusion with mitoPBN demonstrated
significant impairment of respiratory control ratio despite
membrane potential (an index of inner mitochondrial
membrane integrity) was maintained (Table 3). Compared to
control values, these two parameters were maintained by 4b
and 7b possibly because of their lower lipophilicity threshold
and accumulation profile within the cytosol and the
mitochondria (Figure 3).
EXPERIMENTAL SECTION
■
Chemistry. General Methods. Starting materials, reagents, solvents
and the spin trap PBN were obtained from Aldrich Chemical Co. and
were used without further purification. Analytical 1H NMR (operating
at 300.1 MHz), 13C NMR (operating at 75.5 MHz), and 31P NMR
(operating at 121.5 MHz) spectra were recorded in the indicated
solvent on a Bruker AVL300 spectrometer. Chemical shifts were
expressed as δ (ppm) relative to TMS. Coupling constants (J) were
reported in Hertz (Hz) and abbreviations of multiplicity were as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of
doublets. Progress of the reaction was monitored by TLC on silica gel
60 aluminum plates with F254 as indicator (Merck) and products were
purified by flash chromatography on silica gel 60 (230−400 mesh).
Melting points were determined with a Stuart melting point apparatus
SMP30 (Bibby Scientific, U.K.) and are not corrected. High resolution
mass spectrometry (HRMS) in electron spray ionization (ESI) was
performed at the Spectropole (Analytical Laboratory) at Campus St.
́ ̂
Jerome (Marseille, France) on a Q-STAR Elite instrument (Applied
Biosystems, U.S.A.). The purity of key compounds was checked with a
1200 series HPLC apparatus (Agilent; Santa Clara, CA) using a
Beckman Coulter C18 (250 × 10 mm, 5 μm) reverse phase column.
The mobile phase A was 0.01% trifluoroacetic acid in water and the
mobile phase B was acetonitrile, which was increased linearly from
10% to 100% over 21 min, and 100% over the next 8 min with a flow
rate of 2.5 mL/min. Peaks were detected at 230 nm and showed ≥95%
purity for all compounds in this system. The retention times Rt of
selected compounds are reported in minutes. Yields referred to
purified products and were not optimized. Chiral compounds were
CONCLUSIONS
■
This work demonstrates the feasibility of triphenylphospho-
nium salts bearing an α-aminophosphonate scaffold as
nontoxic, poorly metabolizable, specific, and sensitive 31P
NMR probes of mitochondrial pH. Modulating the spacer
length between the two functional groups allowed a fine-tuning
of their pKa value and cytosolic versus mitochondrial
distribution, resulting in the two optimized bromides 4b and
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isolated as racemates. The starting compounds and reagents: (6-
hydroxyhexyl)-carbamic acid 1,1-dimethylethyl ester (1),29 (6-
iodohexyl)-carbamic acid 1,1-dimethylethyl ester (2),29 6-bromohex-
an-1-ol (11),33 12-bromododecan-1-ol (12)33 (for these two latter
compounds, the synthetic procedure is summarized in Scheme 3), 5-
iodopentan-2-one (19),36 IBTP,32 DPP,11 and [4-[4-[[(1,1-
d i m e t h y l e t h y l ) o x i d o i m i n o ] m e t h y l ] p h e n o x y ] b u t y l ] -
triphenylphosphonium bromide (mitoPBN)26 were synthesized
according to reported procedures.
P+Ph3), 22.39 (d, J = 50.6 Hz, −CH2−P+Ph3), 26.38 (−CH2−), 27.15
(d, J = 6.6 Hz, 3 × −CH3), 29.81 (−CH2−), 29.97 (d, J = 16.9 Hz,
−CH2−CH2−CH2−P+Ph3), 34.81 (d, J = 7.1 Hz, −C(CH3)3), 51.09
(d, J = 2.7 Hz, −CH2−), 60.83, 61.30 (2 × −OCH2−), 64.71 (d, J =
140.0 Hz, CH), 117.76 (d, J = 86.2 Hz, −P+Ph3 ipso), 130.15 (d, J =
12.6 Hz, −P+Ph3 meta), 133.18 (d, J = 9.9 Hz, −P+Ph3 ortho), 134.71
+
(d, J = 2.7 Hz, −P+Ph3 para). HRMS-ESI: calcd for C33H48NO3P2
568.3104, found 568.3101.
Synthesis of Aminophosphonates 5a and 5b. Either isobutyr-
aldehyde (synthesis of 5a; 139 mmol) or acetone (synthesis of 5b; 139
mmol) was bubbled with dry ammonia at room temperature for 1 h.
Diethylphosphite (17.8 mL, 139 mmol) was then added slowly, and
the solution was heated at 65 °C for 4 h. After it was cooled, the
mixture was evaporated to dryness in vacuo, and the desired
compound was isolated by distillation under reduced pressure.
Diethyl 1-Amino-2-methylpropylphosphonate (5a). White liquid
(10.0 g, 34%), bp 47−49 °C/3.1 mmHg: 1H NMR (CDCl3) δ 0.94 (d,
3 H, J = 7.0 Hz, −CH3), 0.98 (d, 3 H, J = 7.0 Hz, −CH3), 1.26 (t, 6 H,
J = 7.0 Hz, 2 × −OCH2CH3), 1.97−2.09 (m, 1 H, −CH(CH3)2),
2.74−2.78 (m, 1 H), 4.01−4.12 (m, 4 H, 2 × −OCH2−); 31P NMR
(CDCl3) δ 28.61; 13C NMR (CDCl3) δ 16.33, 16.39 (2 ×
−OCH2CH3), 17.17 (d, J = 4.4 Hz, −CH3), 20.49 (d, J = 13.2 Hz,
−CH3), 28.98 (−CH(CH3)2), 54.10 (d, J = 146.0 Hz, CH), 61.62,
61.69 (2 × −OCH2−). HRMS-ESI: calcd for C8H20NO3P ([M + H]+)
210.1254, found 210.1261.
Diethyl 2-Aminopropan-2-ylphosphonate (5b). White liquid
(10.5 g, 39%), bp 52−54 °C/3.0 mmHg: 1H NMR (CDCl3) δ
1.16−1.30 (m, 12 H), 4.01− 4.12 (m, 4 H, 2 × −OCH2−); 31P NMR
(CDCl3) δ 32.39; 13C NMR (CDCl3) δ 16.54, 16.61 (2 × −CH3),
25.05 (d, J = 3.8 Hz, 2 × −CH3), 49.00 (d, J = 147.7 Hz, C), 62.18,
62.28 (2 × −OCH2−). HRMS-ESI: calcd for C7H18NO3P ([M + H]+)
196.1097, found 196.1085.
S y n t h e s i s o f 6 - [ ( t e r t - B u t o x y c a r b o n y l ) a m i n o ] -
hexyltriphenylphosphonium Iodide (3). A flask charged with 2 (3.0
g, 9.17 mmol) and triphenylphosphine (4.0 g, 15.26 mmol) in dioxane
(40 mL) was heated at 80 °C for 12 h in the dark. The mixture was
cooled and added dropwise to well-stirred Et2O (200 mL). The
resulting white precipitate was separated by filtration, dissolved in
CH2Cl2 (10 mL), and added dropwise to well-stirred Et2O (100 mL).
The resulting white solid was distilled under reduced pressure to give 3
1
(5.0 g, 93%): H NMR (CDCl3) δ 1.29−1.46 (m, 13 H), 1.60−1.75
(m, 4 H), 3.00−3.10 (m, 2 H, −CH2−), 3.55−3.69 (m, 2 H, −CH2−
P+Ph3), 7.60−7.90 (m, 15 H, −P+Ph3); 31P NMR (CDCl3) δ 24.48;
13C NMR (CDCl3) δ 22.32 (d, J = 3.7 Hz, −CH2−CH2−P+Ph3),
22.80 (d, J = 50.6 Hz, −CH2−P+Ph3), 25.86 (−CH2−), 28.18 (3 ×
−CH3), 29.32 (−CH2−), 29.75 (d, J = 16.9 Hz, −CH2−CH2−CH2−
P+Ph3), 39.91 (−CH2−), 78.59 (C), 118.30 (d, J = 85.8 Hz, −P+Ph3
ipso), 130.41 (d, J = 12.6 Hz, −P+Ph3 meta), 133.41 (d, J = 9.9 Hz,
−P+Ph3 ortho), 134.92 (d, J = 2.7 Hz, −P+Ph3 para), 155.89 (CO).
HRMS-ESI: calcd for C29H37NIO2P+ 462.2556, found 462.2555.
Synthesis of Mito-aminophosphonates 4a and 4c. A solution of 3
(1.0 g, 1.69 mmol) in CH2Cl2 (10 mL) was stirred with 4 M HCl in
dioxane (1 mL) for 3 h at room temperature and concentrated under
reduced pressure. The resulting yellow solid was washed with Et2O,
dried under reduced pressure, dissolved in dry CH2Cl2 (15 mL) under
argon, and TEA was added slowly to reach a pH of 6.5−7.0. After the
mixture was stirred for 1 h at room temperature, 2.00 mmol of either
pivaldehyde (synthesis of 4a) or isobutyraldehyde (synthesis of 4c)
was added and the reaction mixture was stirred for 12 h at room
temperature. Diethyl phosphite (540 μL, 4.20 mmol) was then added
dropwise and the mixture was refluxed for 2 days under argon. After
removal of the precipitate by filtration, the mixture was added
dropwise to well-stirred Et2O (50 mL). After cooling, the solvent layer
was decanted and the resulting pale yellow oil was dissolved in CH2Cl2
(5 mL) and added dropwise to well-stirred Et2O (50 mL). The
resulting oil was dissolved in CH2Cl2 (15 mL), washed with water (15
mL), and washed twice with 15% aqueous NaBr solution. The organic
layer was dried with MgSO4 and evaporated to dryness in vacuo to
give the desired compound.
Synthesis of Aminophosphonates 6a and 6b. To a stirred
solution of 4.70 mmol of either 5a (synthesis of 6a) or 5b (synthesis
of 6b) and K2CO3 (1.30 g, 9.42 mmol) in acetonitrile (20 mL) was
added 1,8-dibromooctane (1.3 mL, 7.05 mmol) over 10 min, and the
reaction mixture was refluxed for 24 h. After removal of the precipitate
by suction filtration, the filtrate was evaporated to dryness in vacuo,
and the residue was purified by flash chromatography using CH2Cl2/
EtOH (98/2) as eluent.
Diethyl 1-(8-Bromooctylamino)-2-methylpropylphosphonate
1
(6a). Yellow oil (1.05 g, 56%): H NMR (CDCl3) δ 0.94 (d, 3 H, J
= 7.0 Hz, −CH3), 0.99 (d, 3 H, J = 7.0 Hz, −CH3), 1.20−1.45 (m, 16
H), 1.76−1.83 (m, 2 H, −CH2−CH2−Br), 2.01−2.14 (m, 1 H), 2.53−
2.80 (m, 3 H), 3.35 (t, 2 H, J = 6.8 Hz, −CH2-Br), 4.01−4.12 (m, 4 H,
2 × -OCH2−); 31P NMR (CDCl3) δ 28.82; 13C NMR (CDCl3) δ
16.33, 16.40 (2 × −OCH2CH3), 17.83 (d, J = 4.4 Hz, −CH3), 20.50
(d, J = 12.6 Hz, −CH3), 26.87 (−CH2−), 27.91 (−CH2−), 28.52
(−CH2−), 29.13 (−CH2−), 30.23 (−CH2−), 28.90 (d, J = 4.9 Hz,
−CH(CH3)2), 32.60 (−CH2−), 33.79 (−CH2−), 49.70 (d, J = 4.9 Hz,
−CH2−), 60.59 (d, J = 146.0 Hz, CH), 61.40, 61.60 (2 × −OCH2−).
HRMS-ESI: calcd for C16H35BrNO3P ([M + H]+) 400.1611, found
400.1607.
6 - ( 1 - D i e t h y l p h o s p h o r y l - 2 - m e t h y l p r o p y l a m i n o ) -
hexyltriphenylphosphonium Bromide (4a). Sticky yellow oil (0.43 g,
43%): 1H NMR (CDCl3) δ 0.84 (d, 3 H, J = 7.0 Hz, −CH3), 0.87 (d,
3 H, J = 7.0 Hz, −CH3), 1.25 (t, 6 H, J = 7.0 Hz, 2 × −OCH2CH3),
1.31−1.42 (m, 4 H), 1.50−1.65 (m, 4 H), 1.98−2.13 (m, 1 H,
−CH(CH3)2), 2.50−2.82 (m, 3 H), 3.55−3.70 (m, 2 H, −CH2−
P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15 H,
−P+Ph3); 31P NMR (CDCl3) δ 24.18, 28.73; 13C NMR (CDCl3) δ
16.38, 16.44 (2 × −OCH2CH3), 17.25 (d, J = 3.9 Hz, −CH3), 20.50
(d, J = 14.2 Hz, −CH3), 22.23 (d, J = 3.7 Hz, −CH2−CH2−P+Ph3),
22.52 (d, J = 50.6 Hz, −CH2−P+Ph3), 26.30 (−CH2−), 28.88 (d, J =
4.9 Hz, −CH(CH3)2), 29.62 (−CH2−), 30.01 (d, J = 16.9 Hz, −CH2−
CH2−CH2−P+Ph3), 49.23 (d, J = 5.0 Hz, −CH2−), 60.50 (d, J = 146.0
Hz, CH), 60.70, 61.22 (2 × −OCH2−), 117.15 (d, J = 85.8 Hz,
−P+Ph3 ipso), 130.17 (d, J = 12.6 Hz, −P+Ph3 meta), 133.16 (d, J =
9.9 Hz, −P+Ph3 ortho), 134.83 (d, J = 2.7 Hz, -P+Ph3 para). HRMS-
Diethyl 2-(8-Bromooctylamino)Propan-2-ylphosphonate (6b).
1
Yellow oil (0.87 g, 48%): H NMR (CDCl3) δ 1.15−1.41 (m, 22
H), 1.70−1.80 (m, 2 H, −CH2−CH2−Br), 2.62 (t, 2 H, J = 6.8 Hz,
−CH2−), 3.31 (t, 2 H, J = 6.8 Hz, −CH2−Br), 4.01−4.12 (m, 4 H, 2
× −OCH2−); 31P NMR (CDCl3) δ 31.84; 13C NMR (CDCl3) δ
16.33, 16.40 (2 × −OCH2CH3), 23.67 (d, J = 2.7 Hz, 2 × −CH3),
26.90 (−CH2−), 27.84 (−CH2−), 28.43 (−CH2−), 29.04 (−CH2−),
30.57(−CH2−), 32.53 (−CH2−), 33.71 (−CH2−), 42.74 (d, J = 4.9
Hz, −CH2−), 53.19 (d, J = 146.0 Hz, C), 61.79, 61.89 (2 ×
−OCH2−). HRMS-ESI: calcd for C15H33BrNO3P ([M + H]+)
386.1454, found 386.1450.
+
ESI: calcd for C32H46NO3P2 554.2947, found 554.2947.
6-(1-Diethylphosphoryl-2,2-dimethylpropylamino)-
hexyltriphenylphosphonium Bromide (4c). Sticky yellow oil (0.52 g,
Synthesis of Mito-aminophosphonates 7a and 7b. To a solution
of 1.12 mmol of either 6a (synthesis of 7a) or 6b (synthesis of 7b) in
acetonitrile (15 mL) was added triphenylphosphine (0.50 g, 1.91
mmol), and the reaction mixture was refluxed for 72 h in the dark
under argon. After it was cooled, the reaction mixture was
concentrated under reduced pressure, the residue was dissolved in
1
48%): H NMR (CDCl3) δ 1.00 (s, 9 H, 3 × −CH3), 1.23−1.40 (m,
10 H), 1.55−1.70 (m, 4 H), 2.30−2.85 (m, 3 H), 3.60−3.72 (m, 2 H,
−CH2−P+Ph3), 4.00−4.15 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15
H, −P+Ph3); 31P NMR (CDCl3) δ 23.95, 28.28; 13C NMR (CDCl3) δ
16.09, 16.21 (2 × −OCH2CH3), 22.17 (d, J = 3.7 Hz, −CH2−CH2−
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CH2Cl2 (10 mL), and the resulting solution was added dropwise to
well-stirred Et2O (50 mL). After the mixture was cooled, the solvent
layer was decanted, and the resulting pale yellow oil was dissolved in
CH2Cl2 (5 mL), and added dropwise to well-stirred Et2O (30 mL).
After this CH2Cl2/Et2O extraction was repeated two more times, the
resulting precipitate was redissolved in CH2Cl2 (20 mL), washed twice
with 15% aqueous NaBr solution, dried over MgSO4, and evaporated
to dryness in vacuo to give the desired compound.
134.97 (d, J = 2.9 Hz, −P+Ph3 para). HRMS-ESI: calcd for
+
C30H42NO3P2 526.2634, found 526.2633; Rt 23.65.
4 - ( 2 - D i e t h y l p h o s p h o r y l p r o p a n - 2 - y l a m i n o ) -
butyltriphenylphosphonium Bromide (8b). Sticky yellow oil (2.52 g,
1
72%): H NMR (CDCl3) δ 1.17−1.28 (m, 12 H), 1.68−1.85 (m, 4
H), 2.73 (t, 2 H, J = 6.8 Hz, −CH2−), 3.68−3.78 (m, 2 H, −CH2−
P+Ph3), 4.01−4.08 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15 H,
−P+Ph3); 31P NMR (CDCl3) δ 24.32, 30.79; 13C NMR (CDCl3) δ
16.56, 16.61 (2 × −OCH2CH3), 20.31 (d, J = 4.4 Hz, −CH2−CH2−
P+Ph3), 22.71 (d, J = 50.6 Hz, −CH2−P+Ph3), 23.08 (d, J = 2.9 Hz, 2
× −CH3), 31.02 (d, J = 16.1 Hz, −CH2−CH2−CH2−P+Ph3), 41.90
(d, J = 4.4 Hz, −CH2−), 53.22 (d, J = 149.7 Hz, C), 61.98, 62.05 (2 ×
−OCH2−), 118.19 (d, J = 85.8 Hz, −P+Ph3 ipso), 130.46 (d, J = 12.5
Hz, −P+Ph3 meta), 133.71 (d, J = 10.3 Hz, −P+Ph3 ortho), 134.99 (d,
8 - ( 1 - D i e t h y l p h o s p h o r y l - 2 - m e t h y l p r o p y l a m i n o ) -
octyltriphenylphosphonium Bromide (7a). Sticky yellow oil (0.24 g,
32%): 1H NMR (CDCl3) δ 0.93 (d, 3 H, J = 7.0 Hz, −CH3), 0.98 (d,
3 H, J = 7.0 Hz, −CH3), 1.13−1.40 (m, 14 H), 1.50−1.65 (m, 4 H),
1.98−2.13 (m, 1 H, −CH(CH3)2), 2.50−2.82 (m, 3 H), 3.55−3.70
(m, 2 H, −CH2−P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−), 7.60−
7.90 (m, 15 H, −P+Ph3); 31P NMR (CDCl3) δ 24.06, 28.56; 13C NMR
(CDCl3) δ 15.76, 15.83 (2 × −OCH2CH3), 17.22 (d, J = 4.4 Hz,
−CH3), 19.91 (d, J = 12.6 Hz, −CH3), 21.78 (d, J = 3.8 Hz, −CH2−
CH2−P+Ph3), 21.95 (d, J = 50.5 Hz, −CH2−P+Ph3), 26.20 (−CH2−),
28.25 (d, J = 4.9 Hz, −CH(CH3)2), 28.29 (−(CH2)2−), 29.61
(−CH2−), 29.64 (d, J = 15.9 Hz, −CH2−CH2−CH2−P+Ph3), 49.08
(d, J = 4.9 Hz, −CH2−), 59.80 (d, J = 135.0 Hz, CH), 60.80, 60.90 (2
× −OCH2−), 117.38 (d, J = 86.2 Hz, −P+Ph3 ipso), 129.80 (d, J =
12.6 Hz, −P+Ph3 meta), 132.80 (d, J = 9.9 Hz, −P+Ph3 ortho), 134.32
+
J = 2.9 Hz, −P+Ph3 para). HRMS-ESI: calcd for C29H40NO3P2
512.2478, found 512.2477; Rt 22.33.
Synthesis of Compounds 13 and 14. A mixture of 33.15 mmol of
either 1133 (synthesis of 13) or 1233 (synthesis of 14) and
triphenylphosphine (8.80 g, 33.46 mmol) in acetonitrile (60 mL)
was refluxed for 48 h in the dark under argon. After it was cooled, the
reaction mixture was added dropwise to well-stirred Et2O (200 mL),
the solvent layer was decanted and the resulting white solid was
dissolved in CH2Cl2 (20 mL) and added dropwise to well-stirred Et2O
(100 mL). The resulting white solid was evaporated to dryness in
vacuo to give the desired compound.
+
(d, J = 2.7 Hz, −P+Ph3 para). HRMS-ESI: calcd for C34H50NO3P2
582.3260, found 582.3263; Rt 25.66.
6-Hydroxyhexyltriphenylphosphonium Bromide (13). White solid
(13.52 g, 92%): mp 133 °C; 1H NMR (CDCl3) δ 1.40−1.44 (m, 4 H),
1.60−1.64 (m, 4 H), 3.50−3.54 (t, 2 H, J = 7.0 Hz, HO−CH2−),
3.58−3.65 (m, 2 H, −CH2−P+Ph3), 7.60−7.90 (m, 15 H, −P+Ph3);
31P NMR (CDCl3) δ 24.30; 13C NMR (CDCl3) δ 22.24 (d, J = 3.7 Hz,
−CH2−CH2−P+Ph3), 22.36 (d, J = 50.6 Hz, -CH2−P+Ph3), 24.69
(−CH2−), 29.42 (d, J = 16.9 Hz, −CH2−CH2−CH2−P+Ph3), 31.85
(−CH2−), 61.29 (−CH2−), 118.18 (d, J = 85.8 Hz, −P+Ph3 ipso),
130.46 (d, J = 12.6 Hz, −P+Ph3 meta), 133.54 (d, J = 9.9 Hz, −P+Ph3
meta), 134.96 (d, J = 2.7 Hz, −P+Ph3 para). HRMS-ESI: calcd for
C24H28OP+ 363.1872, found 363.1873.
8 - ( 2 - D i e t h y l p h o s p h o r y l p r o p a n - 2 - y l a m i n o ) -
octyltriphenylphosphonium Bromide (7b). Sticky yellow oil (0.18 g,
1
25%): H NMR (CDCl3) δ 1.10−1.37 (m, 20 H), 1.50−1.65 (m, 4
H), 2.55−2.65 (t, 2 H, J = 6.8 Hz, −CH2−), 3.55−3.70 (m, 2 H,
−CH2−P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15
H, −P+Ph3); 31P NMR (CDCl3) δ 24.42, 31.51; 13C NMR (CDCl3) δ
16.41, 16.48 (2 × −OCH2CH3), 22.42 (d, J = 3.7 Hz, −CH2−CH2−
P+Ph3), 22.50 (d, J = 50.6 Hz, −CH2−P+Ph3), 22.83 (d, J = 3.3 Hz, 2
× −CH3), 26.96 (−CH2−), 28.92 (−(CH2)2−), 30.20 (d, J = 15.9 Hz,
−CH2−CH2−CH2−P+Ph3), 30.80 (−CH2−), 42.70 (d, J = 5.5 Hz,
−CH2−), 53.08 (d, J = 146.0 Hz, C), 61.77, 61.87 (2 × −OCH2−),
118.14 (d, J = 85.6 Hz, −P+Ph3 ipso), 130.36 (d, J = 12.6 Hz, −P+Ph3
meta), 133.44 (d, J = 9.9 Hz, −P+Ph3 ortho), 134.90 (d, J = 2.7 Hz,
12-Hydroxydodecyltriphenylphosphonium Bromide (14). White
1
solid (15.91 g, 91%): mp 99 °C; H NMR (CDCl3) δ 1.10−1.23 (m,
+
−P+Ph3 para). HRMS-ESI: calcd for C33H48NO3P2 568.3104, found
16 H), 1.45−1.60 (m, 4 H), 3.55 (t, 2 H, J = 7.0 Hz, HO-CH2-), 3.64
(m, 2 H, −CH2−P+Ph3), 7.60−7.90 (m, 15 H, −P+Ph3); 31P NMR
(CDCl3) δ 24.26; 13C NMR (CDCl3) δ 22.50 (d, J = 3.7 Hz, −CH2−
CH2−P+Ph3), 22.55 (d, J = 50.6 Hz, −CH2−P+Ph3), 25.59−29.29 (7
× −CH2), 30.25 (d, J = 16.9 Hz, −CH2−CH2−CH2−P+Ph3), 32.62
(−CH2−), 62.64 (−CH2−), 118.20 (d, J = 95.7 Hz, −P+Ph3 ipso),
130.42 (d, J = 12.6 Hz, −P+Ph3 meta), 133.54 (d, J = 9.9 Hz, −P+Ph3
ortho), 134.92 (d, J = 2.7 Hz, −P+Ph3 para). HRMS-ESI: calcd for
568.3104; Rt 25.00.
Synthesis of Mito-aminophosphonates 8a and 8b. To a solution
of 6.00 mmol of either 5a (synthesis of 8a) or 5b (synthesis of 8b) and
K2CO3 (2.20 g, 15.94 mmol) in acetonitrile (25 mL) was added IBTP
(3.37 g, 5.90 mmol) over 10 min. The reaction mixture was refluxed
for 72 h in the dark under argon. After removal of the precipitate by
filtration, the filtrate was evaporated to dryness in vacuo. The residue
was dissolved in CH2Cl2 (10 mL) and added dropwise to well-stirred
Et2O (50 mL). After the mixture was cooled, the solvent layer was
decanted and this CH2Cl2 (5 mL)/Et2O (30 mL) extraction was
repeated. After decantation, the resulting oil was dissolved in water (15
mL), acidified with HCl (1 N), and extracted with ethyl acetate (2 ×
10 mL). The aqueous layer was basified with 10% aqueous NaOH
solution and extracted with CH2Cl2 (2 × 20 mL). The combined
organic layers were washed twice with 15% aqueous NaBr solution,
dried over MgSO4, and concentrated to dryness in vacuo to give the
desired compound.
+
C30H40OP 447.2811, found 447.2808.
Synthesis of Compounds 15 and 16. To a solution of 3.38 mmol
of either 13 (synthesis of 15) or 14 (synthesis of 16) in toluene (15
mL) was added a 48% aqueous HBr solution (565 μL, 5.00 mmol).
The reaction mixture was heated at 85 °C for 12 h. After it was cooled,
the reaction mixture was extracted with CH2Cl2 (15 mL), washed with
1 M NaOH, dried over MgSO4, and evaporated to dryness in vacuo to
give the desired compound.
6-Bromohexyltriphenylphosphonium Bromide (15). Brown oil
1
(1.50 g, 88%): H NMR (CDCl3) δ 1.32−1.40 (m, 2 H), 1.52−1.75
(m, 6 H), 3.25−3.30 (t, 2 H, J = 7.0 Hz, Br−CH2−), 3.62−3.73 (m, 2
H, −CH2−P+Ph3), 7.60−7.90 (m, 15 H, −P+Ph3); 31P NMR (CDCl3)
δ 24.16; 13C NMR (CDCl3) δ 22.29 (d, J = 3.7 Hz, −CH2−CH2−
P+Ph3), 22.51 (d, J = 50.6 Hz, −CH2−P+Ph3), 27.42 (−CH2−), 29.22
(d, J = 16.9 Hz, −CH2−CH2−CH2−P+Ph3), 32.02 (−CH2−), 33.85
(−CH2−), 117.85 (d, J = 85.8 Hz, −P+Ph3 ipso), 130.35 (d, J = 12.6
Hz, −P+Ph3 meta), 133.44 (d, J = 9.9 Hz, −P+Ph3 ortho), 134.88 (d, J
= 2.7 Hz, −P+Ph3 para). HRMS-ESI: calcd for C24H27BrP+ 425.1028,
found 425.1022.
4 - ( 1 - D i e t h y l p h o s p h o r y l - 2 - m e t h y l p r o p y l a m i n o ) -
butyltriphenylphosphonium Bromide (8a). Sticky yellow oil (2.5 g,
70%): 1H NMR (CDCl3) δ 0.91 (m, 6 H, 2 × −CH3), 1.26 (t, 6 H, J =
7.0 Hz, 2 × −OCH2CH3), 1.70−1.89 (m, 4 H), 2.00−2.11 (m, 1 H,
−CH(CH3)2), 2.57−2.95 (m, 3 H), 3.69−3.81 (m, 2 H, −CH2−
P+Ph3), 4.01−4.08 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15 H,
−P+Ph3); 31P NMR (CDCl3) δ 24.41, 28.45; 13C NMR (CDCl3) δ
16.50, 16.55 (2 × −OCH2CH3), 17.81 (d, J = 3.7 Hz, −CH3), 20.08
(d, J = 3.7 Hz, −CH2−CH2−P+Ph3), 20.83 (d, J = 13.2 Hz, −CH3),
22.65 (d, J = 49.9 Hz, −CH2−P+Ph3), 28.99 (d, J = 4.9 Hz,
−CH(CH3)2), 30.40 (d, J = 16.9 Hz, −CH2−CH2−CH2−P+Ph3),
48.43 (d, J = 2.9 Hz, −CH2−), 60.45 (d, J = 143.0 Hz, CH), 61.45,
61.74 (2 × −OCH2−), 118.17 (d, J = 85.8 Hz, −P+Ph3 ipso), 130.45
(d, J = 12.5 Hz, −P+Ph3 meta), 133.71 (d, J = 10.2 Hz, −P+Ph3 ortho),
12-Bromododecyltriphenylphosphonium Bromide (16). Brown
1
oil (1.65 g, 83%): H NMR (CDCl3) δ 1.10−1.20 (m, 12 H), 1.30−
1.35 (m, 2 H), 1.52−1.57 (m, 4 H), 1.72−1.80 (m, 2 H, −CH2−),
3.30−3.35 (t, 2 H, J = 7.0 Hz, Br−CH2−), 3.58−3.68 (m, 2 H,
−CH2−P+Ph3), 7.60−7.90 (m, 15 H, −P+Ph3); 31P NMR (CDCl3) δ
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23.95; 13C NMR (CDCl3) δ 22.48 (d, J = 3.7 Hz, −CH2−CH2−
P+Ph3), 22.67 (d, J = 50.6 Hz, −CH2−P+Ph3), 27.94−29.32 (7 ×
−CH2−), 30.28 (d, J = 16.9 Hz, −CH2−CH2−CH2−P+Ph3), 32.62
(−CH2−), 34.04 (−CH2−), 118.16 (d, J = 85.8 Hz, −P+Ph3 ipso),
130.39 (d, J = 12.6 Hz, −P+Ph3 meta), 133.50 (d, J = 9.9 Hz, −P+Ph3
ortho), 134.92 (d, J = 2.7 Hz, −P+Ph3 para). HRMS-ESI: calcd for
C30H39BrP+ 509.1967, found 509.1966.
Synthesis of Mito-aminophosphonates 4b, 18a, and 18b. To a
solution of 5a (1.00 mmol) and K2CO3 (0.372 g, 2.70 mmol) in
acetonitrile (25 mL) was added 0.90 mmol of either compound 15
(synthesis of 4b) or 16 (synthesis of 18a) over 10 min and the
reaction mixture was refluxed for 5 days in the dark under argon. After
removal of the precipitate by filtration, the filtrate was evaporated to
dryness in vacuo. The mixture was dissolved in CH2Cl2 (5 mL) and
added dropwise to well-stirred Et2O (30 mL). After decantation, the
resulting oil was dissolved in water (10 mL), acidified with HCl (1 M),
and extracted with CH2Cl2 (5 mL). The aqueous layer was basified
with 10% aqueous NaOH solution and extracted with CH2Cl2 (2 × 15
mL). The combined organic layers were washed twice with 15%
aqueous NaBr solution, dried over MgSO4, and evaporated to dryness
in vacuo to give the desired compound. Compound 18b was obtained
using the same procedure used to obtain compound 18a by using 5b
(5.00 mmol) instead of 5a.
Synthesis of Mito-aminophosphonates 21 and 22. The iodide
salt of 5-mitopentan-2-one 20 was prepared from 10.0 g (47.17 mmol)
of 5-iodopentan-2-one 19 in toluene (60 mL) by adding
triphenylphosphine (13.1 g, 50.0 mmol) and refluxing the mixture
for 12 h in the dark under argon. The reaction mixture was cooled,
concentrated under reduced pressure, and the crude solid was
dissolved in CH2Cl2 (20 mL) and added dropwise to well-stirred
Et2O (150 mL). After decantation, the resulting white solid was
1
filtered and evaporated to dryness in vacuo to give 20. The H NMR
and 31P NMR data of 20 were in accordance with the data reported in
the literature for the corresponding bromide salt which was obtained
by another chemical pathway.43
5-(2-Oxo)pentyltriphenylphosphonium Iodide (20). White solid
1
(33.00 mmol, 15.7 g, 70%): mp 205 °C; H NMR (CDCl3) δ 1.75−
1.88 (m, 2 H, −CH2−), 2.13 (s, 3 H, −CH3), 3.03−3.07 (t, J = 6.05
Hz, 2 H, −CH2), 3.67−3.77 (m, 2 H, −CH2−P+Ph3), 7.60−7.90 (m,
15 H, −P+Ph3); 31P NMR (CDCl3) δ 24.26; 13C NMR (CDCl3) δ
16.60 (d, J = 2.8 Hz, −CH2−), 21.68 (d, J = 50.6 Hz, CH2−P+Ph3),
30.19 (−CH3), 42.49 (d, J = 17.1 Hz, −CH2−), 117.87 (d, J = 86.4
Hz, −P+Ph3 ipso), 130.35 (d, J = 12.7 Hz, −P+Ph3 meta), 132.56 (d, J
= 10.5 Hz, −P+Ph3 ortho), 134.95(d, J = 3.3 Hz, −P+Ph3 para), 208.38
(CO). HRMS-ESI: calcd for C23H24OP+ 347.1559, found 347.1555.
To prepare compound 21, compound 20 (1.00 mmol) in ethanol
(10 mL) was saturated with dry ammonia. After the mixture was
stirred for 1 h, diethylphosphite (1.10 mmol) was added slowly, and
the mixture was first stirred at room temperature for 4 h and then
heated at 65 °C during 4 h. After it was cooled, the mixture was
dissolved in water (15 mL), acidified with 1 N HCl, and extracted with
ethyl acetate (2 × 15 mL). The aqueous layer was basified with 10%
aqueous NaOH solution, and extracted with CH2Cl2 (2 × 50 mL).
The combined organic layers were washed twice with 15% aqueous
NaBr solution, dried over MgSO4, and evaporated to dryness in vacuo
to give 21. Compound 22 was obtained using the same procedure as
above except that 2-methylpropan-1-amine (1.00 mmol) was used
instead of ammonia.
6 - ( 2 - D i e t h y l p h o s p h o r y l p r o p a n - 2 - y l a m i n o ) -
hexyltriphenylphosphonium Bromide (4b). Sticky yellow oil (0.14 g,
1
25%): H NMR (CDCl3) δ 1.10−1.25 (m, 16 H), 1.52−1.58 (m, 4
H), 2.50−2.56 (t, 2 H, J = 6.8 Hz, −CH2−), 3.55−3.62 (m, 2 H,
−CH2−P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15
H, −P+Ph3); 31P NMR (CDCl3) δ 24.07, 31.13; 13C NMR (CDCl3) δ
16.42, 16.50 (2 × −OCH2CH3), 22.38 (d, J = 4.4 Hz, −CH2−CH2−
P+Ph3), 22.50 (d, J = 50.6 Hz, −CH2−P+Ph3), 22.76 (d, J = 3.3 Hz, 2
× −CH3), 26.62 (−CH2−), 30.11 (d, J = 16.0 Hz, −CH2−CH2−
CH2−P+Ph3), 30.35 (−CH2−), 42.65 (d, J = 5.2 Hz, −CH2−), 53.12
(d, J = 146.3 Hz, −C−), 62.03, 62.18 (2 × −OCH2−), 118.08 (d, J =
85.8 Hz, −P+Ph3 ipso), 130.33 (d, J = 12.1 Hz, −P+Ph3 meta), 133.48
(d, J = 9.9 Hz, −P+Ph3 ortho), 134.93 (d, J = 3.3 Hz, P+Ph3 para).
4-Amino-4-(diethylphosphoryl)pentyltriphenylphosphonium
1
Bromide (21). Sticky yellow oil (0.42 g, 75%): H NMR (CDCl3) δ
+
HRMS-ESI: calcd for C31H44NO3P2 540.2791, found 540.2783; Rt
1.14−1.18 (d, J = 15.8 Hz, 3 H, −CH3), 1.25 (t, 6 H, J = 7.0 Hz, 2 ×
−OCH2CH3), 1.70−1.85 (m, 4 H, 2 × −CH2−), 3.63−3.85 (m, 2 H,
−CH2−P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15
H, −P+Ph3); 31P NMR (CDCl3) δ 24.19, 30.43; 13C NMR (CDCl3) δ
16.62−16.74 (−CH3 and 2 × −OCH2CH3), 22.54 (d, J = 2.2 Hz,
−CH2−CH2−P+Ph3), 23.25 (d, J = 49.9 Hz, −CH2−P+Ph3), 37.45
(dd, J = 16.9, 4.4 Hz, −CH2−), 51.89 (d, J = 148.2 Hz, C), 62.57,
62.64 (2 × OCH2CH3), 118.12 (d, J = 85.8 Hz, −P+Ph3 ipso), 130.47
(d, J = 12.5 Hz, −P+Ph3 meta), 132.73 (d, J = 9.5 Hz, −P+Ph3 ortho),
135.04 (d, J = 2.9 Hz, −P+Ph3 para). HRMS-ESI: calcd for
23.66.
12-(1-Diethylphosphoryl-2-methylpropylamino)-
dodecyltriphenylphosphonium Bromide (18a). Sticky yellow oil
(0.14 g, 21%): 1H NMR (CDCl3) δ 0.94 (d, 3 H, J = 7.0 Hz, −CH3),
0.99 (d, 3 H, J = 7.0 Hz, −CH3), 1.12−1.30 (m, 22 H), 1.52−1.65 (m,
4 H), 2.02−2.11 (m, 1 H, −CH(CH3)2), 2.52−2.80 (m, 3 H), 3.65−
3.75 (m, 2 H, −CH2-P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−),
7.60−7.90 (m, 15 H, −P+Ph3); 31P NMR (CDCl3) δ 24.09, 28.59; 13
C
NMR (CDCl3) δ 15.78, 15.84 (2 × −OCH2CH3), 17.24 (d, J = 3.9
Hz, −CH3), 19.93 (d, J = 12.6 Hz, −CH3), 21.80 (d, J = 3.8 Hz,
−CH2−CH2−P+Ph3), 21.98 (d, J = 50.0 Hz, −CH2−P+Ph3), 26.36
(−CH2−), 28.48 (d, J = 4.9 Hz, −CH(CH3)2), 28.26−29.78 (7 ×
−CH2−), 29.67 (d, J = 14.8 Hz, −CH2−CH2−CH2−P+Ph3), 49.15 (d,
J = 4.9 Hz, −CH2−), 59.91 (d, J = 146.0 Hz, CH), 60.75, 60.94 (2 ×
−OCH2−), 117.42 (d, J = 85.6 Hz, −P+Ph3 ipso), 129.81 (d, J = 12.0
Hz, −P+Ph3 meta), 132.82 (d, J = 9.9 Hz, −P+Ph3 ortho), 134.33 (d, J
+
C27H36NO3P2 484.2165, found 484.2169.
4 - ( I s o b u t y l a m i n o ) - 4 - ( d i e t h y l p h o s p h o r y l ) -
pentyltriphenylphosphonium Bromide (22). Sticky yellow oil (0.43 g,
70%): 1H NMR (CDCl3) δ 0.77 (d, 6 H, J = 6.7 Hz, 2 × CH3), 1.14−
1.18 (d, J = 16.5 Hz, 3 H, −CH3), 1.25 (t, 6 H, J = 7.0 Hz, 2 ×
−OCH2CH3), 1.37−1.48 (m, 1 H, −CH(CH3)2), 1.70−1.85 (m, 4 H,
2 × −CH2−), 2.32−2.39 (m, 2 H, −CH2−), 3.50−3.86 (m, 2 H,
−CH2−P+Ph3), 4.01−4.12 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15
H, −P+Ph3); 31P NMR (CDCl3) δ 23.93, 29.63; 13C NMR (CDCl3) δ
15.56−16.63 (−CH3 and 2 × −OCH2CH3), 19.59 (2 × −CH3), 19.36
(−CH2−), 22.08 (d, J = 50.6 Hz, −CH2−P+Ph3), 19.98 (−CH-
(CH3)2), 34.38 (dd, J = 16.5, 4.9 Hz, −CH2−), 49.16 (d, J = 4.9 Hz,
−CH2−), 54.79 (d, J = 146.0 Hz, C), 60.20 (d, J = 146.0 Hz, −C−),
60.90, 61.13 (2 × −OCH2CH3), 117.05 (d, J = 85.8 Hz, −P+Ph3 ipso),
129.52 (d, J = 12.7 Hz, −P+Ph3 meta), 132.59 (d, J = 9.9 Hz, −P+Ph3
ortho), 134.14 (d, J = 2.8 Hz, −P+Ph3 para). HRMS-ESI: calcd for
+
= 2.7 Hz, −P+Ph3 para). HRMS-ESI: calculated for C38H58NO3P2
638.3886, found 638.3880.
1 2 - ( 2 - D i e t h y l p h o s p h o r y l p r o p a n - 2 - y l a m i n o ) -
dodecyltriphenylphosphonium Bromide (18b). Sticky yellow oil
1
(0.11 g, 18%): H NMR (CDCl3) δ 1.10−1.60 (m, 32 H), 2.56−2.61
(t, 2 H, J = 6.8 Hz, −CH2−), 3.57−3.63 (m, 2 H, CH2−P+Ph3), 4.01−
4.12 (m, 4 H, 2 × −OCH2−), 7.60−7.90 (m, 15 H, −P+Ph3); 31P
NMR (CDCl3) δ 24.16, 31.27; 13C NMR (CDCl3) δ 16.33, 16.40 (2 ×
−OCH2CH3), 22.08 (d, J = 3.7 Hz, −CH2−CH2−P+Ph3), 22.20 (d, J
= 50.6 Hz, −CH2−P+Ph3), 22.70 (d, J = 2.7 Hz, 2 × −CH3), 26.45
(−CH2−), 28.00−30.00 (7 × −CH2−), 30.70 (d, J = 16.9 Hz, −CH2−
CH2−CH2−P+Ph3), 42.53 (d, J = 5.5 Hz, −CH2−), 53.21 (d, J = 145.9
Hz, C), 60.67, 61.36 (2 × −OCH2−), 117.18 (d, J = 85.8 Hz, −P+Ph3
ipso), 130.10 (d, J = 12.1 Hz, −P+Ph3 meta), 133.12 (d, J = 10.4 Hz,
−P+Ph3 ortho), 134.93 (d, J = 2.7 Hz, −P+Ph3 para). HRMS-ESI:
+
C30H42NO3P2 526.2634, found 526.2631.
31P NMR Titrations. Test compounds (5 mM) were dissolved in a
buffer containing 5 mM KH2PO4 and 25 mM NaHCO3 in doubly
distilled deionized water (Elga, Purelab Option, France), and the pH
was adjusted to at least 10 different values ranging 1.0−12.0 by adding
concentrated HCl or KOH solutions. Titration solutions were
transferred into a 5-mm NMR tube and 1P NMR spectra were
+
calcd for C37H56NO3P2 624.373, found 624.3731.
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acquired on a Bruker AMX 400 spectrometer at 161.98 MHz, with
chemical shifts being referenced to external 85% H3PO4 at 0 ppm. A
small capillary containing D2O was inserted in each sample as a lock
signal. For each compound three independent titrations were
performed. The chemical shifts were plotted against pH to fit the
Henderson−Hasselbach equation (eq 1) using a nonlinear regression
(Prism, GraphPad Software Inc., San Diego, CA, U.S.A.)
supernatant and 100% loss of viability.51 The total LDH amount is the
sum of the enzymatic activity in the lysate and in the culture medium.
Isolated Liver Experiments and Mitochondrial Preparation.
Sprague−Dawley male rats (120−150 g) used for liver perfusion
and mitochondrial preparation were from CERJ (Le Genest St Isle,
France) and fed ad libitum with a standard Teklad 2016 diet (Harlan
Laboratories, Gannat, France). Animals were maintained in the local
animal house under conventional conditions, in a room with
controlled temperature (21−25 °C) and a reverse 12 h light/dark
cycle. All animal procedures used were in strict accordance with the
Directive 2010/63/EU of the European Parliament. The CNRS and
Aix-Marseille University have currently valid licenses for animal
experimentation (agreement C13-055-06) delivered by the French
Government. Animals were deeply anesthetized by intraperitoneal
δ − δA
δB − δ
pH = pKa + log
(1)
where δ is the 31P chemical shift and δA and δB represent the limiting
chemical shift values in acidic and basic conditions, respectively. The
T1 values were determined at pH ≈ pKa using a standard (180°−τ−
90°) inversion/recovery pulse-sequence with a spectral width of 100
ppm (16 spectra, 8 scans). The variable delay between 180° and 90°
pulses ranged 0.01−45 s to obtain a 16-points data set. A recycle delay
of at least 5 times the value of each T1 was used between 180° pulses.
Computation of Lipophilicity. clogD values (at pH 7.4) were
calculated with I-Lab 2.0 prediction software (ACD/Laboratories,
Toronto, ON, Canada).
Biology. Reagents. Doubly distilled deionized water was used
throughout and perfusates were filtered through a 0.2-μm Millipore
filter prior to use. Dulbecco’s modified Eagle’s medium (DMEM),
Eagle’s minimal essential medium (MEM) and fetal calf serum (FCS)
were obtained from Life Technologies Corp. (St Aubin, France). All
other reagents were purchased from Sigma-Aldrich (Saint-Quentin
Fallavier, France). Stock solutions of the test compounds in DMSO
(0.1 M) were diluted in aqueous medium before use.
Toxicity Tests on Chlamydomonas reinhardtii Cultures. Cell wall-
less green algae Chlamydomonas reinhardtii D66 were grown in Tris
acetate phosphate medium44 to densities of 1−5 × 106 cells per mL
under fluorescent light (50 μmol quanta/m2/s) at 25 °C. The cells
were then incubated at 25 °C with aqueous dilutions of stock solutions
of test compounds (to reach a final concentration ranging 0.01−1
mM) for 1 h either under saturating light or in the dark to determine
the photosynthetic and respiratory activities, respectively. At the end of
incubation, 1 mM NaHCO3 was added to the sample (to avoid any
CO2 limitation) and the activities were measured against the
corresponding control containing the same amount of DMSO (0.1%
maximum) by using a Clark-type oxygen electrode. The toxicity of
each test compound was expressed as LD50 (phot. and respectively),
which corresponds to the concentration leading to the loss of 50% of
photosynthetic or respiratory activity, respectively as compared to the
control.
Toxicity Tests on 3T3Murine Fibroblasts. Murine 3T3 fibroblasts
(ATCC-LGC Promochem, Molsheim, France) were cultured at 37 °C
in an atmosphere of 5% CO2 in DMEM containing 1% glucose and
supplemented with 10% FCS, 100 U/mL penicillin and 100 μg/mL
streptomycin as previously described.45 Cells were distributed into 24-
well plates and the medium was replenished every 2−3 days until
confluency, which was checked by microscopic observation and by
measuring protein content in four over 24 randomly selected control
wells by the method of Lowry et al.46 Each well of confluent cells was
then filled with phenol red-free DMEM containing 1% glucose.
Incubation of the cells at 37 °C for 3 h was then carried out by adding
to the culture medium aliquots of the stock solution of each test
compound to reach a final concentration ranging 0.01−5 mM and
0.1% for the test compound and DMSO, respectively, in a final volume
adjusted to 0.5 mL/well. Following incubation the supernatants were
sampled to measure cytoplasmic LDH release (considered as a marker
of cell necrosis47,48) using a commercial kit (Biolabo, Maizy, France),
the adherent cells were reincubated for 2 h in MEM containing neutral
red (50 μg/mL) and 2% FCS, and cell viability was evaluated at 540
nm using a microplate reader (SAFAS, Monaco) as described.49,50 The
medium lethal dose (LD50) and the concentration reducing cell
viability by 10% (LD10) were determined from appropriate dose−
response curves. To estimate the total LDH content a control
measurement was performed for each set of experiments, by treating
cells with 1% Triton X-100 to induce total LDH release in the
́
injection of sodium pentobarbital (100 mg/kg; Ceva Sante Animale,
Libourne, France), and the liver was excised and perfused as previously
described.52,53 Briefly, after the opening of the abdomen wall, the
portal vein was cannulated (antegrade perfusion) and the liver was
perfused at 37 °C in a nonrecirculating mode by a KH bicarbonate
buffer containing (in mM): KH2PO4 (1.2), NaCl (119), KCl (4.8),
MgSO4 (1.2), NaHCO3 (25), and CaCl2 (1.3) and bubbled with a gas
mixture of 95% O2 and 5% CO2 (pH 7.35). Assuming that the liver
weight represents about 4% of the body weight (i.e., 6−8 g), the
starting perfusion rate was set at 20 mL/min. The excised liver was
weighed, and the perfusion was adjusted to a flow rate of 3 mL/min/g
of wet weight. After a 30 min equilibration period, an aliquot of the
stock solution of each test compound in DMSO was added to the
perfusion medium to achieve a final concentration of 0.1 mM, and the
perfusion was extended for 1 h. The perfusate was then switched to an
ice-cold KH buffer for 10 min to completely remove the test
compound from the extracellular space, and the liver was homogenized
in five volumes of a solution containing 0.25 M sucrose and 10 mM
HEPES (pH 7.5). After filtration, the homogenate was centrifuged at
750 × g for 10 min at 2 °C, the mitochondrial and cytosolic fractions
were isolated from the supernatant by differential centrifugation as
previously described,19,20,42 and finally resuspended in 1 mL KH
buffer. These samples were extracted at 20 °C with CH2Cl2 (2 × 5
mL), and the combined organic layers were concentrated under
reduced pressure to give a dry residue, which was first weighed to
determinate the mitochondrial and cytosolic distribution of each test
compound. Data are pooled from 3−4 livers/test compound and are
given in μmoles per gram of fresh tissue. To check for any metabolic
degradation of the perfused compound, each residue was subsequently
redissolved in 1 mL of CDCl3 for 1H and 31P NMR analytical analysis
as described above.
To further evaluate the toxicity of test compounds two additional
sets of livers (3−6 livers per test compound) underwent the same
perfusion protocol as described above. In the first set of livers the end
of perfusion was followed by quickly freezing the organ in liquid
nitrogen and analysis of tissue ATP content of perchloric extracts using
an enzymatic standard procedure.54 In the second set of livers,
mitochondria were isolated from homogenates in ice-cold sucrose (0.3
M) buffer by differential centrifugation and the simultaneous
monitoring of respiration and phosphorylation rates, and membrane
potential were carried out as previously described.20,54 In parallel the
respiratory control ratio was determined by measuring the ratio of the
respiratory rate in the presence of 0.5 mM ADP to that measured after
the cessation of ADP phosphorylation, in a medium containing (in
mM): KH2PO4 (5), EDTA (0.005), MOPS (20), MgCl2 (5),
glutamate (20), and malate (1) maintained at 37 °C (pH 7.2).20,54
Statistics. All experiments were run at least in triplicate. Data are
presented as mean
SD or SEM for the indicated number of
independently performed experiments. Evaluation of statistical
significance was conducted by one-way or two-way analysis of variance
(ANOVA) followed, if significant (P < 0.05), by a Duncan test.
Differences between groups were considered significant when P < 0.05.
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(12) Robitaille, P. M. L; Robitaille, P. A.; Brown, G. G., Jr.; Brown, G.
G. An analysis of the pH-dependent chemical-shift behavior of
phosphorus-containing metabolites. J. Magn. Reson. 1991, 92, 73−84.
(13) Pietri, S.; Martel, S.; Culcasi, M.; Delmas-Beauvieux, M. C.;
Canioni, P.; Gallis, J. L. Use of diethyl(2-methylpyrrolidin-2-
yl)phosphonate as a highly sensitive extra- and intracellular 31P
NMR pH indicator in isolated organs. Direct NMR evidence of acidic
compartments in the ischemic and reperfused rat liver. J. Biol. Chem.
2001, 276, 1750−1758.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sylvia.pietri@univ-amu.fr. Phone: +33 (0)4-91-28-85-
79. Fax: +33 (0)4-91-28-87-58.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(14) Martel, S.; Clement, J. L.; Muller, A.; Culcasi, M.; Pietri, S.
́
■
Synthesis and 31P NMR characterization of new low toxic highly
sensitive pH probes designed for in vivo acidic pH studies. Bioorg. Med.
Chem. 2002, 10, 1451−1458.
The study was supported by the Agence Nationale pour la
Recherche (ANR-09-BLAN-005-03 and ANR-09-BLAN-005-
01-ROS Signal). The authors thank G. Gosset for her valuable
help in NMR experiments and S. Lemaire (IBPC-CNRS, Paris)
and C. Roumestand (CBS-CNRS, Montpellier) for their kind
help.
́
(15) Gosset, G.; Satre, M.; Blaive, B.; Clement, J. L.; Martin, J. B.;
Culcasi, M.; Pietri, S. Investigation of subcellular acidic compartments
using α-aminophosphonate 31P nuclear magnetic resonance probes.
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ABBREVIATIONS USED
■
pHi, intracellular pH; Pi, inorganic phosphate; DEPMPH,
diethyl(2-methylpyrrolidin-2-yl)phosphonate; DPP, diethyl(2-
propylaminoprop-2-yl)phosphonate; PBN, α-phenyl-N-tert-bu-
tylnitrone; IBTP, 4-iodobutyltriphenylphosphonium iodide;
KH, Krebs−Henseleit; LDH, lactate dehydrogenase; mitoPBN,
[4-[4-[[(1,1-dimethylethyl)oxidoimino]methyl]phenoxy]-
butyl]triphenylphosphonium bromide; DMEM, Dulbecco’s
modified Eagle’s medium; MEM, Eagle’s minimal essential
medium; FCS, fetal calf serum
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