Cheletropic traps for the fluorescence spectroscopic detection of nitric oxide (nitrogen monoxide) in biological systems

Article abstract of DOI:10.1002/(SICI)1521-3765(19990604)5:6<1738::AID-CHEM1738>3.0.CO;2-T

The concept of nitric oxide cheletropic traps (NOCT), which relies on the ESR spectroscopic detection of nitroxide radicals formed on trapping of NO by the NOCTs, has been extended to NOCTs that incorporate a fluorophoric system and form a new fluorophore on reaction with NO (FNOCT = fluorescent NOCT). The synthesis of the acetoxymethylesters of two FNOCTs, which can be loaded into cells, is described. Inside the cells the esters are enzymatically hydrolysed, thus allowing intracellular trapping of NO. The primary nitroxides formed on reaction with NO are reduced to the corresponding hydroxylamines under physiological conditions, thus remedying the fluorescence-quenching properties of nitroxide radicals. It is demonstrated that NO formed by lipopolysaccharide(LPS)-activated alveolar macrophages can easily be detected by these FNOCTs on a single cell basis.

Full text of DOI:10.1002/(SICI)1521-3765(19990604)5:6<1738::AID-CHEM1738>3.0.CO;2-T

FULL PAPER
Cheletropic Traps for the Fluorescence Spectroscopic Detection of
Nitric Oxide (Nitrogen Monoxide) in Biological Systems
Petra Meineke,[a] Ursula Rauen,[b] Herbert de Groot,[b] Hans-Gert Korth,[a]
and Reiner Sustmann*[a]
Abstract: The concept of nitric oxide
cheletropic traps (NOCT), which relies
on the ESR spectroscopic detection of
nitroxide radicals formed on trapping of
NO by the NOCTs, has been extended
to NOCTs that incorporate a fluoro-
phoric system and form a new fluoro-
phore on reaction with NO (FNOCTˆ
fluorescent NOCT). The synthesis of the
acetoxymethylesters of two FNOCTs,
which can be loaded into cells, is descri-
bed. Inside the cells the esters are
enzymatically hydrolysed, thus allowing
intracellular trapping of NO. The pri-
mary nitroxides formed on reaction with
NO are reduced to the corresponding
hydroxylamines under physiological
conditions, thus remedying the fluores-
cence-quenching properties of nitroxide
radicals. It is demonstrated that NO
formed by lipopolysaccharide(LPS)-ac-
tivated alveolar macrophages can easily
be detected by these FNOCTs on a
single cell basis.
Keywords: alveolar macrophages ´
cheletropic traps
spectroscopy nitrogen oxides
quinodimethanes
´
fluorescence
´
´
far, they cannot be applied intracellularly and their sensitivity
is limited to micromolar concentrations of NO.
Introduction
Nitric oxide (nitrogen monoxide) is a molecule of great
biological interest. In recent years it has been and still is the
subject of active research.[1, 2] Since its identification as the
endothelium-derived relaxing factor (EDRF)[3±6] an almost
exponential growth of the literature has taken place in this
area. Several methods for the detection and quantitation of
nitric oxide are available. Their characteristics have been
reviewed in recent reference books.[7, 8] Each method has its
merits, but none is without disadvantages and limitations in
terms of specificity, sensitivity, or versatility. Only fluores-
cence methods seem to be ideally suited for the monitoring of
NO production in biological samples with both temporal and
spacial resolution. Recently developed fluorescence ap-
proaches[9, 10] have been applied for the detection of NO in
single cells;[9] however, they require the in-situ transformation
of NO into nitrosation agents and are, therefore, not direct
sensors for NO. Procedures that mimic the biological reaction
of NO with heme proteins use either synthetic iron com-
plexes[11] or cytochrome c[12] labeled with fluorescent dyes. So
In recent years we have been involved in the development
of a family of molecules that specifically trap nitric oxide by
means of a formal cheletropic reaction. By the use of reactive
o-quinodimethanes of type 1 as biradical equivalents persis-
tent nitroxide radicals (2) are produced that can be easily
monitored by ESR spectroscopy.[13±15] The first members of
[a] Prof. R. Sustmann, Dipl.-Chem. P. Meineke, Dr. H.-G. Korth
Institut für Organische Chemie der Universität Essen
D-45117 Essen (Germany)
Fax: (‡49)201-183-3096
E-mail: sustmann@oc1.orgchem.uni-essen.de
the family, which were based on the parent o-quinodimethane,
are characterized by short lifetimes due to secondary reac-
tions and/or low preparative yields. Nevertheless, their use for
[b] Prof. H. de Groot, Dr. U. Rauen
Institut für Physiologische Chemie, Universitätsklinikum Essen
Hufelandstr. 55, D-45122 Essen (Germany)
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1738±1747
Scheme 1. Reaction scheme for the formation of 5 and 6.
the detection of NO could be demonstrated by scavenging NO
produced from rat-liver macrophages (Kupffer cells).[13]
Further developments led to the synthesis of polycyclic
systems containing the o-quinodimethane skeleton; these
proved to be more stable and could be made water soluble
by suitable substitution, for example, 3a.[16] Although
they showed high reactivity towards nitric oxide
troxide radical products 5 with an aromatic phenanthrene unit
as the active fluorophore (Scheme 1).
The parent compounds 4 should be either nonfluorescent or
should show fluorescence at different excitation and emission
wavelengths than products 5. Previous studies revealed that
the fluorescence intensity increased dramatically when the
nitroxide radicals 5 were reduced to the corresponding
hydroxyl amines 6. This results from the fact that nitroxide
radicals are fluorescence quenchers.[21]
In this contribution we report on the synthesis of suitable
FNOCTs and a first application as scavenger of nitric oxide
produced by alveolar macrophages. The production of nitric
oxide in single cells is monitored by means of digital
fluorescence microscopy, following the build up of the
fluorescence of the NO trapping product.
1
(k2 ˆ 103 ± 105 m 1 s in water at 228C) the rather short
lifetime of the corresponding nitroxides put a limit to
their application in physiological environment. Nevertheless,
some successful biological applications have been demon-
strated.[17, 18]
In light of this situation a new concept was developed that
still relies on the same type of reaction but now uses
fluorescence spectroscopy as sensitive detection method
(FNOCTˆ fluorescent nitric oxide cheletropic trap). The
principle has been published in preliminary communica-
tions.[19,20] It is based on the fact that the reaction of o-
quinodimethanes of type 4 with nitric oxide generates ni-
Results
Synthesis and characterization of the fluorescent nitric oxide
cheletropic traps: A biologically useful nitric oxide trap
should show excitation and emission wavelengths that allow
fluorescence microscopy of living cells. If the excitation
wavelength is <340 nm cells might be damaged by irradiation.
Furthermore, most optical systems do not operate at these
wavelengths. We thus reasoned that FNOCT 4a, which has
been described in the preliminary communication, might not
be suited for our purpose owing to its rather short excitation
wavelength of lexc ˆ 315 nm and its major emission between
360 and 420 nm. Therefore, we introduced a dimethylamino
group in 4a in order to shift fluorescence excitation and
emission to longer wavelengths.
Starting materials for the synthesis were the corresponding
phenanthrene-9,10-quinones 7a and 7b. Condensation of 7a
and 7b with 1,3-diphenylpropane-2-one gave 8a and 8b in
90% and 45% yield, respectively, (Scheme 2). Both under-
went smooth Diels ± Alder addition with norborn-5-ene-2,3-
(E)-dicarboxylic acid (9a) and its diacetoxymethylester 9b to
give the polycyclic ketones 10a and 10b. A mixture of two
adducts in a ratio of 2:1 was obtained from the reactions of 8b
with 9a and 9b. As the cycloaddition of 8a to 9a or 9b
produced only one isomer, for which we derived the displayed
stereochemistry from spectroscopic data and semiempirical
quantumchemical calculations, we concluded that a mixture
of regioisomers of 10c and 10d is obtained in the cyclo-
additions of 8b to 9a and 9b. These isomers differ in the
orientation of the dimethylamino group relative to the
Abstract in German: Das Konzept cheletroper Spinfänger
(Nitric Oxide Cheletropic Trap ˆ NOCT), welches auf dem
ESR-spektroskopischen Nachweis von Nitroxid-Radikalen
beruht, die beim Abfang von NO gebildet werden, wurde auf
fluoreszenzfähige cheletrope Fänger (FNOCTˆ Fluorescent
NOCT) ausgedehnt. Bei der Reaktion mit NO entsteht aus dem
fluoreszierenden FNOCT ein neuer Fluorophor mit Fluores-
zenz bei anderen Wellenlängen. Sowohl die Abnahme der
Fluoreszenz der FNOCTs als auch die Zunahme der Fluores-
zenz des nach Abfang von NO durch Reduktion des primären
Nitroxid-Radikals gebildeten Hydroxylamin-Derivates können
für die Erfassung von NO eingesetzt werden. Es wird die
Synthese der Acetoxymethylester von zwei FNOCTs beschrie-
ben, mit denen Zellen beladen werden können. Diese Ester
werden in den Zellen enzymatisch gespalten, so daû die
Säureanionen der FNOCTs nach Zugabe eines Anionenka-
nalhemmers in den Zellen verbleiben und zum Nachweis des
NO genutzt werden können. Die primär entstehenden Nitro-
xid-Radikale werden unter den physiologischen Bedingungen
zu den entsprechenden Hydroxylamin-Derivaten reduziert.
Dadurch wird die fluoreszenzlöschende Eigenschaft der Nitro-
xid-Radikale behoben. Es wird gezeigt, daû NO, gebildet von
Alveolar-Makrophagen nach Stimulation der Zellen mit Li-
popolysacchariden (LPS), in einfacher Weise mit diesen
FNOCTs nachgewiesen werden kann.
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R. Sustmann et al.
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Scheme 2. Reaction scheme for the formation of 4.
Figure 1. Fluorescence spectra of 10a and 10c. Excitation (broken line)
and emission spectra (continuous line) of a) 10a (c ˆ 50mm, lexc ˆ 315 nm
for emission spectrum, lem ˆ 380 nm for excitation spectrum) and b) 10c (B,
c ˆ 50mm, lexc ˆ 380 nm for emission spectrum, lem ˆ 460 nm for excitation
spectrum) in sodium phosphate buffer pH 7.2.
substituents at the norbornane skeleton. As a result of their
similarity, the isomers could not be separated by conventional
techniques. This, however, does not hamper their use. The NO
scavengers 4a ± d finally were obtained by quantitative
photolytic decarbonylation of 10a ± d and were fully charac-
terized by spectroscopic techniques (Scheme 2).
The parent ketones 10a ± d exhibit the same fluorophoric p
system as the NO adducts 5a ± d of the corresponding
FNOCTs 4a ± d. Because nitroxides are fluorescence quench-
ers,[21] reduction of the nitroxides to the corresponding
hydroxylamines 6a ± d is required to obtain the maximum
fluorescence level. With this prerequisite ketones 10a ± d can
serve as spectroscopic models for the reduced NO-trapping
products 6a ± d. Figure 1 shows the fluorescence excitation
and emission spectra of 10a (Figure 1a) and 10c (Figure 1b).
As anticipated, the dimethylamino substituent in 10c shifts
the bands to longer wavelengths, into a region that is well
suited for biological applications.
Figure 2. Fluorescence spectra of 4c. Excitation (broken line) and
emission spectrum (continuous line) of 4c (c ˆ 50mm, lexc ˆ 460 nm for
emission spectrum, lem ˆ 600 nm for excitation spectrum) in sodium
phosphate buffer pH 7.2.
The o-quinoid p systems of 4a and 4c were analyzed with
regard to their fluorescence properties. The fluorescence
excitation and emission spectrum of 4c in phosphate buffer is
displayed in Figure 2. The fluorescence spectrum of 4a is of
negligible intensity compared with that of 4c and therefore
not shown. It is interesting to note that the fluorescence
intensitiy of 4c depends on pH; it decreases with increasing
pH and disappears completely at pH ꢀ 9. The fluorescence
intensity is at a maximum at pH 4 ± 5. The analysis of the
fluorescence intensity as a function of pH yielded a pKa of 6.7,
which is attributed to the protonation equilibrium of the
dimethylamino group. The fluorescence is highest in the fully
protonated form and is lost in the free amino form. At a
physiological pH of 7.2 (phosphate buffer) about 24% of the
dimethylamino group is protonated.
Figure 3. ESR spectrum of 5c. Radical 5c was generated in THF solution
by reaction of 4c with NO at 238C. ESR parameters: g ˆ 2.00610, a(14N) ˆ
2.287 mT, a(exo-Hg) ˆ 0.076 mT (2H), a(H) ˆ 0.048 mT (1H), a(H) ˆ
0.014 mT (4H).
In Figure 3 the ESR spectrum is shown that was recorded
after addition of a solution of NO in THF to a solution of 4c in
the same solvent. The spectrum is easily interpreted in terms
of nitroxide radical 5c. The additional fine structure of the
three nitrogen hyperfine lines results from hyperfine splittings
by the hydrogen atoms of the norbornane moiety. A virtually
identical spectrum was observed for 5a under similar
conditions.[19] The same ESR spectra were also obtained in
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Cheletropic Traps
1738±1747
phosphate buffer. In aqueous solution, however, the radicals
are not very persistent, decaying within 10 ± 30 min to non-
radical products and accompanied by the growth of the typical
fluorescence of the phenanthrene chromophore.
The efficiency of compounds 4 to trap nitric oxide was
determined by the stopped-flow technique by monitoring the
disappearance of the characteristic UV/Vis absorption of the
o-quinoid p system at about 460 nm. The second-order rate
pH dependence of the fluorescence intensity a pKa of 6.7 was
determined, confirming that the protonation of the dimethyl-
amino group is responsible for the pH effect. Even in the
absence of a reducing agent nitroxide 5c has a rather short
lifetime in aqueous solution. The same fluorescence intensity,
as found in the presence of a reducing agent, was reached 10 ±
30 min after the addition of NO. This fact demonstrates that
the degradation of the nitroxide radical does not affect the
fluorophoric system.
The effect of a reducing agent on the fate of nitroxides 5b
and 5d was studied in THF by the use of acetaldehyde in place
of ascorbate. Thus, a freshly prepared solution of 4b or 4d
containing an equimolar amount of acetaldehyde was treated
with an equimolar amount of NO solution and analysed
immediately by 1H NMR and MS spectroscopy. The resulting
spectra were fully consistent with 6b or 6d, respectively, being
the only products formed (40% conversion). As expected, the
1H NMR spectra of 6b and 6d are virtually identical to those
of ketones 10b and 10d, respectively.
1
constant for the reaction of 4a with NO is k2 ˆ 930 Æ 70m 1 s
in benzene at 238C and 60 Æ 8m 1 s in phosphate buffer
1
pH 7.4. The dimethylamino-substituted derivative 4c reacts at
similar rates, namely, k2 ˆ 600 Æ 42m 1 s at room temper-
1
ature in THF (218C) and 170 Æ 10m 1 s 1 in phosphate buffer.
Slightly reduced rate constants in aqueous solution compared
with organic solvents have been observed previously for other
NOCTs too.[19]
In order to allow the FNOCT molecules to permeate cell
membranes the acetoxymethylesters 4b and 4d were synthe-
sized. Inside living cells these esters should be cleaved
enzymatically to produce the free acids 4a and 4c, respec-
tively; this hinders the trap as well as its NO adduct from
leaving the cell. To check on the enzymatic hydrolysis, pig-
Before FNOCTs 4 were applied in a biological system,
further tests were carried out in order to ensure that the
specificity of the reaction with nitric oxide was high compared
with possible reactions with other biologically relevant
reactive molecules. Figure 5 displays the behavior of FNOCT
1
liver esterase (20 unitsmL ) was added to a solution of either
4b or 4d in phosphate buffer at 378C in the presence of 2%
DMSO and 0.2% PluronicꢁR . The progress of ester cleavage
was followed by thin-layer chromatography (on silica gel with
toluene/ethylacetate 3:1). Comparison with authentic samples
of the free acids 4a and 4c, respectively, revealed that for both
esters hydrolysis was complete after 1 h. In the absence of the
enzyme 4b as well as 4d were stable for at least 24 h under
these conditions.
The effect of the controlled addition of ascorbate on the
fluorescence intensity has been described for radical 5a.[19]
The result of a similar trapping experiment with 4c in the
presence of ascorbate is shown in Figure 4. Excitation at
Figure 5. Reactivity of FNOCT 4c towards NO and other biologically
relevant reactive oxygen species. Time dependence of the fluorescence
intensity at 238C monitored from 4c (c ˆ 50mm, lexc ˆ 380 nm, lem
ˆ
!
460 nm) in pure sodium phosphate buffer pH 7.2 ( ) and in the presence
*
of 100mm NO-solution ‡ 100mm ascorbate ( ), 20mm spermine NONOate
.
2‡
~
).
&
&
*
( ), 100mm H2O2 ( ), 100mm H2O2 ‡ 50mm Fe
(
) and 100mm O2
(
4c in phosphate buffer (pH 7.2, 238C, lexc ˆ 380 nm, lem
ˆ
460 nm) against hydrogen peroxide, hydrogen peroxide in
the presence of FeII (Fenton system), and superoxide. No
significant increase in fluorescence intensity was observed in
the presence of these additives compared with the growth
observed in pure buffer solution (which we attribute to a slow
thermal transformation of FNOCT 4c into products also
incorporating the phenanthrene unit). The fastest reaction
was observed with nitric oxide in the presence of ascorbate, in
which the maximum of the fluorescence intensity was reached
within a few seconds.
In another experiment (also shown in Figure 5) the NO
donor spermine NONOate was added to a solution of
FNOCT 4c. A first-order increase of the fluorescence
intensity was observed, which reflects the release of NO
by this molecule. The half-life of fluorescence growth
Figure 4. Fluorescence spectra of 6c. Excitation (broken line) and
emission spectrum (continuous line) of 6c (c ˆ 50mm, lexc ˆ 380 nm for
emission spectrum, lem ˆ 460 nm for excitation spectrum) in sodium
phosphate buffer pH 7.2.
lexc ˆ 380 nm yielded an emission spectrum for 6c (lmax
ˆ
460 nm) that is very similar to the emission spectrum of
ketone 10c (see Figure 1). The fluorescence quantum yields
(QF) of 6a and 6c in phosphate buffer were measured relative
to fluorescein as standard (QF ˆ 0.91[22]), giving QF (6a) ˆ 0.03
and QF (6c) ˆ 0.27. As for 4c, the fluorescence of 6c is pH
dependent, the highest intensity is found at pH 8 ± 9. From the
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(t1/2 ˆ 54 min at 218C) corresponds to the decay rates of
spermine NONOate as reported in the literature.[20, 23, 24]
Aromatic p systems tend to aggregate at higher concen-
trations in aqueous solution.[25] This would lead to a nonlinear
dependence of the fluorescence intensity on the concentration
of the NO trap. However, in the nanomolar to 100-micro-
molar concentration range, that is, in the concentration range
relevant for cellular systems, the fluorescence intensity of 6c
was found to be strictly linearily correlated (r2 ˆ 0.996) with
the concentration of 4c. This is different to 4a/6a system, in
which a deviation from the linear behavior was found for
concentrations of 4a above 20mm.
The sensitivity for NO detection was determined by adding
increasing amounts of NO to a 50mm solution of 4c in
phosphate buffer. As is shown in Figure 6 the fluorescence of
6c can be detected already at nm concentrations of NO. Strict
linearity of the fluorescence intensity versus the NO concen-
tration is observed up to the mm range, indicating that all of
the NO added is trapped by 4c.
the cell nuclei being spared. Cell viability after measurements
was >85%; this was comparable with the respective solvent
control, but was somewhat lower than the viability of un-
treated cells (ꢀ90%). Photobleaching of the fluorescence of
6c was negligible.
The approximate cellular concentration of FNOCT 6c was
determined by fluorescence spectroscopy after lysis of the
cells with triton X-100 (1%). A mean intracellular FNOCT
6
concentration of 100 Æ 5 pmol10
cells was determined
corresponding to an intracellular concentration of 83 Æ
1 [26]
4.2mm, assuming an average cell volume of 1.2 pLcell .
For unstimulated, FNOCT-loaded alveolar macrophages,
fluorescence at lexc ˆ 380 Æ 10 nm and lem ˆ 460 ± 490 nm was
almost identical to the autofluorescence of the cells (as
assessed by an unloaded control incubation; Figure 7). Upon
Figure 7. Effect of NO on the fluorescence of FNOCT-loaded alveolar
macrophages. Rat alveolar macrophages were loaded with FNOCT 4d
(50mm, 90 min) and fluorescence was monitored by digital fluorescence
microscopy at lexc ˆ 380 Æ 10 nm, lem ˆ 460 ± 490 nm. For release of NO
either the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP, 10mm)
was added to the incubation medium (Krebs ± Henseleit buffer containing
2mm l-glutamine and 10mm d-glucose) and supplemented when necessary
with l-arginine (Arg, 0.5mm), or cells were activated by preincubation with
lipopolysaccharides (LPS, 0.5 mgmL 1, 11 h). NG-monomethyl-l-arginine
(NMA, 0.1mm) was added to some incubations in order to inhibit NO
formation. Cellular autofluorescence (autofluoresc.) in the absence of
FNOCT 4d is shown for comparison. Data shown represent mean Æ S. D. of
20 ± 50 cells from 4 preparations.
Figure 6. Dependency of the fluorescence intensity of 6c on the NO
concentration after reaction with 4c (c ˆ 50mm, lexc ˆ 380 nm, lem
ˆ
460 nm) for the micromolar and nanomolar range in sodium phophate
buffer pH 7.2.
Biological applications of FNOCT 4d: Alveolar macrophages
are known to produce relatively large amounts of NO after
activation with lipopolysaccharides and, therefore, were used
to test the applicability of the FNOCT concept in a biological
system. As we aimed at NO measurements on the single-cell
level using digital fluorescence microscopy, conditions for
loading the cells with FNOCT 4d were first investigated. The
best loading was achieved at a FNOCT 4d concentration of
50mm (dissolved in presence of 0.1% PluronicꢁR and 1%
DMSO) and a loading time of 90 min at 378C. Higher
concentrations or longer loading times led to increased cell
death, lower concentrations or shorter loading times yielded
addition of 10mm of the NO donor S-nitroso-N-acetyl-d,l-
penicillamine (SNAP), that is, subjecting an excess amount of
NO to the FNOCT-loaded cells, there was a rapid increase in
fluorescence at these wavelengths. The final fluorescence
level obtained should reflect complete consumption of the
NO trap. When alveolar macrophages had been activated with
LPS for 11 h prior to loading with FNOCT 4d, the fluores-
cence at lexc ˆ 380 Æ 10 nm and lem ˆ 460 ± 490 nm had in-
creased to the same level after the loading period of 90 min.
This increase in fluorescence was observed for all cells with
little cell-to-cell variation. No further increase was monitored
with duration of the experiment, thus, at the present intra-
cellular concentration the dye had been completely consumed
within the loading time of 90 min. Cells treated with the NO
synthase inhibitor NMA, regardless if activated or not, did not
show a fluorescence intensity exceeding the level of auto-
fluorescence, confirming that the foregoing signal was indeed
due to trapping of NO from the NO synthase-catalysed
reaction. For comparison, the Griess and DAN tests were
only suboptimal fluorescence intensities as assessed at lexc
ˆ
488 Æ 10 nm, lem > 520 nm. In the presence of probenecid
(1.4mm) no dye leakage could be detected, whereas in the
absence of probenecid dye leakage occurred. This indicated
that the expected intracellular hydrolysis of ester 4d to the
corresponding acid 4c had taken place. Fluorescence micro-
scopy demonstrated that the dye was distributed almost
homogeneously over the cytosol and the organelles, with only
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Cheletropic Traps
1738±1747
carried out under identical conditions of cell preparation and
stimulation. While FNOCT 4c allowed easy detection
of NO formed 11 ± 12 hours after activation, neither of the
two other methods provided such evidence under these
conditions. The DAN test was positive 12 hours after stim-
ulation when all NO formed during the first 12 hours was
accumulated; for the Griess method 16 h of accumulation
were necessary.
similar to the increase in pure buffer solution, indicating
insensitivity towards these reactive oxygen species. Peroxyni-
trite [oxoperoxonitrate( 1)], another putative reactive oxy-
gen species[32] indeed reacts with FNOCT 4c, as was noticed
by an increase of fluorescence and the disappearance of the
color of FNOCT 4c after addition of a small excess of
authentic peroxynitrite. A large excess of peroxynitrite led to
a decrease of the fluorescence. Details of the reaction of
FNOCT 4c with peroxynitrite will be reported in a forth-
coming publication.[33]
The applicability of the trap for NO detection in single,
viable cells could be demonstrated by the use of isolated rat
alveolar macrophages. FNOCTs 4c/4d were nontoxic in the
final concentration used. FNOCT 4d could be loaded into the
cells and was apparently hydrolyzed to the membrane-
impermeable FNOCT 4c which remained in the cells. The
intracellular trap could be shown to respond in the same way
as in the chemical system to externally applied as well as to
endogeneously produced NO, while no signal was observed in
unstimulated cells or in the presence of an inhibitor of cellular
NO formation. By means of digital fluorescence microscopy,
the signal could be detected on the single-cell level, under-
lining the sensitivity of the method. Together, these data
demonstrate that the approach taken is possible. The method
presented is likely to be useful for studying a broad range of
NO-related questions in cellular systems.
Discussion
A method is presented that permits the sensitive detection of
nitric oxide. The procedure makes use of the fact that
FNOCTs 4 react with NO in a formal cheletropic reaction
to give bridged nitroxide radicals 5. These nitroxides are
immediately reduced under cellular conditions to hydroxyl-
amine derivates 6, thereby generating a phenanthrene-type
fluorophore. This methodology is different to most of the
other known fluorescence assays for NO in the sense that the
fluorophore is produced directly by the reaction of the
reagent with NO, and also leads to a permanent incorporation
of the latter in the fluorescent product. The fluorescence
methods proposed for biological applications so far[8, 9]
produce fluorescence only indirectly and require prior trans-
formation of NO into other more reactive nitrogen ± oxygen
‡
species, for example, NO , NO2, or N2O3. This, of course,
affects the specificity of such methods, always leaving some
ambiguities concerning the truly detected species.
Experimental Section
The second-order rate constants for the reaction of 4a and
Instrumentation: NMR: Bruker AMX300, Varian Gemini200 (signals
indicated with * are attributed to the minor isomer of the compound);
ESR: Bruker ER420 with DigiS data acquisition system (GfS Aachen,
Germany); IR: Perkin ± Elmer Series 1600 FTIR; UV/Vis: Cary 219;
fluorescence spectroscopy: Jobin Yvon JY3 with DigiS data acquisition
system and FL 3095 of J&M; MS: Fisons VG ProSpec 300; elemental
analysis: Carlo Erba Model 1106; melting points: Büchi 510 (uncorrected);
stopped-flow measurements: HiTech Scientific Ltd. SF41 Cryostopped-
flow spectrometer (Salisbury, England).
1
4c with NO are of the order of 100m 1 s . In terms of
quantitation of NO production in biological systems it would
be desirable to have rate constants close to diffusion control in
order to compete efficiently with the physiological decay
reactions of NO. Whereas autoxidation of NO should not
interfere owing to the low steady-state level of O2 in typical
cell systems, NO would react rapidly with iron complexes[27, 28]
or superoxide.[29±31] On the other hand, smaller k2 values might
be more advantageous for physiological studies considering
that not all of the NO being produced will be trapped and,
therefore, interference with NO-dependent cellular responses
will be largely diminished. In other words, a simultaneous
study of NO production and NO-dependent cellular respons-
es remains possible.
The finite lifetimes of our nitroxides 5, which would make
ESR detection in cell systems difficult, were the reason for the
development of the FNOCTs. On reaction with NO these
systems retain the fluorophore regardless of whether the
nitroxide radical decays or not. Even better, as nitroxides are
known to be fluorescence quenchers (see above), it is
desirable to generate nonradical products. This can be
achieved by intentionally adding a suitable reducing agent,
for example, acetaldehyde, or taking advantage of the fact
that reducing agents like ascorbate, glucose, or glutathione are
ubiquituous in living cells.
Materials: Dulbeccoꢁs modified eagle medium (DMEM, low glucose) and
gentamycin sulfate were purchased from GibcoBRL (Eggenstein, Germa-
ny); lipopolysaccharides, probenecid, and fetal calf serum were from Sigma
(Steinheim, Germany); PluronicꢁR F-127, propidium iodide, and spermine
NONOate ((Z)-1-{N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)bu-
tyl]amino}-diazen-1-ium-1,2-diolate) were from Molecular Probes Eu-
rope BV (Leiden, The Netherlands); trypan blue, NG-monomethyl-l-
arginine monoacetate and esterase (pig liver, 200 unitsmg 1) were from
Aldrich (Steinheim, Germany); dimethyl sulfoxide and triton X-100 were
from Merck (Darmstadt, Germany). Falcon 6-well cell-culture plates were
obtained from Becton Dickinson (Heidelberg, Germany) and glass cover-
slips were from Assistent (Sondheim/Röhn, Germany).
2-Dimethylaminophenanthrene-9,10-quinone (7b): A suspension of 2-ami-
no-phenanthrene-9,10-quinone[34, 35] (1.25 g, 5.6 mmol) and sodium carbon-
ate (0.60 g, 5.6 mmol) in methanol (5.0 mL) was heated to 608C and
dimethyl sulfate (1.1 mL, 1.41 g, 10.2 mmol) was added. After 1 h every
30 min sodium carbonate (0.30 g, 2.8 mmol) and dimethyl sulfate (0.55 mL,
5.1 mmol) were added to the reaction mixture five times. The reaction was
stopped after 4 h by addition of chloroform (250 mL). After filtration the
chloroform phase was washed several times with water until the water
phase remained colorless. Removal of chloroform provided a dark-blue
solid consisting of
a mixture of mono- and dimethylated product.
Chromatography (100 g silica gel 60, Merck, 200mm) on a 70 cm column
(diameter 2 cm) with toluene/ethylacetate 3:1 gave 0.80 g (3.2 mmol, 57%)
7b together with 0.33 g (1.4 mmol, 25%) monomethylated product. M.p.
1708C; 1H NMR (200 MHz, CDCl3, TMS): d ˆ 8.06 (dd, 3J(7-H, 8-H) ˆ
7.8 Hz, 4J(6-H, 8-H) ˆ 1.6 Hz, 1H; 8-H), 7.78 (dd, 3J(5-H, 6-H) ˆ 7.8 Hz,
The specificity of our FNOCTS for the trapping of NO is
demonstrated by the fact that the increase in fluorescence
intensity in the presence of hydrogen peroxide, hydrogen
peroxide ‡ FeII (Fenton reaction), or superoxide is very
Chem. Eur. J. 1999, 5, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0506-1743 $ 17.50+.50/0
1743

R. Sustmann et al.
FULL PAPER
4
4J(5-H, 7-H) ˆ 1.0 Hz, 1H; 5-H), 7.76 (d, J(3-H, 4-H) ˆ 9.6 Hz, 1H; 4-H),
7.55 (ddd, 3J(5-H, 6-H) ˆ 7.8 Hz, 3J(6-H, 7-H) ˆ 7.8 Hz, 4J(6-H, 8-H) ˆ
1.4 Hz, 1H; 6-H): 7.37 (d, 4J(1-H, 3-H) ˆ 3.0 Hz, 1H; 1-H), 7.26 (ddd,
3J(6-H, 7-H) ˆ 7.5 Hz, 3J(7-H, 8-H) ˆ 7.6 Hz, 4J(7-H, 5-H) ˆ 1.0 Hz, 1H;
(300 MHz, [D8]THF, TMS): d ˆ 11.28 (brs, 2H; COOH), 8.79 (d, 3J ˆ
8.8 Hz, 2H; 4' H, 5-'H), 8.08 (brd, 3J ˆ 7.5 Hz, 1H; o'-H or o''-H), 7.90
(brd, 3J ˆ 7.8 Hz, 1H; o'-H or o''-H), 7.65 (dd, 3J ˆ 6.5 Hz, 7.5 Hz, 1H; m'-H
3
or m''-H), 7.59 (dd, J ˆ 7.8 Hz, 7.5 Hz, 1H; m'-H or m''-H), 7.47 ± 7.52 (m,
7-H), 6.93 (dd, 3J(3-H, 4-H) ˆ 9.4 Hz, 4J(1-H, 3-H) ˆ 3.0 Hz, 1H; 3-H), 3.06
2H; 3'-H, 6 'H), 7.39 ± 7.45 (m, 2H; p-H), 7.30 ± 7.36 (m, 2H; m-H), 7.10 ±
7.19 (m, 6H; 2'-H, 7'-H), 3.24 (brs, 2H; 2-H, 6-H), 3.14 (dd, 3J ˆ 5.2 Hz,
4.5 Hz, 1H; 8-H), 3.05 (brd, 3J ˆ 5.2 Hz, 1H; 9-H), 2.87 (d, 3J ˆ 3.9 Hz, 1H;
7-H), 2.82 (brs, 1H; 1-H), 0.47 (d, 2J ˆ 10.9 Hz, 1H; 10b-H), 0.51 (d, 2J ˆ
10.0 Hz, 1H; 10a-H); 13C NMR (75 MHz, [D8]THF, TMS, CH-COSY,
13
ˆ
(s, 6H; CH3); C NMR (50 MHz, CDCl3, TMS): d ˆ 180.8 (C O), 150.2
(C-2), 137.4, 131.5, 129.5, 123.3 (C-1a, C-4a, C-5a, C-8a), 135.8, 130.1, 127.2,
125.2, 122.6, 118.5, 112.2 (C-1, C-3, C-4, C-5, C-6, C-7, C-8), 39.91 (CH3); IR
ˆ
ˆ
(KBr): nÄ ˆ 3065 ( C H), 2885 ( CH3), 2801 (N CH3), 1667 (C O)quinone
,
1
‡
ˆ
ˆ
ˆ
1594 cm (C C)quinone ; MS (70 eV, EI, high resolution): m/z: [M ] calcd for
C16H13N1O2: 251.095536; found 251.094629; difference 3.6 ppm; UV/Vis
(THF): lmax (lg e) ˆ 550 (3.28), 330 (4.02, shoulder), 290 nm (4.55); RF ˆ
0.54 (toluene/ethylacetate 3:1).
COLOC): d ˆ 198.99 (C O)ketone, 173.96, 174.65 (C O)acid, 137.94, 138.29
(C-ipso-phenyl), 135.57, 135.73 (C-9', C-10'), 132.44 (C-o), 131.88, 131.92
(C-4'a, C-5'a), 129.56, 129.73 (C-m', C-m''), 128.98, 129.06 (C-m), 128.67 (C-
1'a, C-8'a), 128.56, 128.73 (C-o', C-o''), 128.11, 128.19 (C-p), 127.11 (C-2',
C-7'), 126.89, 126.93 (C-3', C-5'), 126.14 (C-1', C-8'), 124.23 (C-4', C-5'),
63.96, 64.42 (C-3, C-5), 51.65 (C-8), 51.01 (C-9), 48.06 (C-6), 45.39 (C-1),
2,5-Diphenyl-3,4-(2,2'-biphenylene)cyclopenta-2,4-diene-1-one (8a): This
compound was prepared by means of the procedure given in ref. [36].
44.16 (C-2), 43.00 (C-7), 34.60 (C-10); IR (KBr): nÄ ˆ 3350 ± 3490 ( OH),
2,5-Diphenyl-3,4-(4-dimethylamino-2,2'-biphenylene)cyclopenta-2,4-di-
ene-1-one (8b): Compound 7b (0.55 g, 2.2 mmol) and 1,3-diphenyl-
propane-2-one (0.46 g, 2.2 mmol) in ethanol (6.0 mL) were treated with a
20% solution of potassium hydroxide in ethanol (0.6 mL) at 558C
according to the literature.[36] The violet suspension changed to a yellow
solution, from which a black solid precipitated. For completion the reaction
mixture was kept at 08C over night. Filtration gave 0.43 g (1.0 mmol, 45%)
1
ˆ
ˆ
3088, 3058, 3031 ( CH), 2969, 2926 ( CH), 1785 (C Oketone), 1710 cm
ˆ
(C Oacid); UV/Vis (MeOH): lmax (lg e) ˆ 253 (4.45), 260 (4.53), 271 (4.19),
285 (3.80), 297 (3.82), 310 nm (3.82); Fluorescence (phosphate buffer
pH 7.4): lexc ˆ 315 nm, lem ˆ 365, 380, 405 nm; MS (70 eV, EI, high
‡
resolution): m/z: [M ] calcd for C38H28O5: 564.1937; found: 564.1951;
difference 2.5 ppm; C38H28O5 (564.6): calcd C 80.83, H 4.99; found C 80.01,
H 4.68.
1
of 8b. M.p. 200 ± 2058C; H NMR (300 MHz, CDCl3, TMS): d ˆ 7.61 (dd,
3J(5''-H, 6''-H) ˆ 7.6 Hz, J(4''-H, 6''-H) ˆ 1.4 Hz, 1H; 6''-H), 7.60 (d, J(5'-
3,5-Diphenyl-3,5-(9,10-phenanthrylene)tricyclo[5.2.1.02,6]decane-4-one-8-
exo-9-endo-dicarboxylic acid diacetoxymethylester (10b): Compounds 9b
(2.20 g, 12.0 mmol) and 8a (3.83 g, 10.0 mmol) were heated in chloroben-
zene (100 mL) for 15 h at 1308C. After removal of the solvent, the residue
was dissolved in dichloromethane and precipitated by addition of n-
heptane. Recrystallization from ethanol gave 5.24 g (7.40 mmol, 74%) of
10b. M.p. 2258C; 1H NMR (300 MHz, [D8]THF, TMS): d ˆ 8.80 (d, 3J ˆ
8.2 Hz, 2H; 4'-H, 5'-H), 7.97 (brd, 3J ˆ 7.5 Hz, 1H; o'-H or o''-H), 7.90 (brd,
4
4
H, 6'-H) ˆ 9.0 Hz, 1H; 6'-H), 7.45 (dd, 3J(3''-H, 4''-H) ˆ 7.7 Hz, 4J(3''-H, 5''-
3
H) ˆ 1.0 Hz, 1H; 3''-H), 7.43 ± 7.35 (m, 10H; CH-phenyl), 7.16 (ddd, J(3''-
H, 4''-H) ˆ 7.6 Hz, 3J(4''-H, 5''-H) ˆ 7.4 Hz, 4J(4''-H, 6''-H) ˆ 1.5 Hz, 1H; 4''-
H), 6.84 (d, 4J(3'-H, 5'-H) ˆ 3.0 Hz, 1H; 3'-H), 6.77 (ddd, 3J(4''-H, 5''-H) ˆ
7.9 Hz, 3J(3''-H,4''-H) ˆ 7.6 Hz, 4J(3''-H, 5''-H) ˆ 1.0 Hz, 1H; 5''-H), 6.60
(dd, 3J(5'-H, 6'-H) ˆ 9.0 Hz, 4J(3'-H, 5'-H) ˆ 3.0 Hz, 1H; 5'-H), 2.60 (s, 6H;
13
ˆ
CH3); C NMR (75 MHz, CDCl3, TMS): d ˆ 200.6 (C O), 149.6 (C-4'),
3
3
149.3, 148.7 (C-2, C-5), 134.6, 132.9, 132.5, 128.7, 127.0, 122.9, 122.7, 121.9
(C-3, C-4, C-1', C-1'', C-2', C-2'', C-ipso-phenyl), 131.3, 130.1, 130.0, 129.4,
128.5, 128.4, 127.9, 127.8, 126.2, 125.4, 123.1, 115.3, 112.3 (CHarom.), 39.91
3J ˆ 7.5 Hz, 1H; o'-H or o''-H), 7.70 (dd, J ˆ 7.5 Hz, J ˆ 7.5 Hz, 1H; m'-H
or m''-H), 7.68 (dd, 3J ˆ 7.5 Hz, 3J ˆ 7.5 Hz, 1H; m'-H or m''-H), 7.52 (dd,
3J ˆ 7.5 Hz, 3J ˆ 7.5 Hz, 2H; 3'-H, 6'-H), 7.45 (dd, 3J ˆ 7.4 Hz, 3J ˆ 7.4 Hz,
2H; p-H), 7.35 ± 7.32 (m, 2H; m-H), 7.21 ± 7.12 (m, 6H; 1'-H, 2'-H, 7'-H, 8'-
H, o-H), 5.95 (d, 2J ˆ 10 Hz, 1H; OCH2O), 5.92 (d, 2J ˆ 10 Hz, 1H;
OCH2O), 5.65 (d, 2J ˆ 13 Hz, 1H; OCH2O), 5.63 (d, 2J ˆ 13 Hz, 1H;
OCH2O), 3.29 (d, 3J ˆ 9 Hz, 1H; 2-H or 6-H), 3.24 (brs, 2H; 8-H, 9-H), 3.18
(d, 3J ˆ 8.5 Hz, 1H; 2-H or 6-H), 2.99 (brs, 1H; 7-H), 2.82 (brs, 1H; 1-H),
2.01 (s, 3H; CH3), 1.97 (s, 3H; CH3), 0.50 (brd, 2J (10a-H, 10b-H) ˆ 11.7 Hz,
1H; 10a-H), 0.5 (brd, 2J (10a-H, 10b-H) ˆ 13 Hz, 1H; 10b-H); 13C NMR
ˆ
(CH3); IR (KBr): nÄ ˆ 3055 ( C H), 2886 ( CH3), 2790 (N CH3), 1697
1
ˆ
ˆ
(C O), 1599 (C C), 1368 cm (C N); MS (70 eV, EI, high resolution):
‡
m/z: [M ] calcd for C31H23N1O1: 425.185262; found 425.177965; difference
7.2 ppm; UV/Vis (THF): lmax (lg e) ˆ 655 (2.91), 282 (4.49), 320 (4.37,
shoulder), 270 nm (4.45).
5-Norbornene-2-endo-3-exo-dicarboxylic acid diacetoxymethylester (9b):
First sodium hydride (2.4 g, 100 mmol) and then chloromethyl acetate[38]
(5.52 g, 50 mmol) were added to a mixture of 5-norbornene-2,3-(E)-
dicarboxylic acid (9a)[37] (4.55 g, 25 mmol) in absolute dimethyl formamide
(50 mL) at 08C, in analogy to the literature.[39] The mixture was converted
in 38% to the diacetoxymethylester 9b at 408C after 4 h. B.p. 1308C
(10 Pa); 1H NMR (200 MHz, CDCl3, TMS): d ˆ 6.24 (dd, 3J(5-H, 6-H) ˆ
5.8 Hz, 3J(5-H, 4-H) ˆ 3.4 Hz, 1H; 5-H), 6.01 (dd, 3J(6-H, 3-H) ˆ 5.8 Hz,
3J(6-H, 1-H) ˆ 3.0 Hz, 1H; 6-H), 5.74 ± 5.70 (m, 2H; OCH2O), 5.68 ± 5.61
(m, OCH2O), 3.40 (d, 3J(2-H, 3-H) ˆ 4.2 Hz, 1H; 3-H), 3.26 (brs, 1H; 1-H/
4-H), 3.11 (brs, 1H; 1-H/4-H), 2.69 (dd, 3J(2-H, 3-H) ˆ 4.6 Hz, 4J(7'-H,
2-H) ˆ 1.6 Hz, 1H; 2-H), 2.07 (s, 3H; CH3), 2.06 (s, 3H; CH3), 1.57 (d, 2J(7-
H, 7'-H) ˆ 8.6 Hz, 1H; 7-H), 1.41 (dd, 2J(7'-H, 7-H) ˆ 8.8 Hz, 4J(2-H, 7'-
H) ˆ 1.6 Hz, 1H; 7'-H); 13C NMR (50 MHz, CDCl3, TMS): d ˆ 172.8, 171.6,
ˆ
(75 MHz, [D8]THF, TMS): d ˆ 198.7 (C O)ketone, 172.4, 171.6, 169.8, 169.5
ˆ
(C O)ester, 138.0, 137.8, 135.8, 135.5, 132.1, 132.0, 128.8, 128.7 (C-1'a, C-4'a,
C-5'a, C-8'a, C-9', C-10', C-ipso-phenyl), 132.5, 132.4, 130.1, 129.9, 129.2,
129.2, 128.7, 128.6, 128.4, 128.3 (CH-phenyl), 127.3, 127.1, 126.2 (C-2', C-3',
C-4', C-5', C-6', C-7'), 124.4, 124.3 (C-1', C-8'), 80.71, 80.58 (OCH2O), 64.45,
64.07 (C-3, C-5), 51.68, 51.01, 47.89, 45.17, 44.23, 43.16 (C-1, C-2, C-6, C-7,
ˆ
C-8, C-9), 34.76 (C-10), 20.50, 20.28 (CH3); IR (KBr): nÄ ˆ 3029 ( CH), 2924
ˆ
ˆ
ˆ
(CH), 1781 (C Oketone), 1762 (C Oester), 1614 (C C), 1368 (O CO CH3),
1
1228, 1168, 1006 cm (C O); UV/Vis (THF): lmax (lg e) ˆ 360 (282), 350
(2.55), 342 (2.77), 335 (2.62), 320 (4.08), 300 (4.09), 253 (4.79), 245 nm
(4.71); Fluorescence (THF): lexc ˆ 315 nm, lem ˆ 361, 382, 404, 425 nm; MS
‡
‡
(70 eV, EI): m/z (%): ˆ 710 (2) [M ‡2], 680 (100) [M
CO]; C44H36O9
(708.8): calcd C 74.6, H 5.08; found C 73.9, H 4.98.
ˆ
169.4 (C O), 137.6, 134.8 (C-5, C-6), 79.49, 79.24 (OCH2O), 47.66, 47.43,
3,5-Diphenyl-3,5-(2-dimethylamino-9,10-phenanthrylene)tricyclo[5.2.1.02,6]-
decane-4-one-8,9-(E)-dicarboxylic acid (10c): A suspension of 8b (100 mg,
0.235 mmol) was treated with 9a (43 mg, 0.235 mmol) in chlorobenzene
(2.0 mL) at 1308C for 8 h. The white precipitate was removed by filtration,
washed with little chlorobenzene, dissolved in THF and precipitated by
addition of n-heptane. Recrystallization from ethanol yielded 103 mg
(0.17 mmol, 72%) of 10c. M.p. (decomp) 289 ± 2958C; 1H NMR (300 MHz,
[D8]THF, TMS): d ˆ 11.0 (brs, 2H; COOH), 8.58 ± 8.51 (m, 2H; 4'-H, 5'-H),
8.14 ± 8.05 (m, 1H; o'-H or o''-H), 7.97 ± 7.86 (m, 1H; o'-H or o''-H), 7.68 ±
7.53 (m, 2H; m'-H, m''-H), 7.38 ± 6.96 (m, 10H; 1'-H, 6'-H, 7'-H, 8'-H, o-H,
m-H, p-H), 6.40 (dd, 4J ˆ 2.7 Hz, 4J ˆ 2.7 Hz, 1H; 3'-H), [3.19 ± 3.11 (m,
3H), 3.04 ± 3.01 (m, 1H), 2.93 ± 2.91 (m, 1H), 2.87, 2.80* (brs, 1H), 1-H,
47.08, 45.74, 45.63 (C-1, C-2, C-3, C-4, C-7), 20.53 (CH3); IR (film): nÄ ˆ 3067
1
ˆ
ˆ
( C H), 2998, 2954, 2878 (C H), 1762 (C O), 1371 (CH3), 1225, 1157 cm
‡
‡
(C O); MS (70 eV, EI): m/z (%): ˆ 328 (20) [M ‡2], 296 (5) [M
2CH3],
‡
‡
‡
267 (5) [M
OCH2OCOCH3], 207 (15) [M
OCOCH3], 254 (10) [M
CHOCOCH3], 237 (4) [M
‡
‡
2OCOCH3], 194 (8) [M
(60) [C9H8O3], 66 (100) [C5H6]; C15O8H18 (326.3) calcd C 55.2, H 5.52;
found C 54.9, H 5.41.
C5H8O4], 164
3,5-Diphenyl-3,5-(9,10-phenanthrylene)tricyclo[5.2.1.02,6]decane-4-one-8-
exo-9-endo-dicarboxylic acid (10a): Compounds 8a (0.50 g, 1.30 mmol) and
9a (0.27 g, 1.50 mmol) were heated in chlorobenzene (12.0 mL) for 15 h
under reflux in the dark. After the removal of chlorobenzene (6 mL) by
destillation and addition of benzene (10 mL), the precipitated solid was
collected by filtration, dissolved in THF (80 mL) and treated with active
carbon at room temperature for 30 min. The product was precipitated from
the filtrate by addition of n-pentane. Further purification was achieved by
dissolving in THF at 508C and precipitating at room temperature by
addition of water. Yield 0.64 g (1.0 mmol, 84%); m.p. 2828C; 1H NMR
2
2-H, 6-H, 7-H, 8-H, 9-H], 2.61, 2.61* (s, 6H; NCH3), 0.47 (brd, J(10a-H,
10b-H) ˆ 11.8 Hz, 1H; 10a-H), 0.34 (brd, 2J(10a-H, 10b-H) ˆ 11.5 Hz,
13
ˆ
1H; 10b-H); C NMR (75 MHz, [D8]THF, TMS): d ˆ 199.3 (C O)ketone
,
ˆ
174.9, 174.3 (C O)acid, 149.4 (C-2'), 139.2*, 138.9, 138.7, 138.3*,135.7*,
135.5, 134.8, 134.6*, 130.2, 130.1*, 127.1*, 127.1, 122.6 (C-1'a, C-4'a, C-5'a,
1744
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0506-1744 $ 17.50+.50/0
Chem. Eur. J. 1999, 5, No. 6

Cheletropic Traps
1738±1747
1
ˆ
ˆ
C-8'a, C-9', C-10', C-ipso-phenyl), 132.9, 132.6, 129.8*, 129.7, 129.6, 129.5*,
129.2, 129.0*, 129.0, 128.9, 128.8*, 128.7, 128.6*, 128.1*, 128.0, 128.0, 127.9*,
126.9, 126.2, 125.0, 124.9, 124.8*, 123.2, 115.0, 114.9*, 107.2, 107.1* (CHarom.),
64.47*, 64.36, 64.00*, 63.90, (C-3, C-5), 51.97*, 51.86, 51.25, 48.47, 48.24*,
45.44, 44.42*, 44.22, 43.09 (C-1, C-2, C-6, C-7, C-8, C-9), 40.01 (NCH3),
( CH), 2930 ± 2970 ( CH), 1708 cm (C O); UV/Vis (THF): lmax (lg e) ˆ
446 nm (3.91); Fluorescence (phosphate buffer pH 7.4): lexc ˆ 395 ± 400 nm,
‡
lem(max) ˆ 565 nm; MS (70 eV, EI, high resolution): m/z: [M ] calcd for
C37H28O4: 536.1988; found 536.1971; difference 3.2 ppm.
3,6-Diphenyl-4,5-(2,2'-biphenylene)-tricyclo[6.2.1.02,7]undeca-3,5-diene-
9,10-(E)-dicarboxylic acid diacetoxymethylester (4b): M.p. 98 ± 1008C;
1H NMR (300 MHz, [D8]THF, TMS): d ˆ 7.74 (d, 3J ˆ 7.5 Hz, 2H; 6'-H, 6''-
H), 7.44 (dd, 3J ˆ 7.8 Hz, 4J ˆ 1.4 Hz, 2H; o-H or o'-H), 7.35 (dd, 3J ˆ 7.5 Hz,
4J ˆ 1.4 Hz, 2H; o-H or o'-H), 7.30 ± 7.10 (m, 8H; 2', 9', m, m', m'', p-H),
7.10 ± 7.05 (m, 2H; 4'-H, 4''-H), 6.80 ± 6.68 (m, 2H; 5'-H, 5''-H), 5.80 (d, 2J ˆ
5.6 Hz, 1H; OCH2O), 5.70 (d, 2J ˆ 9.0 Hz, 1H; OCH2O), 5.68 (d, 2J ˆ
8.8 Hz, 1H; OCH2O), 5.66 (d, 2J ˆ 5.6 Hz, 1H; OCH2O), 3.35 ± 3.30 (m,
2H; 2-H, 7-H), 3.30 (brs, 1H; 1-H or 8-H), 3.20 (brd, 3J ˆ 4.8 Hz, 1H; 9-H
or 10-H), 2.90 (brs, 1H; 1-H or 8-H), 2.85 (brd, 3J ˆ 3.3 Hz,1H; 9-H or 10-
H), 2.15 (brd, 2J(11a-H, 11b-H) ˆ 10.5 Hz, 1H; 11a-H), 2.02 (s, 3H; CH3),
ˆ
34.84 (C-10); IR (KBr): nÄ ˆ 3068, 3030 ( CH), 2976, 2922, 2801 (CH), 2786
ˆ
ˆ
ˆ
(N CH3), 1780 (C Oketone), 1710 (C Oacid), 1614 (C C), 1365 (C N),
1
1007 cm (C O); UV/Vis (phosphate buffer pH 7.2): lmax (lg e) ˆ 465
(3.48), 325 (4.13), 295 nm (4.37); Fluorescence (phosphate buffer pH 7.2):
‡
lexc ˆ 380 nm, lem ˆ 480 nm; MS (70 eV, EI): m/z (%): ˆ 607 (10) [M ], 579
‡
‡
(100) [M
CO]; MS (70 eV, EI, high resolution): m/z: [M ] calcd for
C40H33N1O5: 607.232730; found 607.235874; difference 5.2 ppm.
3,5-Diphenyl-3,5-(2-dimethylamino-9,10-phenanthrylene)tricyclo[5.2.1.02,6]-
decane-4-one-8,9-(E)-dicarboxylic acid diacetoxymethylester (10d):
A
suspension of 8b (100 mg, 0.235 mmol) and 9b (76.7 mg, 0.235 mmol) in
chlorobenzene (2.0 mL) was heated to 1108C. When the dark-green
solution had turned yellow (4 h) the solvent was removed, the solid was
dissolved in dichloromethane, and the product precipitated by addition of
n-heptane. Recrystallization from ethanol gave 150 mg (0.20 mmol, 85%)
of 10d as a mixture of isomers. M.p.(decomp) 220 ± 2308C; 1H NMR
(300 MHz, [D8]THF, TMS): d ˆ 8.58 (d, 3J ˆ 7.6 Hz, 1H; 4'-H or 5'-H), 8.55
(d, 3J ˆ 8.0 Hz, 1H; 4'-H or 5'-H), 8.01 ± 7.86 (m, 2H; o'-H, o''-H), 7.72 ± 7.62
(m, 2H; m'-H, m''-H), 7.46 ± 6.98 (m, 10H; 1'-H, 6'-H, 7'-H, o-H, m-H, p-H),
2.00 (s, 3H; CH3), 1.60 (brd, 2J(11a-H, 11b-H) ˆ 10.0 Hz, 1H; 11b-H);
13
ˆ
C NMR (75 MHz, [D8]THF, TMS): d ˆ 172.8, 172.1, 169.6, 169.5 (C O),
143.4, 143.1 (C-3,C-6), 135.2, 132.6, 130.6, 130.5, 129.6, 129.4, 128.1, 128.1,
124.3 (CHarom.), 134.9, 134.8, 134.2, 133.9, 128.5, 128.4, 127.3, 127.2 (C-4, C-5,
C-1', C-1'', C-2', C-2'', C-ipso-phenyl), 80.90, 81.27 (OCH2O), 52.36, 51.68,
50.26, 49.72, 48.92, 47.75 (C-1, C-2, C-7, C-8, C-9, C-10), 34.90 (C-11), 20.58,
ˆ
20.35 (CH3); IR (KBr): nÄ ˆ 3068, 3030 ( CH), 2993, 2913, 2880 (CH),
1
ˆ
1757 (C Oester), 1369 (O CO CH3), 1233 , 1153, 1004 cm (C O);
4
4
6.40 (dd, J ˆ 3.0 Hz, J ˆ 3.0 Hz, 1H; 3'-H), 5.95 ± 5.90 (m, 2H; OCH2O),
5.68 ± 5.61 (m, 2H; OCH2O), 3.27 ± 3.19 (m, 3H; 1-H or 7-H, 2-H, 6-H),
3.15, 3.12* (brd, 4J ˆ 3.0 Hz, 1H; 8-H or 9-H), 3.03*, 2.96 (brd, 3J ˆ 3.0 Hz,
1H; 8-H or 9-H), 2.86, 2.80* (brs, 1H; 1-H or 7-H), 2.63 (s, 6H; NCH3),
2.01, 1.98*, 1.97*, 1.95 (s, 6H; CH3), 0.53 (brd, 2J(10a-H, 10b-H) ˆ 12.0 Hz,
UV/Vis (THF): lmax (lg e) ˆ 445 nm (3.69); Fluorescence (THF): negligible;
‡
MS (70 eV, EI): m/z (%): ˆ 680 (100) [M ]; MS (70 eV, EI, high resolution):
‡
m/z: [M ] calcd for C43H36O8: 680.2411; found 680.2449; difference
5.8 ppm.
3,6-Diphenyl-4,5-(4-dimethylamino-2,2'-biphenylene)tricyclo[6.2.1.02,7]un-
deca-3,5-diene-9,10-(E)-dicarboxylic acid (4c): M.p. 230 ± 2358C; 1H NMR
(300 MHz, [D8]THF, TMS): d ˆ 7.60 (d, 3J ˆ 8.2 Hz, 1H; 6'-H or 6''-H), 7.58
(d, 3J ˆ 7.8 Hz, 1H; 6'-H or 6''-H), 7.46 (dd, 3J ˆ 7.8 Hz, 4J ˆ 1.5 Hz, 1H; o'-
H), 7.40 (dd, 3J ˆ 7.8 Hz, 3J ˆ 1.4 Hz, 2H; o-H), 7.33 ± 7.27 (m, 3J ˆ 7.8 Hz,
3J ˆ 1.4 Hz, 2H; o''-H, 3'-H), 7.27 ± 7.14 (m, 5H; 3''-H, m-H, m'-H, m''-H),
7.08 ± 6.98 (m, 2H; CHarom.), 6.67 ± 6.48 (m, 3H; 4''-H, 5'-H, 5''-H), [3.32 ±
3.12 (m, 3H), 3.03 ± 2.95 (m, 1H), 2.82 ± 2.78 (m, 1H), 2.72 ± 2.67 (brd, 3J ˆ
4.2 Hz, 1H), 1-H, 2-H, 7-H, 8-H, 9-H, 10-H], 2.50, 2.48* (s, 6H; CH3), 2.13
1H; 10a-H),
0.31 (brd, 2J(10a-H, 10b-H) ˆ 11.7 Hz, 1H; 10b-H);
13
ˆ
C NMR (75 MHz, [D8]THF, TMS). d ˆ 198.8 (C O)ketone, 172.4, 171.6,
ˆ
169.7, 169.4 (C O)ester, 149.4*, 149.3 (C-2'), 138.7*, 138.5, 138.2, 138.0*,
135.5, 135.2*, 134.6*, 134.3, 132.6, 132.5*, 129.9, 129.7, 122.5 (C-1'a, C-4'a,
C-5'a, C-8'a, C-9', C-10', C-ipso-phenyl), 132.8, 132.7*, 132.5*, 132.4, 130.0*,
130.0*, 129.9, 129.7, 129.2, 129.1*, 129.0*, 129.0, 128.6, 128.0, 126.9, 126.0,
125.0, 124.8, 123.1, 115.0, 114.9, 106.9 (CHarom.), 80.61, 80.54*, 80.48*, 80.42
(OCH2O), 64.29, 64.14, 63.91, 63.77 (C-3,C-5), 51.54, 50.93, 48.05, 47.83*,
45.19, 45.14*, 44.31*, 44.12, 43.10 (C-1, C-2, C-6, C-7, C-8, C-9), 39.94*,
2
2
(brd, J(11a-H, 11b-H) ˆ 10.5 Hz, 1H; 11a-H), 1.60 (brdd, J(11a-H, 11b-
ˆ
39.89 (NCH3), 34.73 (C-10), 20.40 (CH3); IR (KBr): nÄ ˆ 3068, 3030 ( CH),
H) ˆ 10.2 Hz, J(7-H, 11b-H) ˆ 9.3 Hz, 1H; 11b-H); 13C NMR (75 MHz,
ˆ
ˆ
2976, 2922, 2801 (CH), 2793 (N CH3), 1779 (C Oketone), 1764 (C Oester),
ˆ
[D8]THF, TMS): d ˆ 175.2, 175.1*, 174.2 (C O), 149.6, 149.5* (C-4'), 144.8,
1
ˆ
1614 (C C), 1370 (O CO CH3), 1367 (C N), 1007 cm (C O); UV/Vis
144.5*, 143.2, 143.1* (C-3,C-6), 135.6*, 135.6, 135.0, 134.7*, 134.3*, 134.1,
133.7*, 133.5, 133.4*, 133.3, 132.9*, 132.8, 128.0*, 127.9, 127.5, 127.3* (C-4,
C-5, C-1', C-1'', C-2', C-2'', C-ipso-phenyl), 130.6, 130.5*, 130.2, 130.1*,
129.4, 129.2*, 128.8*, 128.7, 125.2*, 125.2, 124.6, 124.0, 123.9*, 127.8, 116.9*,
116.9, 113.3, 113.2* (C-3', C-3'', C-4'', C-5', C-5'', C-6', C-6'', CHarom.), 52.92,
52.32*, 51.99, 50.44, 50.33*, 49.68*, 49.62, 48.25, 48.06*, 47.15 (C-1, C-2, C-7,
C-8, C-9, C-10), 40.00, 39.93* (NCH3), 34.57, 34.40* (C-11); IR (KBr): nÄ ˆ
(THF): lmax (lg e) ˆ 398 (3.53), 369 (3.54), 334 (4.16), 310 nm (4.32);
Fluorescence (THF): lexc ˆ 380 nm, lem ˆ 460 nm; MS (70 eV, EI): m/z
‡
‡
(%): ˆ 751 (27) [M ], 723 (100) [M
CO]; MS (70 eV, EI, high
‡
resolution): m/z: [M ] calcd for C46H41N1O9: 751.277341; found
751.278132; difference 1.1 ppm.
Photolysis of ketones 10a ± d: Ketones 10a--d were photolysed at l ˆ
300 nm in [D8]THF or THF in a Rayonet Photochemical Reactor at
308C. For 1H NMR spectral investigations suprasil NMR tubes were
used, and for preparative purposes quartz tubes of 7 mm diameter and a
volume of 10 mL were used. The concentration of 10a ± d was 0.015m (ca.
ˆ
ˆ
3066, 3029 ( CH), 2977, 2921, 2802 (CH), 2787 (N CH3), 1708 (C Oacid),
1
ˆ
1612 (C C), 1362 (C N), 1006 cm (C O); UV/Vis (THF): lmax (lg e) ˆ
466 (3.30), 295 nm (4.36); Fluorescence (phosphate buffer pH 7.2): lexc
ˆ
‡
460 nm, lem ˆ 600 nm; MS (70 eV, EI): m/z (%): ˆ 579 (100) [M ]; MS
1
‡
10 mgmL 1) for 1H NMR spectroscopical studies and 3 mgmL for
(70 eV, EI, high resolution): m/z: [M ] calcd for C39H33N1O4: 579.132730;
preparations. The solutions of the ketones were purged with nitrogen for
found 579.135874; difference 6.3 ppm.
30 min and photolysed at 308C for up to 100 h. The necessary duration of
the photolysis was determined by running a H NMR spectroscopic and a
preparative sample simultaneously. After termination of the photolysis the
solvent was removed, the o-quinodimethanes characterized by spectro-
scopic techniques, and the samples stored at 788C.
3,6-Diphenyl-4,5-(4-dimethylamino-2,2''-biphenylene)tricyclo[6.2.1.02, 7]-
undeca-3,5-diene-9,10-(E)-dicarboxylic acid diacetoxymethylester (4d):
M.p. 82 ± 838C; 1H NMR (300 MHz, [D8]THF, TMS): d ˆ 7.59 (dd, 3J ˆ
8.4 Hz, 3J ˆ 8.7 Hz, 2H; 6'-H, 6''-H), 7.52 ± 7.46 (m, 1H; o'-H), 7.43 (m, 2H;
o-H), 7.32 ± 7.24 (m, 3H; o''-H, 3'-H, 3''-H), 7.22 ± 7.14 (m, 4H; m-H, m'-H),
7.05 ± 6.97 (m, 2H; p-H), 6.67 ± 6.48 (m, 3H; 4''-H, 5'-H, 5''-H), 5.82 ± 5.56
(m, 4H; OCH2O), [3.35 ± 3.10 (m, 3H), 3.12 ± 3.10 (m, 1H), 2.84 ± 2.82 (m,
1H), 2.74 (brd, 3J ˆ 4.2 Hz, 1H), 1-H, 2-H, 7-H, 8-H, 9-H, 10-H], 2.51, 2.49*
(s, 6H; NCH3), 2.18 (brd, 2J(11a-H, 11b-H) ˆ 9.0 Hz, 1H; 11a-H), 2.03,
2.01* (s, 3H; CH3), 1.98, 1.97* (s, 3H; CH3), 1.60 (brdd, 2J(11a-H, 11b-H) ˆ
9.0 Hz, J(7-H, 11b-H) ˆ 8.4 Hz, 1H; 11b-H); 13C NMR (75 MHz, [D8]THF,
1
3,6-Diphenyl-4,5-(2,2'-biphenylene)tricyclo[6.2.1.02,7]undeca-3,5-diene-
9,10-(E)-dicarboxylic acid (4a): M.p. 265 ± 2678C; 1H NMR (300 MHz,
[D8]THF, TMS, HH-COSY): d ˆ 11.02 (brs, 2H; COOH), 7.74 (d, 3J ˆ
8.1 Hz, 2H; 6'-C, 6''-H), 7.39 ± 7.46 (m, 4H; CHarom.), 7.15 ± 7.26 (m, 6H;
CHarom.), 7.06 ± 7.13 (m, 4H; CHarom.), 6.72 ± 6.78 (m, 2H; 5'-C, 5''-H), [3.34 ±
3.39 (m, 1H), 3.24 ± 3.29 (m, 2H), 3.07 ± 3.10 (m, 1H), 2.90 (brs, 1H), 2.80 ±
2.83 (m, 1H), 1-H, 2-H, 7-H, 8-H, 9-H, 10-H], 2.11 (d, 2J (11a-H, 11b-H) ˆ
10.3 Hz, 1H; 11a-H), 1.57 (d, 2J (11a-H, 11b-H) ˆ 10.3 Hz, 1H; 11b-H);
13C NMR (75 MHz, [D8]THF, TMS, inverse-CH-COSY): d ˆ 175.63, 174.77
ˆ
TMS): d ˆ 172.5, 172.5*, 171.8, 171.8*, 169.3*, 169.2 (C O), 149.7, 149.7*
(C-4'), 144.50*, 144.4, 143.3, 142.9* (C-3, C-6), 135.6*, 135.6, 134.3*, 134.1,
133.8*, 133.7, 133.4*, 133.2, 133.0*, 132.9, 132.5, 132.5*, 128.2*, 128.0,
127.6*, 127.4, (C-4, C-5, C-1', C-1'', C-2', C-2'', C-ipso-phenyl), 130.5*, 130.5,
130.2, 130.0*, 129.9*, 129.4, 129.0, 128.9*, 125.2, 125.1*, 124.7*, 124.7,
123.9*, 123.8, 122.8, 116.9*, 116.8, 113.4, 113.3* (CHarom.), 80.18, 80.06
(OCH2O), 52.52, 51.89*, 51.82, 50.28, 50.06*, 49.51*, 49.26, 48.14*, 47.87,
46.97 (C-1, C-2, C-7, C-8, C-9, C-10), 39.99, 39.90 (NCH3), 34.57, 34.39
ˆ
(C O)acid, 135.74, 135.55, 135.46, 134.70, 134.60, 132.74 (C-3, C-4, C-5, C-6,
C-1', C-1'', C-2', C-2''), 132.93, 130.90, 130.87, 129.67, 129.53, 128.67, 128.56
(C-3', C-3'',CH-phenyl), 128.29, 128.22, 127.56, 127.47 (C-4', C-4'', C-5', C-5''),
124.59 (C-6', C-6''), 52.93, 52.16 (C-9, C-10), 50.85, 50.14, 49.53, 48.22 (C-1,
C-2, C-7, C-8), 35.03 (C-11); IR (KBr): nÄ ˆ 3340 ± 3600 ( OH), 3060, 3021
Chem. Eur. J. 1999, 5, No. 6
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0947-6539/99/0506-1745 $ 17.50+.50/0
1745

R. Sustmann et al.
FULL PAPER
(C-11), 20.13, 20.05 (CH3); IR (KBr): nÄ ˆ 3070, 3033 (CH ˆ ), 2980, 2924,
Statistics: The experiments with alveolar macrophages were performed in
duplicate and repeated 4 times with cells from different rats. Data are
expressed as means Æ standard deviation. Data obtained from two groups
were compared by means of the Studentꢁs t test. A p value of <0.05 was
considered significant.
ˆ
ˆ
2803 (CH), 2795 (N CH3), 1765 (C Oester), 1615 (C C), 1372
1
(O CO CH3), 1369 (C N), 1009 cm (C O); UV/Vis (THF): lmax (lg
e) ˆ 465 (3.40), 298 nm (4.44); Fluorescence (THF): negligible; MS (70 eV,
‡
‡
EI): m/z (%): ˆ 723 (100) [M ]; MS (70 eV, EI, high resolution): m/z: [M ]
calcd for C45H41N1O8: 723.177341; found 723.178132; difference 1.3 ppm.
Formation of hydroxylamines 6b and 6d: An equimolar amount of
acetaldehyde (0.42 mL) was added to a solution of 4b (15mm, 5.1 mg in
0.5 mL) or 4d (5.4 mg in 0.5 mL) in [D8]THF. This solution was mixed with
an equimolar amount of NO dissolved in [D8]THF (0.5 mL). Immediate
analysis by 1H NMR spectroscopy revealed about 40% conversion of 4b
and 4d to 6b and 6d, respectively. The 1H NMR spectra of 4b and 4d are
very similar to those of 10b and 10d, respectively; MS (70 eV, EI) m/z (%):
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891 ± 893.
‡
‡
‡
‡
6b: 711 (4) [M ], 680 (100) [M
NOH] or [M ] of 4b; 6d: 754 (8) [M ],
‡
‡
723 (100) [M
NOH] or [M ] of 4d.
Stopped-flow kinetic measurements: The reactions of FNOCTs with nitric
oxide were carried out at 238C in benzene (c ˆ 90mm, l ˆ 450 nm) and in
phosphate buffer (pH 7.4) for 4a (c ˆ 50mm, l ˆ 450 nm) and at 218C in
THF (c ˆ 100mm, l ˆ 465 nm) and in phosphate buffer (pH 7.3) for 4c (c ˆ
10mm, l ˆ 465 nm) under pseudo-first-order conditions (cNO(benzene) ˆ
12mm, cNO(THF) ˆ 15mm, cNO(water) ˆ 2.0mm).[40]
ESR spectroscopic measurements: Solutions were prepared in septum-
capped, argon-flushed 4 mm o.d. quartz tubes (for organic solvents) or a
0.4 mm quartz flat cell (for aqueous solutions). Deoxygenated solutions
of the FNOCTs (1mm, 0.5 mL) and saturated solutions of NO in the
same solvent (0.5 mL) were injected into the tubes by means of a
gas-tight syringe and briefly mixed by shaking. Recording conditions:
microwave frequency, 9.4 GHz; modulation, 0.03 mT; sweep range, 10 mT;
sweep time, 15 min. The ESR parameters were refined by computer
simulation.
Cell isolation and culture: Alveolar macrophages were isolated from male
Wistar rats (250 ± 310 g) by bronchoalveolar lavage as described previous-
ly.[41] The cells were seeded onto 6.2 cm2 glass coverslips in 6-well cell-
culture plates and cultured in Dulbeccoꢁs modified eagles medium
(DMEM) supplemented with l-glutamine (2mm), gentamycin
(50 mgmL 1), and 10% heat-inactivated fetal calf serum.
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Cell experiments: Experiments with alveolar macrophages were started
15 ± 20 hours after isolation of the cells. Cell density in the experiments was
3.25 Æ 0.5 Â 104 cellscm 2. The cells were used either without further
treatment, or were primed for NO production by 11 hours preincubation
with lipopolysaccharides (LPS; 0.5 mgmL 1), and/or NO production was
inhibited by 11 h preincubation with the NO synthase inhibitor NMA
(0.1mm). The media of stimulated cultures and respective controls were
supplemented with l-arginine (0.5mm). The experiments were performed
in Krebs-Henseleit buffer (NaCl 115mm, NaHCO3 25mm, KCl 5.9mm,
MgCl2 1.2mm, NaH2PO4 1.2mm, Na2SO4 1.2mm, CaCl2 2.5mm, HEPES
20mm, pH 7.4) supplemented with l-glutamine (2mm) and d-glucose
(10mm; modified Krebs-Henseleit buffer). This buffer was equilibrated
with 95% air/5% CO2 throughout the experiments. Cell viability was
determined routinely by trypan blue or propidium iodide exclusion.
Digital fluorescence microscopy: Cells were loaded with FNOCT using the
membrane-permeable acetoxymethyl ester 4d. Retention of the dye in the
cells was improved by the addition of probenecid. In order to evaluate the
optimal loading conditions, the following variations were performed: the
cells were incubated for 10 ± 120 minutes at 378C in the dark in modified
Krebs-Henseleit buffer containing probenecid (1.4mm), FNOCT 4d (10 ±
100mm), NMA (0.1mm) where appropriate, PluronicꢁR F-127 (0.02 ± 0.2%),
and DMSO (0.2 ± 2%), depending on the FNOCT concentration. After this
incubation the glass coverslips were rinsed three times, modified Krebs-
Henseleit buffer supplemented with probenecid (1.4mm), l-arginine
(0.5mm) or NMA (0.1mm) was added, and fluorescence microscopy was
started immediately. Fluorescence microscopy was performed on an
inverted microscope (Axiovert 135TV, Zeiss, Oberkochen, Germany)
equipped with the Attofluor imaging system (Atto Instuments, Rockville,
Maryland, USA).
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Chem. Eur. J. 1999, 5, No. 6

Cheletropic Traps
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Received: October 12, 1998 [F1390]
Chem. Eur. J. 1999, 5, No. 6
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