Convenient synthesis of a [1-14C]diazirinylbenzoic acid as a photoaffinity label for binding studies of V-ATPase inhibitors

Article abstract of DOI:10.1002/ejoc.200700194

Diazirine-tagged systems are considered reliable compounds for photoaffinity labeling (PAL) in biochemical studies as they enable investigation and understanding of biological mechanisms through covalent bonding to the target and subsequent detection. ¹⁴C-labeled 4-(3-trifluoromethyl-3H- diazirin-3-yl)benzoic acid (11) was prepared by a lithium-bromide exchange on the bis-silylated 4-bromophenyldiaziridine 19 with subsequent transformations with electrophiles as key steps of the synthesis. Using ¹⁴CO ₂, which was generated from rather inexpensive Ba¹⁴CO ₃, the desired diaziridinylbenzoic acid 21 was obtained in 78% yield based on the employed radioactive material. Oxidation under mild conditions then yielded diazirine 11 in a 100 mg scale. This versatile photoaffinity label was selectively attached to the tetrahydropyrane ring of bafilomycin A₁ (2) and concanamycin A-derived 3, which both specifically inhibit the V-ATPases. Inhibition assays were performed and revealed that the inhibitory capacities of the labeled derivatives are suitable for PAL studies on this important group of enzymes to elucidate the as yet unknown binding sites. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200700194

FULL PAPER
DOI: 10.1002/ejoc.200700194
Convenient Synthesis of a [1-14C]Diazirinylbenzoic Acid as a Photoaffinity
Label for Binding Studies of V-ATPase Inhibitors
Tobias Bender,[a] Markus Huss,[b] Helmut Wieczorek,[b] Stephanie Grond,*[a] and
Paultheo von Zezschwitz*[a]
Dedicated to Prof. Axel Zeeck
Keywords: Photoaffinity labeling / V-ATPase inhibitors / Carboxylation / 14C-Label / Diazirines / Natural products
Diazirine-tagged systems are considered reliable compounds
for photoaffinity labeling (PAL) in biochemical studies as
they enable investigation and understanding of biological
mechanisms through covalent bonding to the target and sub-
sequent detection. 14C-labeled 4-(3-trifluoromethyl-3H-di-
employed radioactive material. Oxidation under mild condi-
tions then yielded diazirine 11 in a 100 mg scale. This versa-
tile photoaffinity label was selectively attached to the tetra-
hydropyrane ring of bafilomycin A1 (2) and concanamycin
A-derived 3, which both specifically inhibit the V-ATPases.
azirin-3-yl)benzoic acid (11) was prepared by a lithium-bro- Inhibition assays were performed and revealed that the in-
mide exchange on the bis-silylated 4-bromophenyldiazirid-
hibitory capacities of the labeled derivatives are suitable for
PAL studies on this important group of enzymes to elucidate
ine 19 with subsequent transformations with electrophiles as
key steps of the synthesis. Using 14CO2, which was gener- the as yet unknown binding sites.
ated from rather inexpensive Ba14CO3, the desired diazirid-
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
inylbenzoic acid 21 was obtained in 78% yield based on the
Germany, 2007)
fast reactions even with C–H bonds. Additionally, byprod-
ucts formed during irradiation must be chemically inert.
The label should be small to prevent steric perturbations in
the target-binding process, and the activation should occur
at wavelengths which do not cause damage to the examined
biological system (λ Ն 300 nm). Different types of photore-
active moieties generally suitable for PAL are azides, diazo
compounds, benzophenones, diazonium salts, and diazir-
ines. The 3-trifluoromethyl-3-aryldiazirines, which generate
carbenes upon 300 nm irradiation are considered to “meet
most of the criteria of an ideal photoreactive group”.[2d]
Yet, azides have found widespread use since they are readily
obtainable and show a high stability in the dark.[2b] For
tracing and identification of the tagged target, the photo-
phore must bear an easily detectable group like a radioac-
tive label, a biotin tag, magnetic micro spheres, or a fluores-
cent tag.[2h] Although the biotinylation technique was espe-
cially successful in several studies, all but the radioactive
labels suffer from the same potential drawback; the use of
sterically demanding tracer groups causes perturbations be-
tween the biological target and the labeled reagent, and
thus, can inhibit formation of the ligand-receptor com-
plex.[3]
Introduction
Photoaffinity labeling (PAL)[1] is a versatile and reliable
tool in molecular biology and finds a growing interest as
reflected by the ever-increasing number of papers and re-
views on this topic.[2] PAL is needed for the investigation of
molecular machines; for example, for the determination of
the precise binding site of biologically active compounds
and the mode of action of certain peptide domains. The
experimental procedure comprises the attachment of a
photosensitive group to either natural ligands or inhibitors
and the subsequent exposure to the biological target to
form a ligand-receptor complex. This is followed by photo-
activation leading to a highly reactive intermediate, which
then forms a covalent, thus irreversible bond between li-
gand and receptor. The photosensitive molecule has to meet
certain requirements including a high stability of the pre-
cursor prior to activation together with a very high reactiv-
ity of the photolytically released intermediate to allow for
[a] Institut für Organische und Biomolekulare Chemie der Georg-
August-Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
E-mail: sgrond@gwdg.de
Such radioactive labels have been used for PAL studies
on the aglycon 3 (Figure 1) derived from the natural prod-
uct concanamycin A (1), which together with bafilomycin
A1 (2) is one of the most potent inhibitors of vacuolar
pzezsch@gwdg.de
[b] Tierphysiologie - FB Biologie/Chemie, Universität Osnabrück,
Barbarastrasse 11, 49076 Osnabrück, Germany
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Photoaffinity Label for Binding Studies of V-ATPase Inhibitors
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Figure 1. Structures of concanamycin A (1), bafilomycin A1 (2), and concanolides 3 and 4.
ATPase (V-ATPase).[4] This proton-translocating enzyme is a
Results and Discussion
component of almost all eukaryotic cells and is essential in
many processes including bone degradation, acid secretion,
receptor-mediated endocytosis, and cell proliferation.[5] The
malfunction of V-ATPases is considered to cause several
severe diseases such as osteopetrosis, deafness, and male in-
fertility. Furthermore, due to its function in bone metabo-
lism and in invading tumors, the enzyme is a promising
pharmacological target for osteoporosis and cancer ther-
apy.[6,7] Therefore, the determination of the binding sites of
the inhibitors is an important aim. In 2001 Zeeck et al.
reported on the preparation of the concanolide 4, bearing
both a diazirine group and 125I as the tracer,[8] and PAL
studies using this compound[9] as well as genetic approaches
by Bowman and Bowman employing 1 and 2[10] pointed to
the c-subunit of heteromultimeric V-ATPases as the binding
site of both plecomacrolides. Yet, the specific inhibitory po-
tential of derivative 4 on V-ATPase of Manduca sexta is
three orders of magnitude lower than that of the natural
product 1, thus complicating the elucidation of the precise
site of the covalent modification. This decreased potency
might stem from the fact that in 4, hydrophobic substitu-
ents are placed at both C-9 of the macrocyclic ring and
C-23 of the tetrahydropyrane moiety. In contrast, the 21-
deoxyconcanolide 3 itself as well as derivatives modified at
either C-9 or C-23 retain the high inhibition potential of
concanamycin A.[11a] Studies on the structure-activity rela-
tionships of bafilomycin A1 revealed that modifications at
C-21 of the tetrahydropyrane ring but not at the hydroxy
moiety at C-7 of this molecule have no decreasing influence
on the inhibitory activity.[11b] Therefore, we became inter-
ested in the synthesis of a radioactively labeled 3-trifluoro-
methyl-3-aryldiazirine and its attachment to the tetra-
hydropyrane ring of inhibitors 2 and 3 for subsequent de-
tailed PAL studies on the V-ATPase.
Preparation of the Photoaffinity Label
A few syntheses of 3-trifluoromethyl-3-aryldiazirines
containing radioactive isotopes have already been de-
scribed. They incorporate 125I as the tracer in compounds
like 5[12] or tritium in compounds like 6–8[13] and 9[14] (Fig-
ure 2). Both isotopesϪparticularly 125IϪpossess a high spe-
cific activity, an attribute desirable for photoaffinity studies,
but 125I has a half-life of only 60 d, which limits its practical
use. Furthermore, the use of tritium as a marker can lead
to problems due to its low traceability as a consequence of
the low energy of its β radiation.[15] The synthesis of the
14C-labeled compound 10 has already been described,[14]
but as for compounds 5–9, their preparation required the
use of highly toxic thallium reagents and were altogether
quite tedious. In addition, the purification of 10 was per-
formed by preparative HPLC, and the employed 14CH3I is
rather expensive. Since the photoaffinity label for com-
pounds 2 and 3 has to possess a carboxy moiety, we envis-
aged the synthesis of 14C-labeled 4-(3-trifluoromethyl-3H-
diazirin-3-yl)benzoic acid (11) by carboxylation starting
Figure 2. Selected diazirinyl photoaffinity labels bearing a radioac-
tive tracer.
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T. Bender, M. Huss, H. Wieczorek, S. Grond, P. von Zezschwitz
FULL PAPER
from Ba14CO3 as the cheapest source of 14C.[16] The non- the diazirine moiety to the activated metal surface, as pre-
radioactive analogue of 11 has already been used as a reli- viously observed in the case of a cyano group.[20b]
able tool in several PAL studies.
Since diazirine 17 turned out to be unsuitable for metal-
For this purpose inexpensive 1,4-dibromobenzene (12) lation and subsequent carboxylation, this transformation
was chosen as the starting material, which was transformed was attempted at the stage of diaziridine 16. For the inter-
into the trifluoromethyl ketone 13 by the reaction of the mediate protection of the diaziridine moiety, the trimethyl-
corresponding Grignard reagent with ethyl trifluoroacetate silyl group was chosen, which promised to be readily cleav-
according to a reported procedure (Scheme 1).[17] The ob- able upon aqueous work up. While incomplete conversions
tained ketone 13 was efficiently transformed into diaziridine occurred when using TMSCl/NEt3, the more reactive tri-
16 following protocols described in the literature (through methylsilyl triflate (TMSOTf) led to the formation of the
the formation of the oxime 14, its tosylation to furnish 15, bis-silylated 19 in excellent yield (Scheme 2). Though rather
and finally treatment with liquid ammonia in an auto- sensitive to moisture, compound 19 could be stored for up
clave).[18] This led to the isolation of diaziridine 16 in multi- to 10 d at –20 °C. Treatment of 19 with nBuLi (1 equiv.)
gram scale with an overall yield of 48%. Diaziridine 16 was resulted in a smooth lithiation, and the versatility of inter-
successfully dehydrogenated to the corresponding diazirine mediate 20 was examined by its reaction with several elec-
17 using tert-butyl hypochlorite (tBuOCl) as the oxi- trophiles furnishing derivatives 22a–c in reasonable
dant,[18c] yet later on, iodine/triethylamine proved to be a yields.[21] For the carboxylation yielding non-radioactive
more convenient oxidation system and resulted in higher 22d, excess carbon dioxide was generated from dry ice,
yields.[18d] Although the trifluoroethyldiazirine moiety is passed through molecular sieves (3 Å) and then introduced
stable under a broad range of reaction conditions including into the solution of lithium organyl 20. After aqueous
strong bases, the attempted metal-halogen exchange of 17 workup, essentially pure 22d was isolated in high yield
with n-, sec-, or tert-butyllithium as well as with isopro- (89%) and directly oxidized to diazirine 23 using iodine and
pylmagnesium chloride[19] failed. While nBuLi preferen- NEt3. Purification of the product just required a basic ex-
tially underwent nucleophilic attack on the diazirine ring, traction of the reaction mixture and led to the isolation
complex mixtures were obtained from the other reactions. of 23 in quantitative yield. Thus, in addition to a methyl
In addition, no conversion took place when treating aryl substituent,[18b] protected hydroxymethyl groups[18b,22] and
bromide 17 with standard magnesium turnings or highly
reactive Rieke magnesium prepared by the reduction of
MgCl2 with potassium.[20a] This failure of the oxidative ad-
dition and thus formation of the respective Grignard rea-
gent might be caused by coordination of the nitrogens of
Scheme 1. Synthesis of p-bromophenyldiazirine 17.
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Scheme 2. Completion of the synthesis.
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Photoaffinity Label for Binding Studies of V-ATPase Inhibitors
FULL PAPER
an oxazoline ring[18c] a bromo substituent is a suitable “lat- for 15 h and the distillative removal of the latter at 40 mbar.
ent” carboxy moiety for the preparation of diazirinylben- This sophisticated step had to be performed very carefully,
zoic acids.
since on one hand, benzoyl chloride 25 is quite volatile and
Having succeeded with the preparation of unlabeled di- on the other hand, traces of remaining thionyl chloride in
azirine 23 in high yields, the synthesis of the radioactive crude 25 led to the decomposition of macrolide 3. The de-
analogue 11 was started with an extensive testing of dif- sired labeled 21-deoxyconcanolide 26 was obtained in 36%
ferent experimental procedures in order to achieve a maxi- yield (52% yield based on a 69% conversion) with a specific
mum yield based on liberated carbon dioxide. This led to activity of 40.0 mCi/mmol by reacting 3 with freshly pre-
the development of a rather simple setup, as the 14CO2 was pared 25 in the presence of an excess of 4-N,N-dimethylami-
generated by adding concentrated H2SO4 directly to the nopyridine. Thus, both plecomacrolides were selectively
glass vial in which the Ba14CO3 was purchased. The gas tagged with the necessary functionalities for PAL studies in
stream was passed through a cooling bath to freeze out a single operational step and with only one photoaffinity
traces of acid and then introduced into a solution contain- tracer group. This is an apparent experimental advantage
ing only 3 equiv. of the lithium organyl 20.[23] Starting from of radioactively labeled photophores such as 11, especially
50 mCi (0.91 mmol) of Ba14CO3, 165 mg of the 14C-diazir- when working with ligands of limited availability.
idine 21 was thus obtained, which corresponds to a yield of
78%. The oxidation of 21 was performed as described
above affording the pure diazirine 11 in 83% yield after a
basic extraction as the sole purification step. This radioac-
tive photoaffinity label possessed a specific activity of
44.11 mCi/mmol and showed no signs of decomposition
when stored for several months at –18 °C in CH2Cl2 at a
concentration of approximately 4 mCi/mL.
Labeling of the Plecomacrolides and Inhibition Assays
Based on the structure-activity relationships of the pleco-
macrolide derivatives discussed above, the secondary hy-
droxy moieties on the tetrahydropyrane ring in both bafilo-
mycin A1 (2) and 21-deoxyconcanolide A (3) appeared to
be the most promising sites for the attachment of the photo-
affinity label 11. Fortunately, these groups are significantly
more reactive towards esterification than the hydroxy moie-
ties at C-9 in compound 3 and C-7 in metabolite 2, while
all the other hydroxy groups are rather unreactive, presum-
ably due to intramolecular conformational constraints and
hydrogen bonding.[8,11] Thus, regioselective esterification of
2 and 3 seemed to be possible without intermediate protec-
tion. The secondary metabolites 1 and 2 were obtained
Scheme 3. Coupling reaction of diazirine 11 with ATPase inhibitors
2 and 3.
from fermentations of the Streptomyces sp. strains Gö 22/
15 and Gö3822 14F, respectively. Efficient isolation of con-
canamycin A (1) from a 50 L cultivation was achieved by
silica gel chromatography of the acetone cell extract,[24] and
subsequent methylation, reduction, and deglycosylation
were performed as described to yield 3 in multi-mg scale.[8]
Bafilomycin A1 was obtained from shaking-flask cultiva-
tions in yields of 4 mg/L.[24]
The inhibitory potential of the labeled derivatives 24 and
26 on the purified V-ATPase was examined, and the results
are shown in Table 1. Remarkably, the inhibition by the
Table 1. Inhibitory activity of 1, 2, 3, 4, 24, and 26 against purified
V-ATPase form M. sexta. The V-ATPase holoenzyme for the inhi-
bition assays was purified and its activity was determined accord-
ing to the literature.[9]
The transformation of macrolide 2 was performed by a
standard EDCI-based protocol employing only 2.1 equiv.
of 11 (Scheme 3). After aqueous workup and column
chromatography on silica gel, the desired labeled bafilomy-
cin 24 was isolated in 41% yield (59% yield based on a 69%
conversion) with a specific activity of 33.7 mCi/mmol. In
contrast, the attempted esterification of 21-deoxyconcanol-
ide 3 using DCC or EDCI and various bases only led to
decomposition. Therefore, benzoic acid 11 was converted
Entry
Compound
Ki [nM]
I50 [nmol/mg]
1
2
3
4
5
6
1
2
13
1.5
4
0.7
0.08
0.2
600[a]
5.3
1
3
4
24
26
20000[a]
100
20
into benzoyl chloride 25 by a reaction with thionyl chloride [a] Data taken from the literature.[9]
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T. Bender, M. Huss, H. Wieczorek, S. Grond, P. von Zezschwitz
FULL PAPER
tive to CDCl3 (δ = 77.00 ppm), [D6]benzene (δ = 128.0 ppm),
CD2Cl2 (δ = 53.37 ppm), or [D6]acetone (δ = 29.84 ppm). Chemical
shift assignments were made using COSY and HSQC. If not stated
otherwise, spectra were recorded at ambient temperature and with
normal detection (300 MHz) or inverse detection (600 MHz).
600 MHz COSY spectra were collected using a gCOSY pulse se-
quence. HSQC spectra were collected using a gHSQC pulse se-
quence. IR spectra were recorded with a Bruker IFS 66 (FT-IR)
spectrometer. HRMS (ESI) was performed with a Bruker APEX-
Q 7T IV spectrometer with preselected ion peak matching at R ϾϾ
10000 to be within Ϯ2 ppm of the exact masses. Elemental analyses
were performed by Mikroanalytisches Labor des Instituts für Or-
ganische und Biomolekulare Chemie der University of Göttingen,
Germany. Specific radioactivity was measured using a Beckmann
bF Betaszint 5000/300 scintillation counter. Medium-pressure li-
quid-chromatography was performed with a Knauer MaxiStar
pump using a RP18-Lobar LiChroprep column size B. Melting
points are uncorrected. Solvents for extraction and chromatog-
raphy were of technical grade and distilled before use. All moisture-
sensitive reactions were carried out under dry nitrogen or argon in
oven- and/or flame-dried glassware. Column chromatography was
performed with silica gel 60 (0.040–0.063 mm, Machery–Nagel).
Flash column chromatography was performed with silica gel 60
(0.025–0.040 mm, Machery–Nagel). TLC was performed with silica
gel 60 F254 precoated plates (Merck, 0.25 mm). THF was distilled
from sodium benzophenone ketyl, and dichloromethane, NEt3, and
pyridine were distilled from CaH2. 1-(4-Bromophenyl)-2,2,2-tri-
fluoroethanone (13)[15] and the THF solution of formaldehyde[19]
were prepared as described in the literature, and Ba14CO3 was used
as delivered by Moravek Biochemicals and Radiochemicals, 577
Mercury Lane, Brea, CA 92821, USA. Plecomacrolides 1 and 2
were obtained from fermentations of the Streptomyces sp. strains
Gö 22/15 and Gö3822 14F and purified by chromatography as de-
scribed previously.[8,24]
benzoylated 26 is almost equal to the natural concanamycin
A (1) and close to that of concanolide 3, thus representing
a substantial improvement compared to the previously
used, 125I-labeled compound 4.[8,9] In contrast, the labeled
bafilomycin 24 is approximately seventy times less active
than the natural product 2, yet it is still effective enough to
be used for the first PAL studies of this important inhibitor.
Recent reports of 2 increasing the therapeutic effectiveness
of anticancer drugs fuel the interest in elucidating the de-
tailed mode of action of the plecomacrolides.[25]
Conclusions
An effective and convenient synthesis for both the fre-
quently used photoaffinity label 23 and its 14C-labeled ana-
logue 11 has been elaborated, which features high chemical
yields in combination with low prices of all the starting ma-
terials, particularly the 14C source. All transformations can
be performed on a large scale and thus furnish multi-gram
amounts of products. Furthermore, the experimental sim-
plicity of all the procedures including the purifications is
noteworthy. Crucial for this achievement was the observa-
tion that while the diazirine moiety seems to be incompat-
ible with Grignard reagents and lithium organyls, the pro-
tected diaziridine 19 can easily be lithiated and then reacted
with electrophiles in the usual manner. This new reaction
mode of aziridines together with their smooth oxidation to
azirines should provide access to a variety of known and
new photoaffinity labels. The 14C-labeled benzoic acid 11 is
generally suitable for attachment to alcohols and amines,
yet it can also lead to a photoaffinity label for carboxylic
acids, since the carboxy moiety in 11 can easily be reduced
to a hydroxymethyl group.[26] We expect that the convenient
synthesis of 11 together with the long half-life of 14C will
make it the photoaffinity label of choice in several future
binding studies.
For a first application, the photoaffinity label 11 was se-
lectively attached to bafilomycin A1 (2) and the stable con-
canamycin analogue 21-deoxyconcanolide A (3). The pre-
liminary inhibition studies suggest the products 24 and 26
are ideal candidates to further enlighten the precise binding
site of these potent V-ATPase inhibitors. The PAL studies
are still in progress, but first results have already disclosed
that the specific activity of the 14C tracer is sufficient for
easy detection of the photolabeled components. The full re-
sults will be reported in due course.
1-(4-Bromophenyl)-2,2,2-trifluoroethanone Oxime (14): A solution
of
1-(4-bromophenyl)-2,2,2-trifluoroethanone
(13,
1.00 g,
3.95 mmol) and hydroxylamine hydrochloride (0.270 g, 3.89 mmol)
in absolute ethanol (1 mL) and pyridine (2 mL) was heated to 60 °C
for 2 h. After evaporation of the solvents, the residue was parti-
tioned between water (20 mL) and diethyl ether (200 mL). The or-
ganic layer was washed with water (10 mL) and brine (10 mL) and
then dried with Na2SO4. After evaporation of the solvent, the crude
oxime was purified by column chromatography (silica gel, hexane/
EtOAc, 10:1) to furnish 0.870 g (82% yield) of the title compound
as a colorless solid (1:1 mixture of isomers). Rf = 0.57 (hexane/
EtOAc, 3:1); m.p. 75–80 °C. 1H NMR (300 MHz, CDCl3): δ =
7.32–7.42 (m, 2 H, Ar-H), 7.52–7.64 (m, 2 H, Ar-H), 8.89 (s, OH,
isomer 1), 8.92 (s, OH, isomer 2) ppm. 13C NMR (75.5 MHz,
1
CDCl3): δ = 117.2 (q, JC,F = 175.3 Hz, CF3, isomer 1), 121.0 (q,
1JC,F = 166.1 Hz, CF3, isomer 2), 116.2, 118.5, 120.0, 122.2, 124.6,
125.3, 129.9, 130.3, 131.9, 145.9–148.2 (m, CNOH, both isomers)
ppm. IR (KBr): ν = 3287 (OH), 1885, 1620 (C=N), 1587, 1493,
˜
1466, 1000, 806 cm–1. MS (DCI, 70 eV): m/z (%) = 266/268 (50)
[M – H]–, 533/535/537 (100) [2M – H]–. C8H5BrF3NO (268.03):
calcd. C 35.85, H 1.88, N 5.23; found C 35.87, H 2.00, N 5.12.
Experimental Section
General: 1H NMR spectra were recorded at 300 MHz or 600 MHz
using a Varian Unity 300 or a Varian Inova 600 spectrometer.
Chemical shifts in CDCl3, [D6]benzene, CD2Cl2, or [D6]acetone are
reported as δ values relative to CHCl3 (δ = 7.26 ppm), [D5]benzene
(δ = 7.15 ppm), CHDCl2 (δ = 5.32 ppm), or [D5]acetone (δ =
2.05 ppm) as an internal reference unless stated otherwise. 13C
1-(4-Bromophenyl)-2,2,2-trifluoroethanone O-(p-Tolylsulfonyl)oxime
(15): To a solution of oxime 14 (58.1 g, 217 mmol), NEt3 (26.3 g,
260 mmol), and DMAP (2.65 g, 21.7 mmol) in CH2Cl2 (250 mL)
was added in portions p-toluenesulfonyl chloride (47.6 g,
250 mmol) with stirring at 0 °C. When the addition was complete,
NMR spectra were recorded at 75.5 MHz (Unity 300), 125.7 MHz the reaction mixture was stirred at room temp. for 3 h. After evapo-
(Inova 500), or 150.8 MHz (Inova 600). Chemical shifts in CDCl3,
ration of the solvent, diethyl ether (500 mL) was added, and the
[D6]benzene, CD2Cl2, or [D6]acetone are reported as δ values rela-
organic layer was washed with water (3ϫ50 mL) and brine
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Photoaffinity Label for Binding Studies of V-ATPase Inhibitors
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at room temp. The reaction progress was monitored by H NMR
analysis; for this purpose, 0.1 mL samples were taken from the re-
action mixture and concentrated in vacuo. After 3 h, the conversion
was complete, and all volatiles were removed in vacuo. The remain-
ing yellow oil was extracted with benzene (4ϫ10 mL), and the ex-
1
(10 mL). The organic phase was dried with Na2SO4 and concen-
trated to yield 91.0 g (99% yield) of a yellowish solid. The crude
product was of sufficient purity for further transformations; an an-
alytical sample was purified by recrystallization from CHCl3. Rf =
0.43 (hexane/EtOAc, 2:1); m.p. 133–135 °C. 1H NMR (300 MHz,
3
CDCl3): δ = 2.46 (s, 3 H, CH3), 7.26 (d, JH,H = 8.6 Hz, 2 H, Ar- tracts were concentrated in vacuo. For purification of the product
3
3
H), 7.37 (d, JH,H = 8.2 Hz, 2 H, Ar-H), 7.61 (d, JH,H = 8.6 Hz,
this extraction procedure was repeated twice with hexane
2 H, Ar-H), 7.86 (d, JH,H = 8.2 Hz, 2 H, Ar-H) ppm. 13C NMR (3ϫ10 mL each) to furnish 1.97 g (95% yield) of the title com-
(75.5 MHz, CDCl3): δ = 21.8 (CH3), 119.4 (q, JC,F = 277.4 Hz, pound as colorless crystals. H NMR (300 MHz, C6D6): δ = 0.01
3
1
1
3
CF3), 123.3 (C-4), 126.6 (C-1), 129.3 (C-Ar), 129.9 (4 C, C-Ar),
[s, 18 H, Si(CH3)3], 7.15 [d, JH,H = 8.1 Hz, 2 H, 3(5)-H], 7.28 [d,
2
131.0 (C-1Ј), 132.2 (C-Ar), 146.3 (C-4Ј), 153.0 (q, JC,F = 33.8 Hz, 3JH,H = 8.1 Hz, 2 H, 2(6)-H] ppm. 13C NMR (75.5 MHz, C6D6): δ
2
CNO) ppm. IR (KBr): ν = 1927, 1917, 1637 (C=N), 1589, 1489,
= –0.75 [Si(CH3)3], –0.76 [Si(CH3)3], 66.4 [q, JC,F = 35.2 Hz,
˜
1447, 1390, 1179, 901, 820 cm–1. MS (DCI, 70 eV): m/z (%) = 439/
441 (100) [M + NH4]+, 456/458 (20) [M + NH3 + NH4]+, 860/862/
864 (40) [2M + NH4]+. C15H11BrF3NO3S (422.22): calcd. C 42.67,
H 2.63, N 3.32; found C 42.63, H 2.73, N 3.25.
C(NTMS)2], 124.0 (C-4), 125.3 (q, JC,F = 282.4 Hz, CF3), 131.2
1
(C-Ar), 131.4 (C-Ar), 133.1 (C-1).
3-[(4-Hydroxymethyl)phenyl]-3-(trifluoromethyl)diaziridine
(22a):
The protected diaziridine 19 (211 mg, 513 µmol) was dissolved in
THF (15 mL) and cooled to –78 °C. After the addition of nBuLi
(257 µL, 514 µmol, 2.00  in hexane), the orange solution was
stirred at this temperature for 1 h. Freshly prepared formaldehyde
solution (2.93 mL, 2.05 mmol, 0.70  in THF) was then added
slowly, and the resulting mixture was stirred for 6 h and warmed
to –50 °C during this time. After the addition of saturated aqueous
NH4Cl (500 µL), the THF was removed in vacuo. The residue was
diluted with diethyl ether (250 mL), washed with water (15 mL),
saturated aqueous NaHCO3 (10 mL) and brine (10 mL), and dried
with Na2SO4. The crude product (302 mg) was purified by column
chromatography (silica gel, CH2Cl2/MeOH, 20:1) and medium-
pressure liquid-chromatography (CH3CN/H2O, 1:4) to yield
65.0 mg (58%) of the title compound as a colorless oil. Rf = 0.42
(CH2Cl2/MeOH, 20:1). 1H NMR (300 MHz, [D6]acetone): δ = 3.26
3-(4-Bromophenyl)-3-(trifluoromethyl)diaziridine (16): Tosylate 15
(90.9 g, 215 mmol) was placed in an autoclave and dissolved in
CH2Cl2 (200 mL). After cooling to –78 °C, gaseous ammonia was
slowly introduced over a period of 4 h. The resulting mixture was
stirred for 18 h and warmed to room temp. during this time. Excess
ammonia was carefully evaporated, and the residue was partitioned
between water (50 mL) and CH2Cl2 (500 mL). The organic layer
was separated, washed with brine (10 mL), and dried with Na2SO4.
After evaporation of the solvent, the resulting solid was recrys-
tallized repeatedly from a mixture of pentane and CH2Cl2 (1:5) to
give 49.4 g (86% yield) of the title compound as a colorless solid. Rf
= 0.33 (hexane/EtOAc, 2:1); m.p. 47–48 °C. 1H NMR (300 MHz,
3
3
CDCl3): δ = 2.18 (d, JH,H = 8.4 Hz, 1 H, NH), 2.79 (d, JH,H
=
3
8.4 Hz, 1 H, NH), 7.48 (d, JH,H = 8.6 Hz, 2 H, Ar-H), 7.55 (d,
3JH,H = 8.6 Hz, 2 H, Ar-H) ppm. 13C NMR (75.5 MHz, CDCl3):
3
3
(d, JH,H = 8.2 Hz, 1 H, NH), 3.60 (d, JH,H = 8.2 Hz, 1 H, NH),
2
1
δ = 57.6 [q, JC,F = 36.5 Hz, C(NH)2], 123.3 (q, JC,F = 278.2 Hz,
3
4.42 (br. s, 1 H, OH), 4.66 (s, 2 H, CH2OH), 7.43 [d, JH,H
=
CF3), 124.6 (C-4), 129.8 (C-Ar), 130.7 (C-1), 132.0 (C-Ar) ppm. IR
3
8.2 Hz, 2 H, 3(5)-H], 7.58 [d, JH,H = 8.2 Hz, 2 H, 2(6)-H] ppm.
(KBr): ν = 3197 (NH), 1921, 1596 (NH), 1495, 1407, 1398, 1191,
˜
2
13C NMR (75.5 MHz, [D6]acetone): δ = 58.4 [q, JC,F = 35.3 Hz,
1012, 829 cm–1. MS (ESI): m/z (%) = 267/269 (100) [M + H]+;
analysis calcd. for C8H7BrF3N2: 266.97392 [M + H]+ (correct mass
according to ESI-HRMS). C8H6BrF3N2 (267.05): calcd. C 35.98,
H 2.26, N 10.49; found C 35.98, H 2.19, N 10.42.
1
C(NH)2], 64.1 (CH2OH), 125.2 (q, JC,F = 277.4 Hz, CF3), 127.2
(C-Ar), 129.1 (C-Ar), 131.7 (C-1), 145.2 (C-4) ppm. IR (KBr): ν =
˜
3391, 3217, 1601, 1495, 1450, 1416, 1391, 1260, 1234, 1199, 1181,
1147, 1026, 945, 859, 736, 710, 621 cm–1. MS (DCI, 70 eV): m/z
(%) = 219 (100) [M + H]+, 236 (80) [M + NH4]+. C9H9F3N2O
(218.18).
3-(4-Bromophenyl)-3-(trifluoromethyl)-3H-diazirine (17): A solution
of diaziridine 16 (6.01 g, 22.5 mmol), NEt3 (3.14 mL, 22.5 mmol),
and ethanol (22 mL) was cooled to 0 °C. Under vigorous stirring,
a solution of freshly prepared tert-butyl hypochlorite (7.34 g,
67.6 mmol) in tert-butanol (9.5 mL) was slowly added. The mixture
was stirred at 0 °C for 2 h and then quenched with 10% aqueous
Na2S2O5 (150 mL). The aqueous phase was extracted with diethyl
ether (3ϫ50 mL), and the combined organic phases were dried
with Na2SO4. The solvent was evaporated leaving 5.14 g (86%
yield) of a colorless oil, whose purity was Ͼ95%. 1H NMR
3-[4-(2-Hydroxyprop-2-yl)phenyl]-3-(trifluoromethyl)diaziridine
(22b): The protected diaziridine 19 (286 mg, 695 µmol) was dis-
solved in THF (15 mL) and cooled to –78 °C. nBuLi (297 µL,
689 µmol, 2.32  in hexane) was then added, and the resulting mix-
ture was stirred for 40 min at –78 °C. Dry acetone (62 µL, 49 mg,
0.84 mmol) was added, and the mixture was warmed to –45 °C
within 4.5 h and quenched by the addition of H2O (0.5 mL). The
solvents were removed in vacuo, and the residue was diluted with
diethyl ether (150 mL), washed with H2O (10 mL) and brine
(2ϫ10 mL), and dried with Na2SO4. The crude product (165 mg)
was purified by column chromatography (silica gel, hexane/EtOAc,
2:1) to yield 114 mg (67% yield) of the title compound as a color-
less solid. Rf = 0.26 (hexane/EtOAc, 2:1); m.p. 78 °C. 1H NMR
(300 MHz, [D6]acetone): δ = 1.58 (s, 6 H, CH3), 1.66 (br. s, OH),
3
(300 MHz, CDCl3): δ = 7.05 (d, JH,H = 8.4 Hz, 2 H, Ar-H), 7.51
3
(d, JH,H = 8.4 Hz, 2 H, Ar-H) ppm. 13C NMR (75.5 MHz,
2
1
CDCl3): δ = 28.2 (q, JC,F = 43.5 Hz, CN2), 121.9 (q, JC,F
=
274.7 Hz, CF3), 124.2 (C-4), 128.0 (C-Ar), 132.0 (C-Ar) ppm. The
signals of C-1 were not observed. MS (EI, 70 eV): m/z (%) = 71
(82) [HCF3]+, 107 (20) [C7H9N]+, 137 (40), 155/157 (100)
[C6H4Br]+, 171 (20), 236/238 (45) [M – N2]+, 500/502/504 (20)
[C16H8Br2F6N2]+. C8H4BrF3N2 (265.03).
3
3
2.22 (d, JH,H = 8.2 Hz, 1 H, NH), 2.80 (d, JH,H = 8.2 Hz, 1 H,
NH), 7.44–7.68 (m, 4 H, Ar-H) ppm. 13C NMR (75.5 MHz, [D6]-
3-(4-Bromophenyl)-3-(trifluoromethyl)-1,2-bis(trimethylsilyl)diazir- acetone): δ = 31.7 (CH3), 57.8 [q, JC,F = 35.8 Hz, C(NH)2], 72.4
2
1
idine (19): Diaziridine 16 (1.35 g, 5.06 mmol) was added to a solu-
tion of NEt3 (4.2 mL, 30 mmol) in CH2Cl2 (25 mL). After stirring
at room temp. for 1 h, the solution was cooled to –78 °C, and
TMSOTf (2.0 mL, 2.5 g, 11 mmol) was added slowly. After 1 h at
–78 °C, the cooling bath was removed, and the solution was stirred
(COH), 125.2 (q, JC,F = 277.4 Hz, CF3), 127.2 (C-Ar), 129.1 (C-
Ar), 131.7 (C-1), 145.2 (C-4) ppm. IR (KBr): ν = 3371, 3237, 3214,
˜
2983, 2932, 1615, 1515, 1463, 1403, 1236, 1155, 1118, 1020, 952,
832, 693 cm–1. MS (DCI, 70 eV): m/z (%) = 229 (38) [M – OH]+,
246 (100) [M – H2O + NH4]+, 263 (40) [M – H2O + NH3
+
Eur. J. Org. Chem. 2007, 3870–3878
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3875

T. Bender, M. Huss, H. Wieczorek, S. Grond, P. von Zezschwitz
FULL PAPER
NH4]+. C11H13F3N2O (246.23): calcd. C 53.66, H 5.32, N 11.38;
found C 53.76, H 5.07, N 11.12.
was stirred for 30 min at –78 °C. The plastic cap of the glass vial
in which Ba14CO3 was delivered was pierced, and an additional
rubber septum was introduced between the cap and vial. The car-
boxylation was then initiated by the addition of two drops of con-
centrated H2SO4 to the solid Ba14CO3 (50 mCi, 0.91 mmol), and
the gas was introduced into the THF solution of the aryllithium
compound using Teflon tubing. To remove traces of acid in the
14CO2 stream, a 10 cm piece of the Teflon tubing was cooled in
a –78 °C acetone/dry ice bath. When the gas evolution had ceased,
additional concentrated H2SO4 was added dropwise (2 mL). After
all the Ba14CO3 had been consumed, the Teflon tubing was dis-
connected with a clamp, and the solution was stirred for 5 h at
–78 °C. The dark-blue solution was quenched with water (0.5 mL)
and warmed to room temp. The whole apparatus was flushed with
nitrogen, and the gas stream was passed through a wash bottle
containing water and a tube containing soda-lime to remove unre-
acted 14CO2. The reaction mixture was diluted with diethyl ether
(250 mL), and the organic layer was extracted with NaOH solution
(1 , 3ϫ20 mL). The combined aqueous phases were acidified with
hydrochloric acid (6 ) to approximately pH 1. A colorless solid
precipitated, which was extracted with diethyl ether (4ϫ50 mL).
The combined organic phases were washed with water (3ϫ20 mL)
and brine (1ϫ10 mL). Evaporation of the solvent yielded 164 mg
(78% calculated on Ba14CO3) of the title compound as a colorless
solid, which was essentially pure and directly used in the subse-
quent oxidation.
3-[4-(Hydroxyphenylmethyl)phenyl]-3-(trifluoromethyl)diaziridine
(22c): The protected diaziridine 19 (271 mg, 659 µmol) was dis-
solved in THF (15 mL) and cooled to –78 °C. nBuLi (281 µL,
652 µmol, 2.32  in hexane) was added, and the resulting mixture
was stirred for 40 min at –78 °C. A solution of freshly distilled
benzaldehyde (77 mg, 730 µmol) in THF (2 mL) was then added,
and the mixture was warmed to –40 °C within 4.5 h and quenched
by the addition of H2O (0.5 mL). The solvents were removed in
vacuo, and the residue was diluted with diethyl ether (150 mL),
washed with H2O (10 mL) and brine (2ϫ10 mL), and dried with
Na2SO4. The crude product (205 mg) was purified by column
chromatography (silica gel, hexane/EtOAc, 5:1) to yield 130 mg
(67%) of the title compound as a colorless solid. Rf = 0.16 (hexane/
1
EtOAc, 5:1); m.p. 82–84 °C. H NMR (300 MHz, [D6]acetone): δ
3
3
= 3.29 (d, JH,H = 8.1 Hz, 1 H, NH), 3.59 (d, JH,H = 8.1 Hz, 1 H,
3
3
NH), 5.03 (d, JH,H = 2.7 Hz, 1 H, OH), 5.88 (d, JH,H = 2.7 Hz,
1 H, CHOH), 7.18–7.36 (m, 3 H, Ar-H), 7.40–7.60 (m, 6 H, Ar-H)
2
ppm. 13C NMR (125.7 Hz, [D6]acetone): δ = 58.4 [q, JC,F
=
35.2 Hz, C(NH)2], 75.7 (CHOH), 125.1 (q, 1JC,F = 277.7 Hz, CF3),
127.1 (C-Ar), 127.2 (C-Ar), 127.8 (C-Ar), 129.0 (C-Ar), 129.2 (C-
Ar), 131.8 (C-Ar), 146.0 (C-Ar), 148.2 (C-Ar) ppm. IR (KBr): ν =
˜
3434, 3213, 2984, 1615, 1515, 1403, 1236, 1155, 1118, 951, 831, 693
cm–1. MS (DCI, 70 eV): m/z (%) = 229 (100), 246 (90), 264 (10).
C15H13F3N2O (294.28): calcd. C 61.22, H 4.45, N 9.52; found C
60.99, H 4.26, N 9.40.
4-[3-(Trifluoromethyl)-3H-diazirin-3-yl]benzoic Acid (23) and 4-[3-
(Trifluoromethyl)-3H-diazirin-3-yl]-[1-14C]benzoic Acid (11). Gene-
ral Procedure: The diaziridine (21 or 22d, respectively, 0.59 mmol)
was dissolved in MeOH (15 mL), and NEt3 (6 equiv.) was added.
A solution of iodine (30 mg/mL in MeOH) was then added drop-
wise at room temp. to the stirred solution until the orange-red color
of iodine persisted for more than 1 min (ca. 1.2 equiv. of I2). After
the addition of iodine was complete, the solution was stirred for
20–30 min in the dark at room temp. The methanol was removed
under reduced pressure, and the residue was diluted with diethyl
ether. The product was extracted from the organic phase with aque-
ous NaOH (1 ). The combined aqueous phases were acidified
with hydrochloric acid (6 ) to approximately pH 1, and the prod-
uct was extracted with diethyl ether. The combined organic phases
were washed with water and brine. Evaporation of the solvent
yielded the desired products 11 and 23 as colorless solids in yields
ranging from 90% to 99%. The yield in the oxidation of the 14C-
labeled diaziridine 21 was 83%, and the specific activity of the di-
azirine 11 was determined to be 44.11 mCi/mmol using a liquid
scintillation counter. 13C NMR (75.5 MHz, CDCl3): δ = 28.4 (q,
4-[(3-Trifluoromethyl)diaziridin-3-yl]benzoic Acid (22d): The pro-
tected diaziridine 19 (320 mg, 778 µmol) was dissolved in THF
(30 mL) and cooled to –78 °C. After the addition of nBuLi (409 µL,
818 µmol, 2.00  in hexane), the orange-red solution was stirred at
–78 °C for 30 min. CO2 was generated from approximately 3 g of
dry ice, passed through a drying tube containing molecular sieves
(3 Å), and introduced into the solution. After 30 min the addition
was complete, and the solution was stirred for 16 h at –78 °C. The
reaction was quenched with H2O (0.5 mL), and the solvents were
removed in vacuo. Diethyl ether (150 mL) was added, and the or-
ganic phase was extracted with saturated aqueous NaHCO3
(3ϫ15 mL). The combined aqueous phases were acidified with
50% hydrochloric acid to approximately pH 1. A colorless solid
precipitated, which was extracted with diethyl ether (4ϫ50 mL).
The combined organic phases were washed with water (3ϫ20 mL)
and brine (2ϫ10 mL). Evaporation of the solvent yielded 161 mg
(89%) of the title compound as a colorless solid. The product was
essentially pure and was directly used in further transformations.
An analytical sample was purified by recrystallization from ace-
1
2JC,F = 39.6 Hz, CN2), 121.8 (q, JC,F = 274.7 Hz, CF3), 126.4 (C-
1
tone; m.p. 181 °C (decomposition). H NMR (300 MHz, [D6]ace-
Ar), 130.2 (C-1), 130.5 (C-Ar), 134.8 (C-4), 171.3 (COOH) ppm.
C9H5F3N2O2 (230.15): calcd. C 46.97, H 2.19, N 12.17; found C
46.73, H 2.17, N 12.28. All other analytical data and the melting
point were consistent with published data.[18b,18c]
3
3
tone): δ = 3.50 (d, JH,H = 7.6 Hz, 1 H, NH), 3.78 (d, JH,H
=
3
7.6 Hz, 1 H, NH), 7.77 [d, JH,H = 8.3 Hz, 2 H, 2(6)-H], 8.09 [d,
3JH,H = 8.3 Hz, 2 H, 3(5)-H] ppm. 13C NMR (75.5 MHz, [D6]ace-
2
1
tone): δ = 58.4 [q, JC,F = 35.7 Hz, C(NH)2], 125.0 (q, JC,F
=
21-O-[4-(3-Trifluoromethyl-3H-diazirin-3-yl)benzoyl]bafilomycin A1
(24): Bafilomycin A1 (2, 16.4 mg, 26.3 µmol), DMAP (7.8 mg,
64 µmol), diazirine 23 (12.6 mg, 54.7 µmol), and ethyl-[3-(dimeth-
ylamino)propyl]carbodiimide hydrochloride (EDCI, 11.1 mg,
57.9 µmol) were dissolved in dry CH2Cl2 (1.5 mL). The reaction
mixture was stirred for 16 h in the dark at room temp. and moni-
tored by TLC (hexane/EtOAc, 2:1). When the reaction had ceased,
the solution was diluted with diethyl ether (150 mL) and washed
with saturated aqueous NaHCO3 (5 mL), H2O (5 mL), and brine
(5 mL). After drying the organic layer with NaSO4, the solvent
was removed in vacuo. The crude product was purified by column
chromatography (silica gel, hexane/acetone, 6:1) to yield 9.1 mg
277.7 Hz, CF3), 129.6 (C-Ar), 130.5 (C-Ar), 132.8 (C-1), 137.7 (C-
4), 167.0 (COOH) ppm. IR (KBr): ν = 3407, 3192, 3015, 2639,
˜
2518, 1950, 1711, 1617, 1581, 1424, 1386, 1263, 1166, 1110, 1021,
959, 900, 859, 714 cm–1. MS (ESI): m/z (%) = 231 (100) [M – H]–,
485 (45) [2M – 2H + Na]–. C9H7F3N2O2 (232.16): calcd. C 46.56,
H 3.04, N 12.07; found C 46.59, H 2.89, N 12.00.
4-[(3-Trifluoromethyl)diaziridin-3-yl]-[1-14C]benzoic Acid (21):[23]
A
25 mL Schlenk flask with an attached 100 mL glass syringe was
charged with the protected diaziridine 19 (1.23 g, 2.99 mmol) and
THF (20 mL) and cooled to –78 °C. After the addition of nBuLi
(1.23 mL, 2.85 mmol, 2.32  in hexane), the orange-red solution
3876
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 3870–3878

Photoaffinity Label for Binding Studies of V-ATPase Inhibitors
(41%) of the desired product as colorless oil and 3.6 mg (22%) of
FULL PAPER
hexane/acetone, 10:1) to yield 5.0 mg (32%) of the title compound
1
the starting material 2. Rf = 0.25 (hexane/acetone, 6:1). H NMR
as a colorless oil and 4.6 mg (38%) of the starting material 3. Rf =
3
(600 MHz, CD2Cl2): δ = 0.80 (d, JH,H = 7.0 Hz, 3 H, 24-CH3), 0.25 (hexane/acetone, 6:1). 1H NMR (600 MHz, CD2Cl2): δ = 0.80
3
3
0.83 (d, JH,H = 7.9 Hz, 3 H, 16-CH3), 0.83 (d, JH,H = 6.7 Hz, 3
(d, 3JH,H = 7.6 Hz, 3 H, 18-CH3), 0.81 (d, 3JH,H = 6.7 Hz, 3 H, 20-
3
3
3
H, 8-CH3), 0.91 (d, JH,H = 6.1 Hz, 3 H, 24-CH3), 0.92 (d, JH,H
CH3), 0.85 (d, JH,H = 7.6 Hz, 3 H, 24-CH3), 0.85 (mc, 3 H, 8-
3
3
= 6.1 Hz, 3 H, 22-CH3), 0.99 (d, JH,H = 7.2 Hz, 3 H, 18-CH3),
CH2CH3), 1.00–1.10 (m, 3 H, 6-CH3), 1.05 (d, JH,H = 7.0 Hz, 3
1.03 (d, 3JH,H = 7.0 Hz, 3 H, 6-CH3), 1.11 (dd, JH,H = 13.2, 7.4 Hz,
1 H, 20a-H), 1.76–1.81 (m, 1 H, 18-H), 1.82–1.94 [m, 4 H,
H, 10-CH3), 1.13–1.22 (m, 2 H, 8-CH2CH3), 1.22–1.30 (m, 1 H,
24-H), 1.48 (mc, 1 H, 8-H), 1.60 (mc, 1 H, 20-H), 1.64 (dd, JH,H
=
8(9a,22,24)-H], 1.92 (s, 3 H, 10-CH3), 1.98 (d, 3JH,H = 1.1 Hz, 3 H, 6.5, 1.5 Hz, 3 H, 28-H3), 1.64–1.68 (m, 2 H, 22-H2), 1.84 (br. s, 3
3
4-CH3), 2.06–2.16 [m, 2 H, 9b(16)-H], 2.23 (dd, JH,H = 11.8,
4.8 Hz, 1 H, 20b-H), 2.53 (ddq, JH,H = 9.1, 7.1, 2.0 Hz, 1 H, 6-H),
H, 12-CH3), 1.92–1.99 (m, 2 H, 11-H2), 1.96 (s, 3 H, 4-CH3), 2.02
(mc, 1 H, 18-H), 2.23 (mc, 1 H, 10-H), 2.72 (mc, 1 H, 6-H), 3.13–
3
3.22 (s, 3 H, 14-OCH3), 3.27 (mc, 1 H, 7-H), 3.61 (s, 3 H, 2-OCH3), 3.22 (m, 1 H, 9-H), 3.22 (s, 3 H, 16-OCH3), 3.47 (dd, JH,H = 9.7,
3
3.64 (dd, JH,H = 10.3, 2.2 Hz, 1 H, 23-H), 3.88 (t, JH,H = 9.0 Hz,
1 H, 14-H), 4.12 (ddd, JH,H = 10.7, 4.0, 1.9 Hz, 1 H, 17-H), 4.65
(dd, JH,H = 4.0, 0.7 Hz, 1 H, 17-OH), 4.88 (dd, 3JH,H = 8.6, 1.2 Hz,
7.8 Hz, 1 H, 25-H), 3.55 (s, 3 H, 2-OCH3), 3.56–3.61 [m, 2 H, 7(19)-
H], 3.76 (mc, 1 H, 21-H), 3.82 (t, JH,H = 8.9 Hz, 1 H, 16-H), 4.88
(dt, JH,H = 10.8, 4.8 Hz, 1 H, 23-H), 5.14 (mc, 1 H, 17-H), 5.21
(mc, 1 H, 15-H), 5.36 (ddq, JH,H = 15.0, 7.6, 1.6 Hz, 1 H, 26-H),
3
3
3
1 H, 15-H), 5.07–5.17 [m, 2 H, 13(21)-H], 5.46 (d, JH,H = 2.1 Hz,
3
3
3
1 H, 19-OH), 5.77 (d, JH,H = 9.1 Hz, 1 H, 5-H), 5.81 (d, JH,H
10.8 Hz, 1 H, 11-H), 6.53 (dd, JH,H = 15.0, 10.8 Hz, 1 H, 12-H),
=
5.63 (dq, JH,H = 15.0, 6.4 Hz, 1 H, 27-H), 5.67 (mc, 1 H, 5-H),
3
5.79 (d, JH,H = 10.5 Hz, 1 H, 13-H), 6.37 (br. s, 1 H, 3-H), 6.54
4
3
3
3
6.68 (d, JH,H = 0.6 Hz, 1 H, 3-H), 7.26 (d, JH,H = 8.2 Hz, 2 H,
(dd, JH,H = 15.0, 10.5 Hz, 1 H, 14-H), 7.26 (d, JH,H = 8.3 Hz, 2
Ar-H), 8.05 (d, JH,H = 8.2 Hz, 2 H, Ar-H) ppm. 13C NMR
H, Ar-H), 8.05 (d, JH,H = 8.3 Hz, 2 H, Ar-H) ppm. 13C NMR
3
3
(150.8 MHz, CD2Cl2): δ = 7.1 (18-CH3), 9.8 (16-CH3), 12.4 (22- (150.8 MHz, CD2Cl2): δ = 8.0 (20-CH3), 9.4 (18-CH3), 11.5 (8-
CH3), 14.0 (4-CH3), 14.3 (24-CH3), 17.3 (6-CH3), 20.2 (10-CH3), CH2CH3), 13.3 (24-CH3), 14.1 (4-CH3), 16.2 (12-CH3), 16.7 (6-
2
21.2 (24-CH3), 21.7 (8-CH3), 28.3 (C-24), 37.0 (C-6), 37.5 (C-16), CH3), 17.7 (C-28), 21.4 (10-CH3), 22.8 (8-CH2CH3), 28.4 (q, JC,F
40.5 (C-22), 41.5 (C-8), 41.5 (C-9), 42.2 (C-18), 53.4 (C-20), 55.7 = 41.6 Hz, CN2), 34.6 (C-6), 35.3 (C-10), 36.4 (C-22), 37.3 (C-18),
(14-OCH3), 60.2 (2-OCH3), 71.0 (C-17), 75.6 (C-21), 76.0 (C-23), 39.1 (C-20), 40.7 (C-11), 43.4 (C-24), 44.5 (C-8), 55.7 (16-OCH3),
77.0 (C-15), 81.2 (C-7), 82.6 (C-14), 99.2 (C-19), 125.4 (C-11), 126.6 59.1 (2-OCH3), 69.1 (C-19), 74.2 (C-7), 75.7 (C-17), 76.4 (C-21),
1
(C-Ar), 127.1 (C-13), 130.1 (C-Ar), 132.3 (C-Ar), 133.2 (C-4), 133.5 77.1 (C-23), 79.4 (C-9), 82.1 (C-16), 82.6 (C-25), 121.8 (q, JC,F
=
(C-12), 133.6 (C-Ar), 134.0 (C-3), 141.5 (C-2), 143.5 (C-5), 143.6 274.8 Hz, CF3), 122.9 (C-13), 126.1 (C-Ar), 127.1 (C-15), 129.1 (C-
(C-10), 165.2 (CO2), 167.6 (C-1) ppm, signals of CF3 and CN2 were 27), 129.8 (C-Ar), 130.1 (C-Ar), 130.2 (C-26), 131.4 (C-3), 132.0
not observed due to low intensity. IR (KBr): ν = 3422, 2930, 1718,
(C-4), 133.1 (C-Ar), 133.4 (C-14), 139.6 (C-12), 141.97 (C-2),
˜
1275, 1194, 1159, 1101 cm–1. MS (ESI): m/z (%) = 853 (34) [M +
142.02 (C-5), 164.9 (CO ), 165.7 (C-1) ppm. IR (KBr): ν = 3446,
˜
2
N H 4
]
+ , 8 5 8 ( 1 0 0 ) [ M + N a ] + ; a n a l y s i s c a l c d . f o r
2967, 2930, 1718, 1700, 1458, 1382, 1343, 1276, 1196, 1159, 1106
C44H61F3N2NaO10: 857.41705 [M + Na]+ (correct mass according cm–1. MS (ESI): m/z (%) = 825 (20) [M–2 MeOH]+, 858 (100) [M –
to ESI-HRMS).
MeOH + H]+ , 912 (30) [M + Na]+ ; analysis calcd. for
C48H67F3N2NaO10: 911.46455 [M + Na]+ (correct mass according
to ESI-HRMS).
21-O-[4-(3-Trifluoromethyl-3H-diazirin-3-yl)-[1-14C]benzoyl]bafilo-
mycin A1 (24): The reaction was performed as described above em-
ploying bafilomycin A1 (2, 15.7 mg, 25.2 µmol) and label 11
(11.6 mg, 50.4 µmol). The product yield was 8.6 mg (41%), and
4.9 mg (31%) of bafilomycin A1 (2) were re-isolated. The 1H NMR
spectroscopic data were consistent with the data of the unlabeled
compound 24. The specific activity was determined to be 33.7 mCi/
mmol using a liquid scintillation counter.
23-O-[4-(3-Trifluoromethyl-3H-diazirin-3-yl)-[1-14C]benzoyl]-21-de-
oxyconcanolide A (26): The reaction was performed as described
above employing 21-deoxyconcanolide A (3, 12.5 mg, 18.5 µmol)
and label 25 (7.17 mg, 28.9 µmol). The product yield was 4.6 mg
(36%), and 3.9 mg (31%) of 21-deoxyconcanolide A (3) were re-
isolated. The 1H NMR spectroscopic data were consistent with the
data of the unlabeled compound 26. The specific activity was deter-
mined to be 40.0 mCi/mmol using a liquid scintillation counter.
23-O-[4-(3-Trifluoromethyl-3H-diazirin-3-yl)benzoyl]-21-deoxycon-
canolide A (26): Diazirine 23 (36.0 mg, 156 µmol) was dissolved in
thionyl chloride (0.50 mL), and the resulting solution was stirred
for 15 h at room temp. in the dark. Then, the thionyl chloride was
carefully removed under reduced pressure (40 mbar) to yield
38.5 mg (99 %) of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzoyl
chloride (25) which was dissolved in CH2Cl2 (3 mL). 21-Deoxycon-
canolide A (3, 12.0 mg, 17.7 µmol) and DMAP (11.5 mg,
94.1 µmol) were dissolved in CH2Cl2 (1.5 mL), and NEt3 (50 µL)
was added. Then, a solution of benzoyl chloride 25 (5.3 mg,
21 µmol) in CH2Cl2 (0.41 mL) was added dropwise and the re-
sulting mixture was stirred at room temp. in the dark. Due to in-
complete conversion after 1.5 h as detected by TLC, more benzoyl
chloride 25 (2.7 mg, 11 µmol) dissolved in CH2Cl2 (0.21 mL) was
added. After 4.5 h, another portion of DMAP (11.0 mg, 90.0 µmol)
was added, and the reaction was stopped after 7 h by the addition
of water (1 mL). The mixture was diluted with diethyl ether
(150 mL), and the organic layer was washed with aqueous
NaHCO3 (1 , 5 mL), H2O (5 mL), and brine (5 mL). Drying with
Na2SO4 and evaporation of the solvent yielded a crude product
(51 mg) which was purified by column chromatography (silica gel,
Supporting Information (see also the footnote on the first page of
this article): A figure showing the experimental setup for the car-
1
boxylation using Ba14CO3 and H NMR spectra of all new com-
pounds.
Acknowledgments
This work was financially supported by the Bundesministerium für
Bildung und Forschung (BMBF) (GenoMik+: MetabolitGeno-
Mik) and the Deutsche Forschungsgemeinschaft (DFG) (SFB 431,
Project P3). The authors are indebted to Dr. Michael Gründel, Dr.
Friedrich Güthoff, and Michael Schlote of the isotope laboratory
at the faculty of chemistry of the Georg August University of
Göttingen for helpful discussions and technical assistance. S. G.
and P. v. Z. are grateful to Prof. Axel Zeeck and Prof. Armin de
Meijere, respectively, for their continuing support. M. H. and H. W.
would like to thank Martin Dransmann for his excellent technical
assistance.
Eur. J. Org. Chem. 2007, 3870–3878
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3877

T. Bender, M. Huss, H. Wieczorek, S. Grond, P. von Zezschwitz
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3878
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