Synthesis of penicillenol C1 and of a bis-azide analogue for photoaffinity labeling

Article abstract of DOI:10.1021/jo3026737

Two diasteroisomers of the Penicillium metabolite penicillenol C ₁ were synthesized for the first time by 3-acylation of an l-threonine-derived tetramic acid with enantiopure 2-methyloct-(6E)-enoic acids. The 5S,6R,9S isomer has NMR spectra and optical rotation identical with those of the natural compound. A bis-azide-tagged penicillenol analogue was also synthesized for photoaffinity labeling of target proteins. The photolysis of the bis-azide in the presence of methanol as a protein-mimicking nucleophile led to reaction only of the aryl azide, while leaving the benzyl azide available for pull-downs or the attachment of fluorescent tracers. As a proof of concept, the distribution of this bis-azide-tagged tetramic acid in living cells was visualized via a Staudinger ligation between the azide tag and a phosphane fluorophore.

Full text of DOI:10.1021/jo3026737

Article
pubs.acs.org/joc
Synthesis of Penicillenol C1 and of a Bis-Azide Analogue for
Photoaffinity Labeling
Karl Kempf,† Aruna Raja,‡ Florenz Sasse,‡ and Rainer Schobert*,†
†Organic Chemistry Laboratory, University Bayreuth, 95440 Bayreuth, Germany
‡Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
S
* Supporting Information
ABSTRACT: Two diasteroisomers of the Penicillium metab-
olite penicillenol C1 were synthesized for the first time by 3-
acylation of an L-threonine-derived tetramic acid with
enantiopure 2-methyloct-(6E)-enoic acids. The 5S,6R,9S
isomer has NMR spectra and optical rotation identical with
those of the natural compound. A bis-azide-tagged penicillenol
analogue was also synthesized for photoaffinity labeling of
target proteins. The photolysis of the bis-azide in the presence
of methanol as a protein-mimicking nucleophile led to reaction
only of the aryl azide, while leaving the benzyl azide available
for pull-downs or the attachment of fluorescent tracers. As a
proof of concept, the distribution of this bis-azide-tagged
tetramic acid in living cells was visualized via a Staudinger ligation between the azide tag and a phosphane fluorophore.
nucleophilic residues in the protein, while visualization of the
resulting protein−tetramate conjugate becomes possible via a
Staudinger reaction of the second azide with a fluorescent
phosphane. We provide evidence for the feasibility of this
approach.
INTRODUCTION
■
The penicillenols 1 represent a small family of pyrrolidine-2,4-
diones (aka tetramic acids)1 that were isolated from Penicillium
sp. GQ-7, an endophytic fungus associated with Aegiceras
corniculatum.2 They share the structural motif of an N-
methylated tetramic acid derived from threonine and bearing
an α-methyl branched C8-fatty acyl residue at C-3 (Figure 1).
The penicillenols A1 (1a) and A2 (1b) were recently
synthesized by Yoda et al. from L-threonine and D-
allothreonine, respectively.3 By comparison with the reported
NMR and optical rotation data of the natural products, their
configurations were inferred to be (5S,6R,9S)-1a and
(5R,6R,9S)-1b. The penicillenols B1 (1c) and B2 (1d) also
carry an α-methyloctanoyl residue of hitherto unspecified
configuration yet have lost the stereogenic centers C-5 and C-6
due to elimination of water. For the penicillenols C1 (1e) and
C2 (1f) the discovering group assumed configurations at C-5 to
be S for 1e and R for 1f on the grounds of CD and NMR
spectra. So far, the penicillenols have been only cursorily tested
for biological activity, which is somewhat surprising, given the
high incidence of biological effects reported for the closely
related melophlins, metabolites of bacteria dwelling on the
marine sponge Melophlus sarasinorum.4 Likewise, there are no
data available for penicillenols concerning their uptake by and
distribution in cells.
RESULTS AND DISCUSSION
■
Syntheses of Penicillenol C1 ((5S,6R)-1e). We intended
to synthesize 1e by 3-acylation of the tetramic acid 12 (Scheme
1). Preliminary studies5 had indicated that its benzyl ether 11
should be accessible by a Wittig-type cyclization reaction of the
O-protected N-methylated L-threoninate 9 with the ylide
Ph3PCCO (10).6 This strategy differs from Yoda’s synthesis
of penicillenols A, which was based on the cyclization of N,O-
protected threonines with Meldrum’s acid and an N-
methylation postponed until the very last step.3 We now
optimized the synthesis of the immediate cyclization precursor,
benzyl (2S,3R)-2-methylamino-3-((triisopropylsilyl)oxy)-
butanoate (9), so that only two intermediates, namely 4 and
6, required purification. L-Threonine was N-protected with N-
(9-fluorenylmethoxycarbonyloxy)succinimide (=FmocONSu)
to give carboxylic acid 3. This was converted to its Cs salt
and treated with benzyl bromide to give the ester 4 in 91% yield
after column chromatography. When it is prepared on a large
scale, ester 4 can be purified conveniently by recrystallization
from ethyl acetate. The β-hydroxy group of 4 was silylated with
triisopropylsilyl triflate/NEt3, affording the benzyl N-Fmoc-O-
TIPS-threoninate 5 in 84% yield. Cleavage of the Fmoc group
Herein we describe a total synthesis of penicillenol C1 (1e)
that differs from Yoda’s approach to the penicillenols A by an
early N-methylation of threonine and by the method of ring
closure. We also report the synthesis of a bis-azide-tagged
penicillenol analogue that should allow the identification of
target proteins. Photolysis of one azide enables attachment to
Received: December 10, 2012
Published: February 25, 2013
© 2013 American Chemical Society
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Figure 1. Structures of penicillenols 1 and melophlins 2.
a
comprised of amine addition to the CC bond of 10 followed
by an intramolecular Wittig olefination of the so-formed amide
ylide. Hydrogenolysis of benzyl tetramate 11 eventually
liberated tetramic acid 12.
Scheme 1. Synthesis of Tetramic Acid 12
The (6E)-2-methyl-octenoic acid required for the 3-acylation
of 12 was prepared by a stereoselective α-methylation of (6E)-
octenoic acid 137 (Scheme 2). Given the ambiguity of the
a
Scheme 2. Synthesis of (6E)-2-Methyloctenoic Acids 16
a
a
Reagents and conditions: (i) (a) Me3CCOCl, NEt3, THF, −10 °C,
Reagents and conditions: (i) FmocONSu, dioxane, room temper-
(b) Evans auxiliary, NEt3, LiCl, −10 °C (1 h) → 0 °C (2 h), 87%; (ii)
NaHMDS, THF, −78 °C, then MeI, 3 h, 89% (95% de); (iii) LiOH,
H2O2, THF, H2O, 0 °C, 87%.
ature, 15 h; (ii) (a) Cs2CO3, MeOH; (b) BnBr, DMF, room
temperature, 91% (based on L-threonine); (iii) TIPSOTf, NEt3,
CH2Cl2, room temperature, 2 h, 84%; (iv) piperidine, CH2Cl2, 0 °C,
15 min, 81%; (v) (o-NO2)C6H4SO2Cl, NEt3, CH2Cl2, room
temperature, 99%; (vi) MeI, K2CO3, DMF, room temperature, 94%
(crude); (vii) PhSH, K2CO3, DMF, room temperature, 70% (based on
6).
configuration at C-9 in the natural 1e, we synthesized both
enantiomers by applying the appropriate Evans (R)- and (S)-4-
benzyloxazolidin-2-one auxiliaries. Their attachment afforded
the enantiomeric imides 14a,b, which were diastereoselectively
(95% de) methylated to the corresponding α-methylamides
15a,b. These were hydrolyzed to the (6E)-2-methyloctenoic
acids 16 in 67% overall yield based upon 13.
Tetramic acid 12 was then acylated with the carboxylic acids
16 under modified Yoshii8−Yoda3 conditions (Scheme 3).
Treatment of the acids 16 with ethyl chloroformate/NEt3 to
give the mixed anhydride followed by addition of tetramic acid
12 led to the tetramates 17. The 4-O → C-3 acyl shift in the
presence of CaCl2 as recommended by Yoda proceeded readily,
with piperidine gave the α-amino ester 6 in 81% yield upon
chromatographic purification. For the mono-methylation of the
amino group of 6, it was first nosylated to the sulfonamide 7
and then methylated with MeI/K2CO3 to afford the tertiary
sulfonamide 8. This was N-deprotected with PhSH/K2CO3 to
furnish the benzyl N-methyl-O-TIPS-threoninate 9 in 70% yield
(over three steps) after chromatographic purification. Refluxing
of a mixture of amino ester 9 and ylide 10 in toluene gave
benzyl tetramate 11 in 55% yield via a domino sequence
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downs that melophlin A (2: R1−3 = H, n = 12, m = 0) targets
dynamins, GTPases crucial for endocytosis, cytokinesis, and
signaling in eukaryotic cells.9 While studies with melophlins are
problematic due to their high cytotoxicities, penicillenol C1 is
far less cytotoxic and so lends itself ideally to protein target
identification in living cells. In cytotoxicity (MTT) assays with
cells of murine L929 fibrosarcoma, human KB-3-1 cervix
carcinoma, and nonmalignant PTK2 rat kangaroo kidney cells
both diastereomers of 1e were antiproliferative only at very
high concentrations, with IC50 (72 h) > 50 μM (cf. the
Supporting Information).
Scheme 3. Synthesis of Penicillenols C1 (9R)-1e and (9S)-1e
For a study of the protein targets of the penicillenols, we now
prepared a derivative 23 that bears a known10 bis-azide
photoaffinity label, allowing a two-step target identification
(Scheme 4). Incubation of 23 with cell lysates and subsequent
irradiation at ca. 250 nm should cleave the aryl azide to a
reactive intermediate that covalently links the tetramic acid to
its target protein. The remaining benzyl azide may then be
attached via click or Staudinger reactions to fluorescence labels
or an affinity compound such as biotin. Scheme 4 depicts the
synthesis of the bis-azide-tagged tetramic acid 23 by 3-acylation
of tetramic acid 12 with the carboxylic acid 21 under Yoda
conditions, followed by O-desilylation of the intermediate 22
with HF in pyridine. The acid 21 was prepared by amidation of
the known 3-azido-5-(azidomethyl)benzoic acid 1911 with
methyl 11-aminoundecanoate12 in the presence of propylphos-
phonic acid cyclic anhydride (T3P) and subsequent saponifi-
cation of the ester 20. Scheme 4 also shows a photochemical
trapping reaction of the bis-azido carboxylic acid 21 with
methanol as a nucleophilic protein surrogate to prove the
validity of step 1 of the concept. Acid 21 was used in lieu of the
tetramic acid 23, since it is available in larger amounts, is easier
to purify, and precludes the possibility of unwanted side
reactions between the tetramate and an excess of the
nucleophile. This reaction was complete after 28 h of
irradiation with a 150 W mercury lamp. For real qualitative
protein labeling, far shorter irradation times will suffice.10 The
primary product imidate, resulting from loss of N2 and trapping
of the intermediate seven-membered cyclic azaallene with
methanol, could not be isolated but was readily hydrolyzed
upon workup to leave the stable β-azidomethyllactam 24, which
was purified by HPLC and fully characterized. The bottom line
is that the bis-azide label can be photolyzed to an intermediate
that will link covalently to nucleophilic groups of proteins while
retaining the benzyl azide for attachment of secondary
fluorescence or affinity labels.
Finally, we tested whether the bis-azide-tagged tetramic acid
23 can be used to address particular subcellar components in
living cells and if these can then be visualized by a Staudinger
ligation between the azide(s) of 23 and a commercially
available fluorescent dye−phosphane conjugate (DyLight488-
phosphine, Thermo Scientific). PTK2 rat kangaroo kidney cells,
which are flat and thus easy to observe, were incubated with 23
for 12 h and then fixed, permeabilized, and incubated with
DyLight488-phosphine at 37 °C for 3 h. The images we
obtained with a fluorescence microscope show an accumulation
of green fluorescent dots, representing the tetramic acid 23, in
the vicinity of the cell nuclei (Figure 2). For a reliable
identification of the subcellular target component, a colocaliza-
tion with specific antibodies would be required. However, the
important outcome of this test is that we can use bis-azide
labels to trace the intracellular distribution of tetramic acids. In
combination with the photolabeling of individual proteins we
affording the O-protected target compounds 18. The cleavage
of the silyl ether of 18 required exceptionally drastic conditions:
namely, exposure to 4 equiv of 70% HF in pyridine for more
than 12 h. Gratifyingly, deprotection went to completion
without any signs of decomposition. The pure diasteroisomeric
penicillenols C1 (9R)-1e and (9S)-1e were obtained in almost
80% yield. This synthetic approach should be flexible enough to
allow the preparation of structurally modified penicillenol
analogues for an in-depth study of the biological properties of
this family of 3-acyltetramic acids.
For the assignment of the configuration at C-9 of the natural
penicillenol C1 we compared the known chemical shifts in its
13C NMR spectrum2 with those of our synthetic diastereomers.
Table 1 shows a perfect match of indicative carbon signals for
a
Table 1. Chemical Shifts (δ, ppm) of Indicative Carbon
Atoms of Natural Penicillenol C1 and Deviations of the
Corresponding Shifts of Synthetic Penicillenols (9S)-1e and
(9R)-1e
no. of C
b
atom
type
δ(natural
1e)
δ(natural 1e) −
δ(9S-1e)
δ(natural 1e) −
δ(9R-1e)
atom
3
Cq
101.1
66.7
192.7
36.3
33.0
27.1
32.4
130.8
17.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
−0.2
0.1
6
CH
Cq
8
0.1
9
CH
CH2
CH2
CH2
CH
CH3
0.2
10
11
12
13
16
−0.3
0.1
0.1
0.1
0.2
a
13C NMR spectra were recorded in CDCl3 at 150 MHz (natural 1e)2
and 126 MHz (synthetic (9S)- and (9R)-1e). For a comparison of all
b
signals cf. the Supporting Information. Cf. Figure 1.
the isomer (9S)-1e. A comparison of the optical rotations
further corroborates this assignment. Natural 1e and synthetic
(9S)-1e both show an optical rotation of [α]24 = −47° (c =
D
0.125, MeOH), while isomer (9R)-1e has [α]24 = −12° (c =
D
0.125, MeOH).
Synthesis and Preliminary Tests of a Bis-Azide-
Tagged Penicillenol Photolabel. Little is known, yet,
about the cellular targets of 3-acyltetramic acids. The
Waldmann group showed by means of protein affinity pull-
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a
Scheme 4. Synthesis of Bis-Azide-Tagged 3-Acyltetramic Acid 23 and Photoreaction of the Bis-Azide 21
a
Reagents and conditions: (i) T3P, NEt3, ClH3N(CH2)10CO2Me, CH2Cl2, 0 °C, 1 h, 96%; (ii) 2 NaOH, MeOH, room temperature, 18 h, 85%.
analogue. Its phenyl azido group was selectively cleaved by light
irradiation, and the resulting intermediate was trapped with
methanol, representing the nucleophilic moieties within every
protein. The remaining benzyl azide may then be used for the
attachment of fluorescent tracers or bioaffinity groups. As an
example, we tracked the distribution of the bis-azide-tagged
tetramic acid in PTK2 kidney cells by a Staudinger ligation with
a fluorophore−phosphane conjugate under a fluorescence
microscope. We are now preparing structural variants of bis-
azide-tagged tetramic acids, and we are also running the first
actual photolabeling experiments with whole cell lysates to find
optimum irradiation conditions.
EXPERIMENTAL SECTION
■
General Remarks. IR spectra were recorded with an FT-IR
spectrophotometer equipped with an ATR unit. Chemical shifts of
NMR signals are given in parts per million (δ) downfield from
tetramethylsilane for 1H and 13C. Mass spectra were obtained under EI
Figure 2. Distribution of bis-azide-tagged tetramic acid 23 (50 μg/
mL) in PTK2 cells, visualized with a Zeiss Axioplan fluorescence
microscope (63× magnification) after Staudinger ligation with
fluorescent DyLight488-phosphine.
(70 eV) conditions. High-resolution mass spectra were obtained with a
UPLC/Q-TOF MS system in ESI mode. For chromatography silica
gel 60 (230−400 mesh) was used. HPLC was performed on Prontosil
RP 200-5-C18, 5 μm, 250 × 4 mm (analytic) and 250 × 20 mm
(preparative) columns.
should now be in a position to visualize and identify the
molecular targets of tetramic acids. It should be noted that the
bis-azide 21 did not accumulate in specific compartments of
PTK2 cells but rather gave a faint greenish background
indicative of a negligible uptake and an unspecific distribution.
(5S,6R)-4-(Benzyloxy)-1-methyl-5-(1-((triisopropylsilyl)oxy)-
ethyl)-1H-pyrrol-2(5H)-one (11). A solution of benzyl (2S,3R)-2-
methylamino-3-((triisopropylsilyl)oxy)butanoate (9;5 560 mg, 1.48
mmol) and Ph3PCCO (10; 530 mg, 1.65 mmol) in toluene (10 mL)
was refluxed for 18 h and then concentrated under vacuum. The
remainder was purified by column chromatography (cyclohexane/
ethyl acetate 1/1, Rf = 0.38) to leave 11 (330 mg, 55%) as a colorless
CONCLUSIONS
oil. [α]20 = 46° (c = 1, CH2Cl2). IR (ATR): νmax 2942, 2865, 1689,
■
D
The first synthesis of natural penicillenol C1 (5R,6S,9S)-1e and
its 9R diastereomer was based on the 3-acylation of a tetramic
acid obtained by Wittig cyclization of a protected benzyl L-N-
methylthreoninate with the phosphorus ylide Ph3PCCO. The
synthesis of the 3-acyl side chain used Evans auxiliaries for the
introduction of the α-methyl branch. We also assigned the
configuration at the carbon atom C-9 of the natural penicillenol
C1 to be S by comparison of NMR and optical rotation data.
Since the cytotoxicity of penicillenol C1 is low, it may be used
as a probe to identify the proteins 3-acyltetramic acids interact
with in living cells. We have demonstrated the feasibility of this
strategy by test reactions with a bis-azide-tagged penicillenol C
1620, 1499, 1462, 1422, 1377, 1351, 1308, 1225, 1200, 1141, 1071,
1
989, 881, 846, 802, 755, 737, 716, 676 cm−1. H NMR (CDCl3, 300
MHz): δ 0.93−1.02 (m, 21 H), 1.09 (d, J = 6.3 Hz, 3 H), 2.87 (s, 3
H), 3.86 (d, J = 3.0 Hz, 1 H), 4.32 (qd, J = 6.3, 3.0 Hz, 1 H), 4.84 (d, J
= 11.8 Hz, 1 H) 4.89 (d, J = 11.8 Hz, 1 H), 5.09 (s, 1 H), 7.25−7.33
(m, 5 H). 13C NMR (CDCl3, 75 MHz): δ 12.1, 17.8, 18.3, 27.5, 66.4,
67.4, 72.5, 95.6, 127.6, 128.2, 128.4, 134.8, 171.9, 173.3. MS (EI) m/z
403 [M+], 359, 268, 226, 203, 201, 157, 115, 91. HRMS: calcd for
C23H38NO3Si+ 404.2615, found 404.2626 [M + H]+.
(5S,6R)-5-(1-((triisopropylsilyl)oxy)ethyl)-1-methylpyrroli-
dine-2,4-dione (12). A mixture of tetramate 11 (430 mg, 1.1 mmol),
dry MeOH (50 mL), and 5% Pd on charcoal (170 mg) was flushed
and then pressurized (1 bar, balloon) with H2 gas and stirred at room
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temperature for 8 h. After filtration over Celite and evaporation of the
volatiles the residue was taken up in ethyl acetate. The solution was
filtered over a short pad of silica and concentrated under reduced
pressure to leave the product as a colorless oil. Yield: 320 mg (93%),
(4S,2′S,6′E)-4-Benzyl-3-(2′-methyloctenoyl)oxazolidin-2-one
(15b). This compound was obtained from 14b as a colorless oil
analogously to 15a. Yield: 89%. [α]28 = 79° (c = 1.0, CH2Cl2).
D
(2R,6E)-2-Methyloctenoic Acid (16a). A solution of amide 15a
(0.70 g, 2.2 mmol) in THF/H2O (88 mL, 10/1) at 0 °C was treated
with LiOH (0.35 g, 8.4 mmol) and H2O2 (30% in H2O, 1.2 mL, 11.3
mmol) and stirred for 2 h at room temperature. Me2S (0.9 mL, 11.3
mmol) was added, and the resulting mixture was stirred for another 10
min. It was acidified with 1 M NaHSO4 solution and extracted three
times with ethyl acetate. The organic extracts were washed with brine,
dried, and concentrated. Column chromatography (cyclohexane/
diethyl ether 7/1) left 16a (0.30 g, 87%) as a colorless oil with Rf =
0.42 (cyclohexane/ethyl acetate/acetic acid 100/33/1). [α]28D = −17°
(c = 1.0, CH2Cl2). IR (ATR): νmax 2933, 2858, 1702, 1464, 1416,
Rf = 0.38 (cyclohexane/ethyl acetate 1/1). [α]28 = −20° (c = 1,
D
CH2Cl2). IR (ATR): νmax 2943, 2892, 2867, 1771, 1694, 1463, 1374,
1
1333, 1244, 1141, 1089, 1067, 998, 882, 811, 750, 717, 679 cm−1. H
NMR (CDCl3, 300 MHz): δ 1.00−1.05 (m, 21 H), 1.40 (d, J = 6.7 Hz,
3 H), 2.93 (t, J = 0.8 Hz, 1 H), 2.95 (dd, J = 1.1, 0.8 Hz, 1 H), 3.11
(dd, J = 1.1, 0.8 Hz, 3 H), 3.68 (ddquin, J = 2.0, 1.1, 0.8 Hz, 1 H), 4.50
(qd, J = 6.7, 2.0 Hz, 1 H). 13C NMR (CDCl3, 75 MHz): δ 12.6, 17.8,
21.7, 30.1, 41.2, 68.8, 74.6, 169.8, 205.6. MS (EI): m/z 270 (100) [M−
C3H7]+, 228 (11), 226 (9), 213 (7), 201 (45), 184 (15), 171 (10), 157
(34), 129 (6), 124 (11), 115 (26), 87 (13), 75 (13), 73 (14), 59 (19),
43 (9). HRMS: calcd for C16H32NO3Si+ 314.2151, found 314.2141 [M
+ H]+.
1
1378, 1289, 1237, 1076, 963 cm−1. H NMR (CDCl3, 300 MHz): δ
1.18 (d, J = 7.0 Hz, 3 H), 1.33−1.48 (m, 3 H), 1.64 (d, J = 4.7 Hz, 3
H), 1.66−1.75 (m, 1 H), 1.93−2.04 (m, 2 H), 2.45 (qt, J = 7.0, 6.3 Hz,
1 H), 5.29−5.53 (m, 2 H), 11.22 (br. s, 1 H). 13C NMR (CDCl3, 75
MHz): δ 16.8, 17.9, 27.0, 32.4, 33.0, 39.3, 125.2, 130.8, 183.3. MS
(EI): m/z 156 (15) [M + H]+, 138 (21), 110 (9), 101 (6), 95 (5), 87
(22), 83 (75), 74 (100), 69 (27), 68 (32), 55 (88). Anal. Calcd for
C9H16O2: C, 69.19; H, 10.32. Found: C, 68.89; H, 10.35.
(2S,6E)-2-Methyloctenoic Acid (16b). This compound was
obtained from 15b as a colorless oil analogously to 16a. Yield: 87%.
[α]28D = 17° (c = 1.0, CH2Cl2). HRMS: calcd for C9H16O2+ 155.1078,
found 155.1068.
(R)-4-Benzyl-3-((6E)-octenoyl)oxazolidin-2-one (14a). A sol-
ution of (6E)-octenoic acid (13;7 0.48 g, 3.4 mmol) in dry THF (20
mL) at −10 °C was treated with NEt3 (1.18 mL, 8.5 mmol) followed
by Me3CCOCl (0.42 mL, 3.4 mmol). After the mixture was stirred at
−10 °C for 1 h, LiCl (0.22 g, 5.1 mmol) and (R)-4-benzyloxazolidin-2-
one (0.60 g, 3.4 mmol) were added at this temperature. Stirring was
continued for 1 h at −10 °C and then for a further 2 h at 0 °C.
Saturated aqueous NH4Cl solution (20 mL) was added, and the
mixture was extracted twice with ethyl acetate. The combined organic
layers were washed with brine, dried over Na2SO4, and concentrated.
The crude product was purified by column chromatography
(cyclohexane/diethyl ether 7/1, Rf = 0.24). Yield: 0.89 g (87%) as a
(5S)-3-((2′R,6′E)-1′-Hydroxy-2′-methylocten-1′-ylidene)-5-
((R)-1″-((triisopropylsilyl)oxy)ethyl)-1-methylpyrrolidine-2,4-
dione (18a). A solution of acid 16a (0.35 g, 2.2 mmol) and NEt3
(0.70 mL, 5.1 mmol) in THF (20 mL) was cooled to 0 °C and treated
dropwise with ethyl chloroformate (272 μL, 2.8 mmol). After 1 h
tetramic acid 12 (0.63 g, 2.0 mmol) was added and the resulting
mixture was stirred at room temperature for 12 h and then
concentrated under vacuum. The residue was taken up in CH2Cl2
and washed with dilute NaHSO4 solution. The water phase was re-
extracted with CH2Cl2, and the combined organic layers were dried
and concentrated under reduced pressure. The crude 4-O-acyltetramic
acid 17a was dissolved in CH2Cl2 (20 mL), cooled to 0 °C, and
treated with DMAP (0.49 g, 4.0 mmol) and dry CaCl2 (0.44 g, 4.0
mmol). The mixture was stirred at room temperature for 8 h and then
washed with 0.05 M Na2-EDTA solution. The water phase was re-
extracted with CH2Cl2, and the combined organic layers were dried
and concentrated. The crude product was dissolved in MeOH (17
mL), treated with water (3 mL) to give a milky emulsion, and
centrifuged to leave 18a as a red oil. Purification by HPLC (rinsing
with 85% MeOH, then elution with 100% MeOH) afforded 468 mg
(55%) of 18a as a red oil consisting of a 5.6/1 mixture of the two
tautomers A/B. [α]28D = −63° (c = 1, CH2Cl2). IR (ATR): νmax 2939,
2866, 1710, 1651, 1614, 1461, 1376, 1329, 1264, 1213, 1139, 1096,
1069, 997, 965, 921, 881, 809, 778, 755, 704 cm−1. 1H NMR (CDCl3,
300 MHz): δ 0.96−1.04 (m, 21 H), 1.16 (d, J =6.9 Hz, 3 H), 1.27−
1.39 (m, 3 H), 1.36B/1.43A (d, J = 6.6 Hz, 3 H), 1.59−1.72 (m, 1 H),
1.62 (dt, J = 4.7, 1.2 Hz, 3 H), 1.90−2.02 (m, 2 H), 3.05B/3.10A (s, 3
H), 3.47A (d, J = 1.9 Hz, 1 H), 3.55A (ddq, J = 7.4, 7.1, 6.9 Hz, 1 H),
3.70B (d, J = 2.5 Hz, 1 H), 3.77B (tq, J = 7.7, 6.9 Hz, 1 H), 4.56 (qd, J =
6.6, 1.9 Hz, 1 H), 5.30−5.49 (m, 2 H). 13C NMR (CDCl3, 75 MHz): δ
12.5B/12.6A, 17.4, 17.9B/18.0A, 22.8, 27.2/27.3, 28.9, 32.56, 32.62,
35.6B/36.1A, 67.6B/67.9A, 69.4B/72.4A, 100.8, 125.0, 130.9, 174.4,
190.8, 193.0. MS (EI): m/z 451 (<1) [M]+, 408 (100) [M − C3H7]+,
394 (10), 364 (17), 251 (17), 201 (33), 157 (32), 149 (15), 115 (9).
HRMS: calcd for C25H46NO4 Si+ 452.3196, found 452.3192 [M + H]+.
(5S)-3-((2′S,6′E)-1′-Hydroxy-2′-methylocten-1′-ylidene)-5-
((R)-1″-((triisopropylsilyl)oxy)ethyl)-1-methylpyrrolidine-2,4-
dione (18b). A 160 mg amount (55%) was obtained as a red oil,
consisting of a 7/1 mixture of the two tautomers A/B, analogously to
18a, from 12 (207 mg, 0.66 mmol) and 16b (113 mg, 0.73 mmol).
[α]28D = −104° (c = 1.0, CH2Cl2). IR (ATR): νmax 2938, 2866, 1712,
1647, 1615, 1488, 1462, 1375, 1328, 1262, 1214, 1140, 1096, 1069,
967, 924, 882, 797, 753, 702, 679 cm−1. 1H NMR (CDCl3, 300 MHz):
δ 0.97−1.02 (m, 21 H), 1.13A/1.17B (d, J = 6.7 Hz, 3 H), 1.27−1.34
colorless oil. [α]28 = −65° (c = 1.0, CH2Cl2). IR (ATR): νmax 3028,
D
2919, 2856, 1777, 1697, 1604, 1497, 1481, 1384, 1350, 1288, 1196,
1
1099, 1050, 1013, 965, 921, 843, 761 cm−1. H NMR (CDCl3, 300
MHz): δ 1.43−1.50 (m, 2 H), 1.58−1.83 (m, 5 H), 1.96−2.13 (m, 2
H), 2.77 (dd, J = 13.4, 9.3 Hz, 1 H), 2.82−3.05 (m, 2 H), 3.30 (dd, J =
13.4, 3.3 Hz, 1 H), 4.08−4.25 (m, 2 H), 4.67 (ddt, J = 9.3, 6.7, 3.3 Hz,
1 H), 5.34−5.55 (m, 2 H), 7.14−7.40 (m, 5 H). 13C NMR (CDCl3, 75
MHz): δ 17.9, 23.7, 29.0, 32.3, 35.4, 37.9, 55.1, 66.1, 125.1, 127.3,
128.9, 129.4, 130.9, 135.3, 153.4, 173.3. MS (EI): m/z 301 (2) [M+],
246 (5), 190 (8), 178 (27), 159 (8), 142 (23), 134 (43), 125 (26), 117
(35), 97 (43), 92 (22), 91 (33), 86 (20), 81 (13), 69 (20), 75 (74).
+
HRMS: calcd for C18H24NO3 302.1751, found 302.1756 [M + H]+.
(S)-4-Benzyl-3-((6E)-octenoyl)oxazolidin-2-one (14b). This
compound was obtained from (S)-4-benzyloxazolidin-2-one as a
colorless oil analogously to 14a. Yield: 87%. [α]28 = 65° (c = 1.0,
D
CH2Cl2).
(4R,2′R,6′E)-4-Benzyl-3-(2′-methyloctenoyl)oxazolidin-2-
one (15a). A solution of amide 14a (0.45 g, 1.5 mmol) in THF (15
mL) at −78 °C was treated dropwise with NaHMDS (1.5 M solution
in THF, 1.5 mL, 2.3 mmol) and stirred for 30 min at this temperature.
MeI (0.3 mL, 4.6 mmol) was added, and the reaction mixture was
stirred for a further 3 h at −78 °C. Saturated aqueous NH4Cl solution
(20 mL) was added, and the mixture was warmed to room
temperature and then extracted twice with ethyl acetate. The organic
extracts were washed with brine, dried, and concentrated. Column
chromatography (cyclohexane/diethyl ether 10/1) left 0.44 g (89%) of
15a as a colorless oil with Rf = 0.55 (cyclohexane/ethyl acetate 3/1).
[α]28D = −79° (c = 1.0, CH2Cl2). 95% de (GC). IR (ATR): νmax 2927,
2855, 1776, 1695, 1604, 1497, 1454, 1383, 1348, 1289, 1238, 1208,
1
1195, 1100, 1075, 1015, 966, 921, 838, 761 cm−1. H NMR (CDCl3,
300 MHz): δ 1.26 (d, J = 6.9 Hz, 3 H), 1.34−1.46 (m, 3 H), 1.67 (dt, J
= 4.9, 1.0 Hz, 3 H), 1.73−1.86 (m, 1 H), 1.95−2.10 (m, 2 H), 2.81
(dd, J = 13.3, 9.6 Hz, 1 H), 3.30 (dd, J = 13.3, 3.3 Hz, 1 H), 3.75 (qt, J
= 6.9, 6.5 Hz, 1 H), 4.15−4.27 (m, 2 H), 4.71 (ddt, J = 9.6, 6.7, 3.3 Hz,
1 H), 5.34−5.54 (m, 2 H), 7.18−7.44 (m, 5 H). 13C NMR (CDCl3, 75
MHz): δ 17.4, 17.9, 27.1, 32.5, 32.9, 37.6, 37.9, 55.3, 66.0, 125.1, 127.3,
128.9, 129.4, 130.9, 135.3, 153.0, 177.3. MS (EI): m/z 315 (10) [M+],
233 (100), 218 (10), 190 (6), 178 (72), 159 (5), 139 (81), 117 (55),
+
111 (29), 91 (53), 82 (42). HRMS: calcd for C19H26NO3 316.1907,
found 316.1920 [M + H]+.
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The Journal of Organic Chemistry
Article
(m, 2 H), 1.36B/1.44A (d, J = 6.6B/6.9A Hz, 3 H), 1.39−1.49 (m, 1 H),
1.62 (dt, J = 4.7, 1.1 Hz, 3 H), 1.64−1.74 (m, 1 H), 1.88−2.01 (m, 2
H), 3.05B/3.11A (s, 3 H), 3.48A (d, J = 1.6 Hz, 1 H), 3.59A (dqd, J =
8.5, 6.7, 6.3 Hz, 1 H), 3.70B (d, J = 2.5 Hz, 1 H), 3.76B (q, J = 6.7 Hz, 1
H), 4.57 (qd, J = 6.9, 1.6 Hz, 1 H), 5.27−5.48 (m, 2 H). 13C NMR
(CDCl3, 75 MHz): δ 12.5B/12.6A, 16.7, 17.94B/17.97A, 23.0, 27.1,
29.0, 32.5, 33.5, 35.7, 67.5B/68.1A, 68.4B/72.5A, 101.2, 125.1, 130.9,
174.4, 190.5, 193.2. MS (EI): m/z 451 (<1) [M]+, 408 (100) [M −
C3H7]+, 394 (4), 364 (16), 251 (10), 201 (29), 157 (28), 115 (9).
HRMS: calcd for C25H45KNO4Si+ 490.2749, found 490.2762 [M +
K]+.
11-(3-Azido-5-(azidomethyl)benzamido)undecanoic Acid
(21). A mixture of ester 20 (0.75 mg, 1.8 mmol), MeOH (40 mL),
water (10 mL), and NaOH (0.14 g, 3.6 mmol) was stirred at room
temperature for 18 h and then concentrated under reduced pressure.
The remainder was extracted three times with CH2Cl2, and the
extracts were dried, concentrated, and purified by column chromatog-
raphy (cyclohexane/ethyl acetate/HOAc 1/1/0.01, Rf = 0.32). Yield:
0.61 g (85%) as a white solid with mp 78 °C. IR (ATR): νmax 3292,
2918, 2851, 2107, 2081, 1694, 1631, 1592, 1534, 1468, 1439, 1410,
1330, 1317, 1299, 1278, 1267, 1241, 1215, 1190, 1118, 1078, 1019,
1
920, 879, 795, 766 cm−1. H NMR (CDCl3, 300 MHz): δ 1.23−1.40
(m, 12 H), 1.53−1.66 (m, 4 H), 2.32 (t, J = 7.4 Hz, 2 H), 3.41 (td, J =
7.1, 5.9 Hz, 2 H), 4.37 (s, 2 H), 6.43 (t, J = 5.9 Hz, 1 H), 7.06 (t, J =
1.8 Hz, 1 H), 7.38 (t, J = 1.8 Hz, 1 H), 7.41−7.47 (m, 1 H). 13C NMR
(CDCl3, 75 MHz): δ 24.6, 26.9, 28.9, 29.05, 29.13, 29.2, 29.3, 29.4,
34.0, 40.3, 53.9, 117.5, 120.9, 122.6, 137.1, 138.0, 141.3, 166.2, 179.2.
MS (EI): m/z 401 (24) [M+], 375 (9), 345 (5), 342 (32), 331 (27),
313 (45), 303 (35), 289 (10), 275 (9), 261 (8), 231 (16), 201 (22),
190 (19), 184 (20), 175 (28), 164 (50), 162 (48), 147 (40), 145 (48),
136 (35), 122 (40), 106 (20), 90 (33), 83 (26), 69 (48), 63 (42), 56
(9R)-Penicillenol C1 ((5S,6R,9R)-1e). A solution of 18a (136 mg,
0.30 mmol) in THF (500 μL) in a plastic vial was treated with HF
(70% in pyridine, 33 μL, 1.2 mmol, 4 equiv) and stirred overnight. The
reaction was quenched by stirring with Et3SiH (191 μL, 1.2 mmol) for
30 min (Caution! the vessel must not be tightly sealed!) and
subsequent pouring into MeOH/water (77/23, 8 mL). Centrifugation
and purification by HPLC (MeOH/H2O 77/23 + 0.1% HCO2H, flow
rate 10 mL/min, tR of major isomer 29 min) left 69 mg (77%) of
(5S,6R,9R)-1e as a red oil. [α]28 = −12° (c = 0.125, MeOH). IR
D
+
(100). HRMS: calcd for C19H27N7NaO3 424.2068, found 424.2067
(ATR): νmax 3448 br, 2972, 2934, 2157, 1698, 1602, 1453, 1408, 1377,
[M + Na]+. Anal. Calcd for C19H27N7O3: C, 56.84; H, 6.78; N, 24.42.
Found: C, 56.96; H, 6.80; N, 24.13.
1
1339, 1259, 1212, 1121, 1087, 965, 851, 810, 797, 766, 710 cm−1. H
NMR (CDCl3, 300 MHz): δ 1.11 (d, J = 6.3 Hz, 3 H), 1.16 (d, J = 6.9
Hz, 3 H), 1.27−1.38 (m, 2 H), 1.40−1.53 (m, 1 H), 1.61 (d, J = 5.2
Hz, 3 H), 1.64−1.73 (m, 1 H), 1.90−1.99 (m, 2 H), 2.97 (s, 3 H), 3.57
(ddq, J = 8.0, 6.9, 6.6 Hz, 1 H), 3.78 (d, J = 4.4 Hz, 1 H), 4.17 (qd, J =
6.9, 4.4 Hz, 1 H) 5.26−5.47 (m, 2 H). 13C NMR (CDCl3, 126 MHz):
δ 17.0, 17.5, 17.9, 27.0, 27.1, 32.3, 33.3, 36.1, 66.6, 68.5, 101.3, 125.2,
130.7, 174.1, 192.6, 194.9. MS (EI): m/z 295 (58) [M]+, 277 (10),
251 (100), 233 (11), 213 (53), 204 (17), 182 (13), 169 (40), 151 (9),
(5S)-3-(1′-Hydroxy-11′-(3-azido-5-(azidomethyl)-
benzamido)undecan-1′-ylidene)-5-((R)-1″-((triisopropylsilyl)-
oxy)ethyl)-1-methylpyrrolidine-2,4-dione (22). Analogously to
18, 3-acyltetramic acid 22 (244 mg, 54%) was obtained as a red oil
from bis-azido acid 21 (0.26 g, 0.65 mmol) and tetramic acid 12 (0.24
g, 0.77 mmol). [α]25D = −59° (c = 0.8, CH2Cl2). IR (ATR): νmax 3330,
2926, 2864, 2105, 1712, 1607, 1540, 1461, 1374, 1324, 1281, 1214,
1
1139, 1096, 1068, 975, 921, 882, 780, 754 cm−1. H NMR (CDCl3,
+
140 (78), 139 (55), 113 (34), 112 (11). HRMS: calcd for C16H26NO4
300 MHz): δ 0.95−1.03 (m, 21 H), 1.23−1.38 (m, 12 H), 1.42 (d, J =
6.8 Hz, 3 H), 1.53−1.69 (m, 4 H), 2.77 (m, 2 H), 3.09 (s, 3 H), 3.43
(q, J = 6.3 Hz, 2 H), 3.47 (d, J = 1.6 Hz, 1 H), 4.39 (s, 2 H), 4.54 (qd,
J = 6.8, 1.6 Hz, 1 H), 6.20−6.38 (m, 1 H), 7.06 (s, 1 H) 7.38 (s, 1 H)
7.45 (s, 1 H). 13C NMR (CDCl3, 75 MHz): δ 12.5, 17.9, 22.8, 25.7,
26.9, 29.0, 29.16, 29.18, 29.21, 29.25, 29.3, 29.5, 32.5, 40.3, 53.9, 68.1,
72.4, 101.6, 117.5, 120.9, 122.6, 137.3, 138.0, 141.3, 166.1, 174.1,
186.7, 193.3. MS (EI): m/z 653 (100) [M − C3H7]+, 582 (4), 553 (6),
496 (72), 479 (25), 453 (29), 435 (12), 392 (3), 322 (10), 270 (22),
201 (54), 157 (62), 147 (12), 131 (56), 115 (29), 103 (43), 87 (15),
75 (40), 73 (22), 61 (22), 55 (12), 43 (60). HRMS: calcd for
296.1862, found 296.1856 [M + H]+.
(9S)-Penicillenol C1 ((5S,6R,9S)-1e). A 38 mg amount (77%) was
obtained analogously to (5S,6R,9R)-1e from 18b (51 mg, 0.17 mmol).
[α]28 = −47.0° (c = 0.125, MeOH). IR (ATR): νmax 3454 br, 2971,
D
2932, 2858, 1702, 1602, 1453, 1407, 1376, 1338, 1258, 1212, 1121,
1087, 965, 851, 811, 796, 768, 710 cm−1. 1H NMR (CDCl3, 300
MHz): δ 1.13 (d, J = 6.6 Hz, 3 H), 1.17 (d, J = 6.9 Hz, 3 H), 1.27−
1.40 (m, 2 H), 1.41−1.52 (m, 1 H), 1.61 (d, J = 4.7 Hz, 3 H), 1.63−
1.74 (m, 1 H), 1.90−2.00 (m, 2 H), 2.98 (s, 3 H), 3.55 (ddq, J = 8.0,
6.9, 6.6 Hz, 3 H), 3.78 (d, J = 4.5 Hz, 1 H), 4.18 (qd, J = 6.6, 4.5 Hz, 2
H), 5.24−5.50 (m, 2 H). 13C NMR (CDCl3, 126 MHz): δ 17.2, 17.7,
17.9, 27.1, 27.2, 32.4, 33.0, 36.3, 66.7, 68.6, 101.1, 125.2, 130.8, 174.1,
192.7, 194.8. MS (EI): m/z 295 (20) [M]+, 277 (8), 251 (63), 233
(9), 213 (37), 204 (19), 182 (16), 169 (36), 151 (13), 140 (100), 139
+
C35H56N8NaO5 719.4041, found 719.4023 [M + Na]+.
(5S)-3-(1′-Hydroxy-11′-(3-azido-5-(azidomethyl)-
benzamido)undecan-1′-ylidene)-5-((R)-1″-hydroxyethyl)-1-
methylpyrrolidine-2,4-dione (23). Analogously to 1e, 3-acyltetr-
amic acid 23 (65 mg, 75%) was obtained from 22 (111 mg, 0.16
+
(62), 113 (32), 112 (14). HRMS: calcd for C16H26NO4 296.1862,
found 296.1863 [M + H]+.
mmol) as a red oil. [α]25 = −31° (c = 1.0, MeOH). IR (ATR): νmax
D
Methyl 11-(3-Azido-5-(azidomethyl)benzamido)-
undecanoate (20). An ice-cold solution of 3-azido-5-(azidomethyl)-
benzoic acid (19;11 1.07 g, 4.9 mmol), methyl 11-aminoundecanoate
hydrochloride12 (1.85 g, 7.36 mmol), and NEt3 (2.73 mL, 19.6 mmol)
in CH2Cl2 (60 mL) was treated dropwise with propanephosphonic
acid anhydride (T3P; 50% in THF, 3.38 mL, 5.6 mmol) and stirred for
1 h. Water was added, the phases were separated, the aqueous layer
was re-extracted twice with CH2Cl2, and the combined organic phases
were dried and concentrated. Column chromatography (cyclohexane/
ethyl acetate 3/1, Rf = 0.24) left 20 (1.95 g, 96%) as a light brown
solid with mp 52 °C. IR (ATR): νmax 3305, 2926, 2854, 2100, 1736,
1640, 1592, 1538, 1437, 1344, 1312, 1278, 1209, 1170, 1110, 998, 859,
764, 698 cm−1. 1H NMR (CDCl3, 300 MHz): δ 1.19−1.38 (m, 12 H),
1.58 (m, 4 H), 2.27 (t, J = 7.5 Hz, 2 H), 3.40 (q, J = 7.0 Hz, 2 H), 3.63
(s, 3 H), 4.36 (s, 2 H), 6.47 (t, J = 6.3 Hz, 1 H), 7.05 (dd, J = 2.1, 1.5
Hz, 1 H), 7.38 (t, J = 1.9 Hz, 1 H), 7.43 (t, J = 1.5 Hz, 1 H). 13C NMR
(CDCl3, 75 MHz): δ 24.5, 24.7, 26.9, 28.7, 29.1, 29.2, 29.3, 29.5, 34.0,
40.2, 53.9, 63.9, 118.8, 121.4, 124.0, 131.7, 140.7, 143.3, 166.2, 174.3.
MS (EI): m/z 415 (21) [M]+, 384 (19), 345 (44), 317 (29), 313 (25),
285 (10), 231 (20), 201 (27), 175 (42), 161 (24), 145 (43), 118 (33),
3305 br, 2926, 2854, 2105, 1706, 1637, 1606, 1541, 1455, 1373, 1313,
1
1260, 1213, 1088, 1025, 945, 860, 801, 763 cm−1. H NMR (CDCl3,
300 MHz): δ 1.14 (d, J = 6.6 Hz, 3 H), 1.24−1.40 (m, 12 H), 1.56−
1.71 (m, 4 H), 2.82 (td, J = 7.5, 1.6 Hz, 2 H), 2.98 (s, 3 H), 3.44 (td, J
= 7.0, 6.0 Hz, 2 H), 3.79 (d, J = 4.7 Hz, 1 H), 4.19 (qd, J = 6.6, 4.7 Hz,
1 H), 4.40 (s, 2 H), 6.15−6.22 (m, 1 H), 7.06−7.11 (m, 1 H), 7.40 (t,
J = 1.6 Hz, 1 H), 7.42−7.48 (m, 1 H). 13C NMR (CDCl3, 75 MHz): δ
17.8, 26.9, 27.2, 29.01, 29.04, 29.18, 29.21, 29.3, 29.6, 32.8, 40.3, 54.0,
66.7, 68.6, 101.8, 117.5, 121.0, 122.6, 127.9, 137.3, 141.4, 166.0, 173.9,
+
188.7, 194.9; HRMS: calcd for C26H37N8O5 541.2887, found
541.2920 [M + H]+.
11-((2E,4E)-5-Azidomethyl-7-oxo-1H-azepine-3-
carboxamido)undecanoic Acid (24). A solution of bisa-zide 21
(148 mg, 0.37 mmol) in MeOH (3 mL) was irradiated for 28 h with a
150 W Mercury UV lamp in a quartz cuvette from a distance of 10 cm.
Purification by HPLC (MeOH/H2O 65/35 + 0.1% HCOOH) gave
three main fractions (tR = 22, 26.5, 39 min, flow rate 10 mL/min). The
third fraction contained the main product imidate, which hydrolyzed
upon evaporation to leave amide 24. The latter was also found in the
first fraction. The second fraction contained a different unspecified
+
1
83 (30), 69 (51), 56 (100). HRMS: calcd for C20H29N7NaO3
azide. The first and third fractions had identical IR and H NMR
438.2230, found 438.2238 [M + Na]+.
spectra as well as identical retention times in analytical HPLC. They
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Article
were pooled to afford 30 mg (21%) of 24 as a white solid with mp 76
°C. IR (ATR): νmax 2926, 2853, 2103, 1703, 1657, 1627, 1585, 1541,
(9) Knoth, T.; Warburg, K.; Katzka, C.; Rai, A.; Wolf, A.;
Brockmeyer, A.; Janning, P.; Reubold, T. F.; Eschenburg, S.;
1
1436, 1367, 1295, 1263, 1181, 1110, 1015, 885, 799, 759 cm−1. H
Manstein, D. J.; Hubel, K.; Kaiser, M.; Waldmann, H. Angew. Chem.,
̈
NMR (CDCl3, 300 MHz): δ 1.26−1.38 (m, 12 H), 1.52−1.66 (m, 4
H), 2.34 (t, J = 7.4 Hz, 2 H), 2.94 (s, 2 H), 3.35 (dd, J = 7.1, 6.0 Hz, 2
H), 4.03 (s, 2 H), 5.65−5.79 (m, 1 H), 6.33−6.54 (m, 1 H), 7.33 (d, J
= 5.2 Hz, 1 H), 8.21−8.37 (m, 1 H). 13C NMR (CDCl3, 126 MHz): δ
24.6, 26.7, 28.8, 28.90, 28.95, 29.0, 29.1, 29.4, 33.7, 39.4, 40.1, 56.1,
119.6, 121.5, 129.3, 131.5, 165.9, 166.6, 177.7. MS (EI): m/z 392 (80)
Int. Ed. 2009, 48, 7240−7245.
(10) Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.;
Aoyama, H.; Hosoya, A.; Iwata, T.; Maruyama, K.; Endo, M.; Suzuki,
M. Org. Biomol. Chem. 2004, 2, 637−641.
(11) Neelarapu, R.; Holzle, D. L.; Velaparthi, S.; Bai, H.; Brunsteiner,
M.; Blond, S. Y.; Petukhov, P. A. J. Med. Chem. 2011, 54, 4350−4364.
+
[M + H]+, 349 (10), 331 (10). HRMS: calcd for C19H30N5O4
́
(12) Leydet, A.; Barragan, V.; Boyer, B.; Montero, J.-L.; Roque, J.-P.;
392.2292, found 392.2304 [M + H]+.
Witvrouw, M.; Este, J.; Snoeck, R.; Andrei, G.; De Clercq, E. J. Med.
Chem. 1997, 40, 342−349.
Distribution of Bis-Azide-Tagged Tetramic Acid 23 in PTK2
Cells. PTK2 cells were incubated overnight with compound 23 (50
μg/mL) and then fixed and permeabilized with formaldehyde (4%)
and Triton X-100. The cells were then treated with DyLight488-
phosphine (Thermo Scientific), incubated at 37 °C for 3 h, irradiated
at 245 nm for 15 min, and finally photographed through a Zeiss
Axioplan fluorescence microscope at 63× magnification.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Tables and figures giving H and 13C NMR spectra of 11−16,
18, 20−24, (9R)-1e, and (9S)-1e, details of the cytotoxicity of
(9S)-1e, (9R)-1e, and 21, and chemical 13C shifts of natural and
synthetic 1e. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail for R.S.: Rainer.Schobert@uni-bayreuth.de.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (Scho 402/9-2). We are indebted to
Dr. Holger Schmidt, Department of Plant Physiology,
University Bayreuth, for high-resolution mass spectra.
REFERENCES
■
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