Novel antifungal polyene amides from the myxobacterium Cystobacter fuscus: Isolation, antifungal activity and absolute structure determination

Article abstract of DOI:10.1016/j.tet.2004.09.013

Three new unstable metabolites, (6E,10Z)-2′-O-methylmyxalamide D (1), 2′-O-methylmyxalamide D (2) and (6E)-2′-O-methylmyxalamide D (3) were isolated from the myxobacterium Cystobacter fuscus. The planar structures were elucidated by spectroscopic analyses to be geometrical isomers of a polyene amide related to a myxobacterial metabolite, myxalamide D (4). Their absolute stereochemistry was determined by synthesis of degradation products. Antifungal activities of 1-3 as well as their acetates were evaluated against the phythopathogenic fungus Phythopthora capsici. Three new polyene antibiotics 1-3, structurally related to myxalamide D, were isolated as unstable geometrical isomers. The absolute stereochemistry was elucidated by synthesis of degradation products.

Full text of DOI:10.1016/j.tet.2004.09.013

Tetrahedron 60 (2004) 10217–10221
Novel antifungal polyene amides from the myxobacterium
Cystobacter fuscus: isolation, antifungal activity and absolute
structure determination
Bangi A. Kundim,a Yu Itou,a Youji Sakagami,a Ryosuke Fudou,b Shigeru Yamanakac
and Makoto Ojikaa,
*
aGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
bInstitute of Life Science, Ajinomoto Co. Inc., Kawasaki-ku, Kawasaki 210-0801, Japan
cFaculty of Textile Science and Technology, Shinshu University, Tokida, Ueda 386-8567, Japan
Received 20 July 2004; accepted 1 September 2004
Abstract—Three new unstable metabolites, (6E,10Z)-20-O-methylmyxalamide D (1), 20-O-methylmyxalamide D (2) and (6E)-20-O-
methylmyxalamide D (3) were isolated from the myxobacterium Cystobacter fuscus. The planar structures were elucidated by spectroscopic
analyses to be geometrical isomers of a polyene amide related to a myxobacterial metabolite, myxalamide D (4). Their absolute
stereochemistry was determined by synthesis of degradation products. Antifungal activities of 1–3 as well as their acetates were evaluated
against the phythopathogenic fungus Phythopthora capsici.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Myxobacteria are unique gram-negative bacteria character-
ized by the gliding and fruiting body forming nature. They
have recently been well established as a new and potent
source for natural products with biological activities
because of their potential to produce a considerable variety
of metabolites.1–3 Polyene antibiotics are a group of
metabolites characteristic of the myxobacteria. Myxal-
amides, examples of such antibiotics from myxobacteria,
have been discovered from Myxococcus xanthus Mx X12.4,5
In the course of our screening for bioactive metabolites from
myxobacteria, we previously discovered a series of
antifungal metabolites named cystothiazoles from a myxo-
bacterium species, Cystobacter fuscus AJ-13278.6–8 On the
other hand, a further search for additional antifungal agents
from C. fuscus has resulted in the isolation of three new
polyene amides that are structurally related to myxalamide
D (4),5 namely, (6E,10Z)-20-O-methylmyxalamide D (1),
20-O-methylmyxalamide D (2) and (6E)-20-O-methyl-
myxalamide D (3) (Fig. 1). This paper reports the isolation,
Keywords: Myxobacteria; Cystobacter fuscus; Antifungal; Polyene
antibiotics.
* Corresponding author. Tel./fax: C81 52 789 4284;
e-mail: ojika@agr.nagoya-u.ac.jp
Figure 1. Structures of myxalamide D (4) and its new derivatives 1–3.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.09.013

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B. A. Kundim et al. / Tetrahedron 60 (2004) 10217–10221
Table 2. 13C NMR chemical shifts for 1–3 in CDCl3 (150 MHz)
structure elucidation, antifungal activity and absolute
stereochemistry for these new metabolites.
Position
1a
2a
3
1
2
3
4
5
6
7
8
168.3
129.8b
133.6
127.7
138.1
132.8
136.1
130.0b
131.4
134.0
135.2
36.3
168.3
130.5
133.5
128.0
133.2
128.4
132.5
122.3
139.8
136.0
137.6
37.3
168.3
129.6
133.7
127.3b
138.3
132.0
136.0
127.2b
139.2
136.1
137.1
37.2
2. Results and discussion
Production of the antibiotics was performed as reported
previously for the isolation of the cystothiazoles.6,7 The
bacterial cells and adsorbent resin obtained from a 150-L
fermentation broth of C. fuscus were extracted with acetone
and the extract was subjected to solvent partition. The non-
polar fraction was chromatographed twice on silica gel to
afford less polar fractions containing cystothiazoles6–8 and a
relatively polar fraction containing polyene metabolites.
The latter fraction was further subjected to reversed-phase
HPLC to give the myxalamide D derivatives 1 (0.6 mg), 2
(0.6 mg) and 3 (2.4 mg). The content of these metabolites
should be potentially much higher (more than 10 times) than
the isolated amounts, because the more they were purified,
the more they decomposed due to their instability.
9
10
11
12
13
14
15
16
17
18
19
20
10
82.7
82.7
82.8
135.6
123.6
13.1
10.7
17.8
20.7
13.1
45.0
75.6
135.7
123.4
13.1
10.7
17.3
13.1
13.1
45.0
75.6
135.6
123.5
13.1
10.6
17.3
12.9
13.1
45.0
75.6
20
30
17.8
59.1
17.8
59.1
17.8
59.1
All three compounds 1–3 showed similar spectroscopic data
to each other. The molecular formula of C24H37NO3 for all
three compounds were determined by HRMS measurement.
The resonances at 168.3 ppm in the 13C NMR spectra
indicated the presence of an amide carbonyl carbon in all
compounds, which was confirmed by the characteristic IR
absorption band at 1653 cmK1. Based on these findings, 2D
NMR analysis was then performed to determine the planar
structures of the compounds as discussed below.
20-OCH3
a Chemical shifts were partially determined by HMQC and HMBC data.
b Interchangeable signals.
connectivities in 1 were first determined by the HMQC
spectrum. The chemical shifts are summarized in Tables 1
and 2. The partial structures of H3–H9, H11–H12(H18)–
H13, H15–H16 and NH–H10(H30)–H20 were revealed by the
DQF-COSY data (Fig. 2). These were then successfully
connected to each other by the HMBC spectrum to give the
planar structure of 1 (Fig. 2). The singlet signal observed at
d 3.38 (3H) in the 1H NMR spectrum was easily determined
to be the 20-O-methyl group from the HMBC correlation to
C20. The geometry of the double bonds at C4, C6, and C8
were determined from the proton coupling constants of
J4,5Z14.6 Hz, J6,7Z14.5 Hz, J8,9Z15.0 Hz as 4E, 6E, 8E,
respectively. The geometry of the trisubstituted olefins at
C2, C10, and C14 was determined from the NOESY
correlations of H4/H20, H9/H12, H11/H19, and H13/H15 as
2E, 10Z, 14E, respectively (Fig. 2). Although the molecular
formula of 1 is the same as myxalamide C,5 which is the
C16-methylated homologue of myxalamide D (4), these
were unambiguously distinguished by the NMR analysis,
especially the HMBC correlation of OCH3/C20, Thus, the
Many olefinic signals observed between 5 and 7 ppm in the
1H NMR spectrum of 1 (Table 1) are characteristic of
polyenes like the myxalamides.5,9 The proton–carbon direct
Table 1. 1H NMR spectral data for 1–3 in CDCl3 (600 MHz)
Position
1
2
3
dH (m, J in Hz)
dH (m, J in Hz)
dH (m, J in Hz)
3
4
6.96 (d, 11.0)
6.47 (dd, 14.6,
11.0)
7.00 (d, 11.0)
6.48 (dd, 14.5,
11.0)
6.95 (d, 11.3)
6.45 (dd, 14.6,
11.3)
5
6
7
8
6.54 (dd, 14.6,
10.6)
6.39 (dd, 14.5,
10.6)
6.48 (dd, 14.5,
10.8)
6.36 (dd, 15.0,
10.8)
6.99 (dd, 14.5,
11.0)
6.10 (dd, 11.0,
11.0)
6.16 (dd, 11.3,
11.0)
6.69 (dd, 15.1,
11.3)
6.53 (dd, 14.6,
10.0)
6.35 (dd, 15.0,
10.0)
6.40 (dd, 15.0,
10.0)
6.26 (dd, 15.0,
10.0)
9
6.73 (d, 15.0)
5.28 (d, 10.0)
2.83 (m)
3.64 (d, 8.9)
5.49 (q, 6.5)
1.64 (d, 6.5)
1.66 (s)
0.83 (d, 6.7)
1.91 (s)
1.98 (s)
4.25 (m)
3.43 (dd, 9.5,
4.0)
6.36 (d, 15.1)
5.46 (d, 9.8)
2.75 (m)
3.69 (d, 8.7)
5.49 (q, 6.3)
1.64 (d, 6.3)
1.64 (s)
0.84 (d, 6.7)
1.91 (s)
1.99 (s)
4.25 (m)
3.43 (dd, 9.5,
4.0)
6.36 (d, 15.0)
5.43 (d, 9.7)
2.73 (m)
3.68 (d, 8.8)
5.49 (q, 6.0)
1.63 (d, 6.0)
1.64 (s)
0.83 (d, 6.7)
1.86 (s)
1.97 (s)
4.25 (m)
3.42 (dd, 9.5,
4.0)
11
12
13
15
16
17
18
19
20
10
20
3.39 (dd, 9.5,
4.0)
1.23 (d, 6.8)
3.38 (s)
5.96 (d, 7.6)
3.39 (dd, 9.5,
4.0)
1.23 (d, 6.8)
3.38 (s)
5.98 (d, 7.6)
3.39 (dd, 9.5,
4.0)
1.22 (d, 6.2)
3.37 (s)
5.96 (d, 7.8)
30
20-OCH3
NH
Figure 2. Gross structure of 1 determined by the 2D NMR correlations
shown.

B. A. Kundim et al. / Tetrahedron 60 (2004) 10217–10221
10219
compound 1 was determined to be (6E,10Z)-20-O-methyl-
myxalamide D.
mixture of the corresponding acetates, which was subjected
to oxidative cleavage of the double bonds followed by
esterification to yield p-bromophenacyl ester 5 (Scheme 1).
The spectral data including specific rotation for 5 was
identical to those reported,9 revealing the 12R, 13R
configuration of 1–3. To determine the stereochemistry at
C10 a mixture of the acetates of 1–3 was treated with ozone
followed by Me2S to obtain keto amide (K)-6 (Scheme 1).
The configuration of (K)-6 was determined by the synthesis
of (S)-6 from phthalimide 710 in four steps as shown in
Scheme 2. Methylation of the hydroxyl group in 7 afforded
methyl ether 8. Deprotection of the phthalimide group of 8
with hydrazine gave the corresponding free amine, which
was treated with methacryloyl chloride and triethylamine to
give acrylamide 9. Finally, ozonolysis of 9 gave (S)-
ketoamide 6: [a]2D3 K138 (c 0.3, CHCl3) [natural: K178 (c
0.03, CHCl3)]. The NMR spectra and other spectral data
including specific rotations of both synthetic and natural 6
were identical, indicating the 10S configuration of 1–3. The
stereochemistry of 1–3 is therefore the same as that of the
known myxalamides.
The NMR data of 2 (Tables 1 and 2) were quite similar to
those for 1, suggesting that it was a stereoisomer of 1. The
planar structure of 2 was determined by 2D NMR in a
similar manner to that for 1. The 4E, 6Z, 8E geometry was
determined from the coupling constants of J4,5Z14.5 Hz,
J6,7Z11.0 Hz, J8,9Z15.1 Hz, respectively. The 2E, 10E,
14E geometry of the trisubstituted double bonds was
determined from the NOESY correlations of H4/H20, H9/
H11, H12/H19, and H13/H15. These findings revealed that
the compound 2 was 20-O-methylmyxalamide D.
Similarly, the NMR data of 3 (Tables 1 and 2) suggested that
this compound was a stereoisomer of 1 and 2. The same
planar structure as those of 1 and 2 was then determined by
2D NMR analysis. The 4E, 6E, 8E geometry were
determined from the coupling constants as shown in
Table 1, and the 2E, 10E, 14E geometry of the trisubstituted
double bonds were determined from the NOESY corre-
lations of H4/H20, H12/H19, and H13/H15. Thus, the
compound 3 was determined to be (6E)-20-O-methyl-
myxalamide D. The steric repulsion effect in the 13C
NMR data, that is, high-field shifts of the carbons adjacent
to a cis olefin, also supported the geometry of these polyene
metabolites 1–3. Thus, high-field shifts compared to the all
E compound 3 were observed for the following carbons of 1
and 2: C9 (Dd K7.8) in 1, C5 (Dd K5.1) and C8 (Dd K4.9)
in 2.
To determine the absolute stereochemistry of the myxal-
amide D derivatives 1–3, we followed the methodology
described by Jansen et al.9 with a slight modification. A
mixture of 1–3 was used for degradation to obtain the
fragments that contained asymmetric centers because 1–3
easily isomerized to each other under light and decomposed
during purification. Acetylation of a mixture of 1–3 gave a
Scheme 2. Synthetic route to (S)-6.
The antifungal activities of the compounds 1–3 as well as
their acetates was evaluated by a paper disc assay method
against the phytopathogenic fungus Phytophthora capsici.
The minimum dose to form a recognizable inhibition zone
in the neighborhood of the paper disc was determined to be
2 mg/disc for all three compounds. The acetates of 1–3 as a
mixture showed antifungal activity at a minimum dose of
5 mg/disc.
Discovery of the myxalamide D derivatives 1–3 from the
myxobacterium C. fuscus suggests that polyene amides such
as the myxalamides seem to be fairly common secondary
metabolites in the myxobacteria. The myxalamides were
also discovered from Stigmatella aurantiaca11 and others2
after the original discovery from M. xanthus,4,5 although
methyl ether-type derivatives such as 1–3 were the first
examples in the myxalamides. C. fuscus seems to possess a
metabolic system that is similar to that of the other
myxalamide producers but is unique, because myxal-
amide-related metabolites other than 1–3 were not detected
in photodiode array HPLC analysis of the extracts. Since all
three isomers 1–3 showed the same antifungal activity
against the fungus P. capsici, the olefin geometry plays no
crucial role for their antifungal activity. The activity of the
acetates (mixture) suggests that the free 13-OH group is not
essential. In addition, the free 20-hydroxyl group of the
known myxalamides seems not to be very important, though
a direct comparison of the activity was not performed
Scheme 1. Degradation routes to determine the absolute stereochemistry of
1–3.

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B. A. Kundim et al. / Tetrahedron 60 (2004) 10217–10221
between the myxalamides and our compounds. Based on a
structural similarity to the myxalamides the mode of action
of our new myxalamide derivatives could be blocking the
mitochondrial respiratory chain by inhibition of NADH
oxidation at complex I.4,12 It should be noted that more than
one species of the myxobacteria can produce similar
polyene amides with stereochemical homogeneity.
3.2.1. (6E,10Z)-20-O-Methylmyxalamide D (1). Pale
yellow oil; [a]D21 K128 (c 0.03, MeOH); IR (film) nmax
3370 (br), 1653, 1522, 1108, 1000 cmK1; UV (MeOH) lmax
260 (3 6000), 340 (sh), 355 (30,000), 370 (sh) nm; MS
(ESIC) m/z 388 [MCH]C, 410 [MCNa]C. HRMS found
388.2794, calcd for C24H38NO3 [MCH]C 388.2846. For
NMR data refer to Tables 1 and 2.
3.2.2. 20-O-Methylmyxalamide D (2). Pale yellow oil;
[a]2D1 K278 (c 0.04, MeOH); IR (film) nmax 3367 (br), 1653,
1522, 1107, 1000 cmK1; UV (MeOH) lmax 260 (3 6000),
340 (sh), 355 (30,000), 370 (sh) nm; MS (ESIC) m/z 388
[MCH]C, 410 [MCNa]C. HRMS found 388.2805, calcd
for C24H38NO3 [MCH]C 388.2846. For NMR data refer to
Tables 1 and 2.
3. Experimental
3.1. General
Thin-layer chromatography (TLC) was performed by using
pre-coated silica gel 60 F254 plates (Art. 5715, Merck) or
RP-18 F254 plates (Art. 15389, Merck). Open column
chromatography was performed using silica gel BW-300
(Fuji Silysia) or Cosmosil 75C18-OPN (Nacalai Tesque).
HPLC was performed on a high-pressure gradient system
equipped with PU-980 pumps and UV-970 detector or an
MD-915 photodiode array UV detector (JASCO). Specific
rotations were obtained by using a DIP-370 digital
polarimeter (JASCO). FT-IR spectra were recorded on a
FT-IR-7000S spectrometer (JASCO). UV spectra were
recorded on a Ubest-50 UV/VIS spectrophotometer
(JASCO). Mass spectra (MS) were recorded on a Mariner
Biospectrometry Workstation (Applied Biosystems) in the
positive ESI mode. Residual phthalic acid anhydride (m/z
149.0233) and a peptide mixture (angiotensin I, bradykinin
and neurotensin) were used as internal standards for high-
resolution MS analysis. NMR spectra were recorded on an
ARX 400 (400 MHz) or an AMX2 600 (600 MHz)
spectrometer (Bruker). The NMR chemical shifts (ppm)
were referenced to the solvent peaks of dH 7.26 (residual
CHCl3) and dC 77.0 for CDCl3 solutions or dH 3.30 (residual
CHD2OD) and dC 49.0 for CD3OD solutions. The assay
method was the same as the previously reported one for the
cystothiazoles.6,7
3.2.3. (6E)-20-O-Methylmyxalamide D (3). Pale yellow
oil; [a]2D1 K318 (c 0.017, MeOH); IR (film) nmax 3365 (br),
1653, 1522, 1106, 998 cmK1; UV (MeOH) lmax 257 (3
3000), 340 (sh), 356 (30,000), 370 (sh) nm; MS (ESIC) m/z
388 [MCH]C, 410 [MCNa]C. HRMS found 388.2807,
calcd for C24H38NO3 [MCH]C 388.2846. For NMR data
refer to Tables 1 and 2.
3.3. Degradation
3.3.1. 13-O-Acetyl derivatives of 1–3. A mixture of 1–3
(46.1 mg, 0.12 mmol) was treated with a mixture of
pyridine (4 mL), DMAP (22 mg, 0.18 mmol) and Ac2O
(2 mL) at room temperature for 3 h. The mixture was
concentrated and the residue was chromatographed on silica
gel (hexane/EtOAc 2:1) to obtain a mixture of acetates as a
pale yellow oil (20.7 mg, 40%): 1H NMR (400 MHz,
CDCl3) d 6.90–7.05 (1H, m, H-3), 6.05–6.68 (6H, m, H-4–
H-9), 5.90–6.00 (1H, m, NH), 5.50–5.60 (1H, m, H-15),
5.37 (1H, m, H-11), 4.98 (1H, m, H-13), 4.25 (1H, m, H-10),
3.35–3.45 (2H, m, H-20), 3.33, 3.37 and 3.38 (total 3H, s
each, 20-OCH3), 2.83–2.93 (1H, m, H-12), 1.97 and 1.98
(total 3H, s each, H-20), 1.94 and 1.95 (total 3H, s each,
acetate), 1.86, 1.81 and 1.72 (total 3H, s each, H-19), 1.65
and 1.58 (total 6H, m, H-16,017), 1.21, 1.22 and 1.23 (total
3H, d each, JZ6.8 Hz, H-3 ), 0.87 and 0.88 (total 3H, d
each, JZ6.8 Hz, H-18).
3.2. Isolation of 1–3
A large scale (150 L) fermentation of C. fuscus followed by
extraction and silica gel chromatography was described
previously.6,7 The first chromatography of the crude hexane/
EtOAc (3:1)–soluble material (14.2 g) on silica gel afforded
five fractions. The third fraction yielded cystothiazole A as
described in our reference (see Ref. 6). A portion (770 mg)
of the fourth fraction (2.5 g) eluted with EtOAc was
chromatographed on silica gel [Develosil LOP60 (: 20!
300 mm), Nomura Chemical; 160 min linear gradient from
2 to 42% acetone in benzene, 5 mL/min]. The sixth fraction
(42.8 mg) eluted from 8.25 to 8.5% acetone in benzene was
applied to HPLC [TSK gel ODS-120T (: 20!250 mm),
TOSOH, 72% aq MeOH, 5 mL/min, detected at 380 nm] to
obtain (6E,10Z)-200-O-methylmyxalamide D (1) (0.6 mg,
tRZ101.0 min), 2 -O-methylmyxalamide D (2) (0.6 mg,
tRZ105.4 min), and (6E)-20-O-methylmyxalamide D (3)
(2.4 mg, tRZ114.6 min) as pale yellow oils. The rest
(1.73 g) of the fourth fraction eluted with EtOAc was used
for obtaining a crude mixture of 1–3 (287 mg), which was
used for degradation without further purification (see
Section 3.3.1).
3.3.2. N-(2-Methoxy-1-methylethyl)-2-oxopropanamide
(6). The above acetate mixture (20.7 mg, 0.048 mmol)
was dissolved in MeOH (1.5 mL) and cooled to K78 8C. A
stream of ozone (8%) in oxygen was passed through this
solution for 1 h. The solution was flushed with oxygen,
treated with Me2S (0.5 mL) and then allowed to warm to
room temperature with stirring for 3 h. Solvent evaporation
gave an oily residue (4.9 mg), which was chromatographed
on silica gel (hexane/acetone 10:1) to obtain pure 6 (0.5 mg,
7%) as a colorless oil: [a]2D3 K178 (c 0.03, CHCl3). The
NMR and other spectroscopic data were identical to
synthetic (S)-6 (refer to Section 3.4).
3.3.3. 3-Acetoxy-2-methyl-4-oxopentanoic acid p-bromo-
phenacyl ester (5). The reaction was carried out in a
manner similar to that described in Ref. 6. Briefly, a mixture
of the above acetates (24.7 mg, 0.057 mmol) was subjected
to ozonolysis followed by oxidative treatment with H2O2
and subsequent esterification of the resulting carboxylic

B. A. Kundim et al. / Tetrahedron 60 (2004) 10217–10221
10221
acid with p-bromophenacyl bromide (26 mg, 0.094 mmol)
to give 5 (0.7 mg, 3%): [a]2D3 C108 (c 0.06, CHCl3) (lit.:9
[a]2D0 C118 (c 0.3, CHCl3)). 1H NMR spectrum was
identical to that reported in the literature.9
(2H, dd, JZ1.0, 4.4 Hz), 3.35 (3H, s), 2.46 (3H, s), 1.21
(3H, d, JZ6.8 Hz); 13C NMR (100 MHz, CDCl3) d 197.2,
159.6, 75.1, 59.1, 45.1, 24.4, 17.2; UV (MeOH) lmax 230 (3
1440, sh) nm; IR (film) nmax 3400, 1718, 1684, 1522, 1169,
1110, 669 cmK1; HRMS m/z 182.0778 (MCNa)C, calcd
for C7H13NO3Na 182.0788. It must be noted that product
(S)-6 (both natural and synthetic) is a very volatile
compound and most product was lost mainly during
concentration between spectroscopic measurements.
3.4. Synthesis of ketoamide (S)-6
3.4.1. (S)-2-(2-Methoxy-1-methylethyl)-1H-isoindole-
1,3(2H)-dione (8). To a solution of phthalimide 710
(592 mg, 2.9 mmol) in CH3CN (7.5 mL) was added Ag2O
(2 g, 8.7 mmol) and the mixture was heated at reflux
temperature for 5 h in the dark. Ag2O was filtered off by
using a pad of celite and the filtrate was concentrated to
afford 8 (602 mg, 95%) as chromatographically pure
Acknowledgements
This work was financially supported by KAKENHI
(12480172) from JSPS and the TAKEDA SCIENCE
FOUNDATION. We are grateful to Mr. Masayuki Hirata
for his help in an antifungal test. B. A. Kundim is grateful
for the Japanese Government Foreign Student Scholarship
through the Ministry of Education, Culture, Sports, Science
and Technology.
material: colorless oil, [a]2D3 C208 (c 0.83, CHCl3); H
1
NMR (400 MHz, CDCl3) d 7.81 (2H, m), 7.69 (2H, m), 4.61
(1H, m), 3.97 (1H, t, JZ9.8 Hz), 3.53 (1H, dd, JZ5.4,
9.8 Hz), 3.32 (3H, s), 1.44 (3H, d, JZ7.1 Hz); 13C NMR
(100 MHz, CDCl3) d 168.5, 133.8, 132.1, 123.1, 72.9, 58.7,
46.3, 15.0; UV (MeOH) lmax 219 (3 31,000), 240 (7600, sh),
295 (1600) nm; IR (film) nmax 2984, 2939, 2894, 1775,
1714, 1705, 1468, 1394, 1373, 1337, 1111, 1042, 878, 720,
532 cmK1; HRMS m/z 220.0967 (MCH)C, calcd for
C12H14NO3 220.0968.
References and notes
3.4.2. (S)-N-(2-Methoxy-1-methylethyl)-2-methyl-2-pro-
penamide (9). Phthalimide 8 (96.4 mg, 0.44 mmol) was
dissolved in EtOH (0.1 mL) in a sealed tube. To this
solution was added hydrazine monohydrate (25 mL,
0.48 mmol), and the tube was sealed and heated at 75 8C
for 5 h. After cooling to room temperature, Et3N (0.61 mL,
4.4 mmol) was added and stirred for 5 min before cooling to
K10 8C. Then methacryloyl chloride (0.43 mL, 4.4 mmol)
was added dropwise and the mixture was stirred at 0 8C for
5 h. The reaction mixture was diluted with water and
extracted with EtOAc three times. The combined organic
layers were dried (anhyd Na2SO4) and concentrated to give
a crude oil, which was chromatographed on silica gel
(hexane/acetone 4:1) to give acrylamide 9 (21.6 mg, 31%):
colorless oil, [a]2D3 K108 (c 0.23, CHCl3); 1H NMR
(400 MHz, CDCl3) d 6.02 (NH, br s), 5.65 (1H, s), 5.29
(1H, s), 4.19 (1H, m), 3.39 (1H, dd, JZ4.2, 9.4 Hz), 3.36
(1H, dd, JZ4.1, 9.4 Hz), 3.35 (3H, s), 1.94 (3H, s), 1.19
(3H, d, JZ6.8 Hz); 13C NMR (100 MHz, CDCl3) d 167.8,
140.2, 119.2, 75.4, 59.0, 44.8, 18.6, 17.6; UV (MeOH) lmax
203 (3 8700) and 296 (350, sh) nm; IR (film) nmax 3312,
2981, 2930, 1721, 1678, 1659, 1624, 1530, 1455, 1154,
1111, 1038, 935 cmK1; HRMS m/z 180.0991 (MCNa)C,
calcd for C8H15NO2Na 180.0995.
¨
1. Reichenbach, H.; Gerth, K.; Irschik, H.; Kunze, B.; Hofle, G.
Trends Biotechnol. 1998, 6, 115–121.
¨
2. Reichenbach, H.; Hofle, G. Production of Bioactive Secondary
Metabolites. In Myxobacteria II; Dworkin, M., Kaiser, D.,
Eds.; American Society for Microbiology: Washington, DC,
1993; pp 347–397; Chapter 16.
3. Gerth, K.; Pradella, S.; Perlova, O.; Beyer, S.; Mu¨ller, R.
J. Biotechnol. 2003, 106, 233–253.
¨
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Kunze, B.; Reichenbach, H.; Thierbach, G. J. Antibiot. 1983,
36, 1150–1155.
¨
5. Jansen, R.; Reifenstahl, G.; Gerth, K.; Reichenbach, H.; Hofle,
G. Liebigs Ann. Chem. 1983, 1081–1095.
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275–281.
7. Suzuki, Y.; Ojika, M.; Sakagami, Y.; Fudou, R.; Yamanaka, S.
Tetrahedron 1998, 54, 11399–11404.
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Sakagami, Y.; Ojika, M.; Fudou, R.; Yamanaka, S. Tetra-
hedron 2004, 60, 4735–4738.
¨
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2117–2124.
3.4.3. (S)-N-(2-Methoxy-1-methylethyl)-2-oxopropana-
mide [(S)-6)]. A stream of ozone (8% in O2) was passed
through a solution of 9 (26.3 mg, 0.17 mmol) in MeOH
(1.5 mL) at K78 8C for 1 h. After removal of ozone, the
reaction mixture was concentrated to obtain (S)-6 (6.6 mg,
¨
11. Hofle, G.; Kunze, B.; Zorzin, C.; Reichenbach, H. Liebigs Ann.
Chem. 1984, 1883–1904.
12. Friedrich, T.; Van Heek, P.; Leif, H.; Ohnishi, T.; Forche, E.;
1
25%): colorless oil, [a]2D3 K138 (c 0.3, CHCl3); H NMR
Kunze, B.; Jansen, R.; Trowitzsch-Kienast, W.; Hofle, G.;
Reichenbach, H. Eur. J. Biochem. 1994, 219, 691–698.
¨
(400 MHz, CDCl3) d 7.06 (NH, br s), 4.10 (1H, m), 3.37