Cyrmenins, Novel Antifungal Peptides Containing a Nitrogen-Linked β-Methoxyacrylate Pharmacophore: Isolation and Structural Elucidation

Article abstract of DOI:10.1002/ejoc.200300367

The novel antifungal metabolites cyrmenin A, B₁, and B ₂ (1-3) were isolated from Archangium gephyra and Cystobacter armeniaca strains (myxobacteria). The cyrmenins are modified N-acyldipeptide esters containing a didehydroalanine, a 3-O-methyl-didehydroserine and a (2E,4Z)-undecadienoic or -dodecadienoic acid residue. These compounds represent the first bacterial counterparts of strobilurins that are characterized by an α-substituted β-methoxyacrylate pharmacophore. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300367

FULL PAPER
Cyrmenins, Novel Antifungal Peptides Containing a Nitrogen-Linked
β-Methoxyacrylate Pharmacophore: Isolation and Structural Elucidation[‡]
Thomas Leibold,*[a] Florenz Sasse,[a] Hans Reichenbach,[a] and Gerhard Höfle[a]
Dedicated to Professor Wolfgang Steglich on the occasion of his 70th birthday
Keywords: Dehydropeptides / Fungicides / Myxobacteria / Natural products / Structure elucidation
The novel antifungal metabolites cyrmenin A, B1, and B2
(1−3) were isolated from Archangium gephyra and Cysto-
bacter armeniaca strains (myxobacteria). The cyrmenins are
modified N-acyldipeptide esters containing a didehydro-
represent the first bacterial counterparts of strobilurins that
are characterized by an α-substituted β-methoxyacrylate
pharmacophore.
alanine, a 3-O-methyl-didehydroserine and a (2E,4Z)-unde- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
cadienoic or -dodecadienoic acid residue. These compounds
Germany, 2004)
Introduction
terial was partitioned between methanol and heptane to re-
move the lipophilic components. Further purification and
isolation of 1Ϫ3 was accomplished by three consecutive
chromatographic steps on silica gel, RP-18 silica gel, and
Sephadex LH-20. Because of the considerable decompo-
sition of the cyrmenins that occurs under basic conditions,
all extractive steps and chromatographic separations had to
be performed with control of pH (6.5). When these pre-
cautions were taken, the pure cyrmenins 1Ϫ3 were obtained
as colorless oils: cyrmenin A (1; 440 mg), cyrmenin B1 (2;
492 mg), and cyrmenin B2 (3; 60 mg).
In the course of our screening of myxobacteria for bio-
logically active metabolites, we noticed that strains of
Cystobacter armeniaca and Archangium gephyra had strong
antifungal activity. According to HPLC, UV spectroscopy,
and MS analyses of crude extracts, the compounds respon-
sible for this activity were not related to the antifungal com-
pounds isolated previously from myxobacteria.[2,3] Here we
report the isolation and structural elucidation of the major
components from A. gephyra, named cyrmenins A (1), B1
(2), and B2 (3). In their pure states, the cyrmenins exhibit
high activity against, for example, Botrytis cinerea, Phy-
thium debaryanum, Hansenula anomala, Metschnikowia
pulcherrima, but they are inactive against bacteria.[1b] Like
the strobilurin[4] and myxothiazol[5] groups of fungicides,
the cyrmenins[1b] are inhibitors of mitochondrial respiration
at the cytochrome bc1 complex. In spite of this general
mechanism of action, the cyrmenins exhibit an exception-
ally low toxicity for animal cell cultures.[1b]
The structural elucidation of the cyrmenins was mainly
based on data obtained from cyrmenin B2 (3). High-resolu-
tion (ϩ)-DCI MS of the protonated molecular ion at m/z ϭ
393 suggested the elemental composition C21H32N2O5,
which implies the presence of seven double-bond equiva-
lents. The UV spectrum of 3 shows one intense, broad band
at 259 nm, while the IR spectrum indicates ester and amide
groups by bands at 1720, 1658, and 3360 cmϪ1. All the
1
signals in the H and 13C NMR spectra were assigned and
correlated by 1H,1H-COSY and direct 1H,13C-correlation
(HMQC) NMR spectroscopy (Table 1). By this means, the
constitution of the hydrocarbon fragment, which is marked
by bold bonds in Figure 1, was derived straightforwardly.
1H,13C long-range correlations between C-7 and both 8-Me
and 6-H completed the 2,10-dimethylundeca-2,4-dienamide
partial structure. Its (8E) configuration and preferred con-
formation were verified by the observation of strong NOEs
between protons 6-H and 8-Me and protons 8-Me and 10-
H. The configuration of the ∆10 unit was deduced to be (Z)
by the coupling constant between protons 10-H and 11-H
(10.9 Hz), which is in the range typical for (Z) double
bonds, and by an NOE between protons 9-H and 12-H.[6]
Results and Discussion
The cyrmenins were recovered by methanol extraction of
the amberlite XAD 16 adsorbent resin present during a
300-L fermentation of A. gephyra. Evaporation of the meth-
anol from the extract and extraction of the aqueous residue
with ethyl acetate gave 80 g of the crude product. This ma-
[‡]
Antibiotics from Gliding Bacteria, 96. Part 95: Ref.[1a]
[a]
GBF, Gesellschaft für Biotechnologische Forschung mbH,
Mascheroder Weg 1, 38124 Braunschweig, Germany
E-mail: tleibold@gbf.de
Eur. J. Org. Chem. 2004, 431Ϫ435
DOI: 10.1002/ejoc.200300367
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
431

T. Leibold, F. Sasse, H. Reichenbach, G. Höfle
FULL PAPER
Table 1. NMR spectroscopic data (CDCl3, 400/100 MHz) for cyrmenin B2 (3)
Position
δH
M[a]
J [Hz]
δC
M[a]
Position
δH
M[a]
J [Hz]
δC
M[a]
1
Ϫ
3.74
Ϫ
7.33
3.90
7.18
Ϫ
Ϫ
s
Ϫ
s
s
br. s
Ϫ
Ϫ
d
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
1.8
1.5
Ϫ
Ϫ
165.2
52.0
106.7
155.3
62.4
Ϫ
162.8
134.2
102.4
Ϫ
s
q
s
d
q
Ϫ
s
s
t
Ϫ
Ϫ
s
8
Ϫ
Ϫ
d
Ϫ
1.5
129.6
12.5
129.9
123.5
139.8
28.2
29.8
27.1
38.9
28.0
s
1-OMe
2
2Ј
2Ј-OMe
3
4
5
5Јa
5Јb
6
8-Me
9
1.99
7.30
6.24
5.80
2.28
1.39
1.28
1.15
1.49
0.84
q
d
d
d
t
t
t
t
d
q
tq
ddt
dt
dq
m
m
m
m
t
1.3/11.7
1.7/10.9/11.7
7.8/10.9
1.7/7.4
Ϫ
Ϫ
Ϫ
Ϫ
6.6
10
11
12
13
14
15
16
17
Ϫ
6.66
5.41
8.51
Ϫ
t
br. s
Ϫ
Ϫ
167.9
22.6
7
[a]
Multiplicity.
Scheme 1. Photoisomerization of model compound 4
(Z) isomers of the β-methoxyacrylate, 4 and 4Ј (see
Scheme 1).
Figure 1. Structural elements of cyrmenin B2 (3) and selected corre-
Stereochemical assignment of the double bond configu-
ration was accomplished by a ROESY NMR spectrum of
this mixture.[10] The major isomer 4 displayed strong inter-
actions between the 2Ј-H proton and the 1-OMe and 2Ј-
OMe units, whereas only one interaction, between 2Ј-H and
2Ј-OMe, was observed for the minor isomer 4Ј. These fea-
tures proved the (Z) configuration for 4 and the (E) con-
figuration for 4Ј. Based on these observations, cyrmenin B2
(3) should have the (E) configuration because we observed
only one NOE interaction between the 2Ј-H proton and the
2Ј-OMe group (see above). Inspection of the NMR spectro-
scopic chemical shifts, however, clearly indicates the op-
posite assignment (Table 2).
lations deduced from NMR spectra
Extending this partial structure to include a dipeptide
formed from didehydroalanine and 3O-methyl-didehydro-
1
serine methyl ester was deduced by H,13C long-range cor-
relation (HMBC) NMR spectroscopy (Figure 1). Strong
long-range interactions between the amide proton 6-H and
C-5Ј and between C-5 and both protons 5Ј-Ha and 5Ј-Hb,
as well as the interaction between each of the methylene
protons 5Ј-H and carbonyl C-4, substantiated the presence
of the didehydroalanine fragment. Carbonyl atom C-4, on
the other hand, is linked to the nitrogen atom of an α-
aminoacrylate fragment, as was indicated by long-range
correlations between 3-H and each of the carbonyl atoms
C-1 and C-4 of the carboxy groups. Similarly, the methoxy
group and hydrogen atom in the β-position
exhibited the expected C,H correlations with the C-1, C-2
and C-2Ј atoms.
Table 2. Selected NMR spectroscopic chemical shifts of cyrmenin
B2 (3) and model compounds 4 and 4Ј
1H NMR
13C NMR
3
4
4Ј
3
4
4Ј
Remarkably, only one geometric isomer of the enol ether
of cyrmenin was isolated from the fermentation, and at-
tempts to determine its configuration by NOE failed. Un-
der a variety of conditions no NOE was observed between
the ester methoxy group and either the 2Ј-H or 2Ј-OMe
units. Therefore, we synthesized two model compounds, 4
and 4Ј, of the corresponding (E)/(Z) isomers from 4-meth-
oxymethylene-2-phenyl-4H-oxazol-5-one.[7] Methanolysis
1-OMe 3.74
3.72
Ϫ
7.31
3.86
3.83
Ϫ
8.08
3.89
52.0
106.7
155.3
62.4
51.8
107.5
154.8
62.1
52.3
108.7
155.3
62.8
2
2Ј
Ϫ
7.33
2Ј-OMe 3.90
Whereas the 13C NMR spectroscopic chemical shifts are
of the oxazolone in the presence of DMAP furnished two essentially identical for 4, 4Ј, and 3, and, thus, are not useful
products: the desired methyl 2-benzoylamino-3-methoxy- for an assignment, the 2Ј-H chemical shifts are significantly
acrylate (4) and methyl 2-benzoylamino-3,3-dimethoxypro- different. In the (E) isomer 4Ј, the 2Ј-H proton is strongly
panoate.[8,9] After chromatographic separation, 4 was pho- deshielded by the C-4 carbonyl group and its signal is
toisomerized to yield a 4:1 equilibrium mixture of the (E)/ shifted to δ ϭ 8.08 ppm. In the (Z) isomer 4 and cyrmenin
432
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2004, 431Ϫ435

Cyrmenins, Novel Antifungal Peptides
FULL PAPER
B2 (3), the 2Ј-H signals are located at δ ϭ 7.31 and are the first α-linked β-methoxyacrylates produced by bac-
7.33 ppm, respectively. Therefore, disregarding the lack of teria that use an entirely different biosynthetic route. Occu-
an NOE between the 2Ј-H and 1-OMe units, we assign cyr- pying the same binding site on the cytochrome bc1 complex,
menin B2 (3) and its relatives 1 and 2 to have a (2Z) con- the strobilurins, myxothiazoles, and cyrmenins are examples
figurations.[11] This assignment is in line with the biological of convergent evolution on the molecular level (see
activity observed for cyrmenins; during SAR studies of Figure 3).
strobilurin derivatives, the (E) configuration [corresponding
to the (Z) configuration in cyrmenins] was found to be
essential[4b,12] for biological activity.
From the 1H and 13C NMR spectroscopic data, cyrmenin
A (1) is characterized as a higher homologue of B2 (3) be-
cause it contains an additional methylene group in the fatty
acid part. Cyrmenins B1 (2) and B2 (3) are isomers and
were separated only with difficulty by chromatography. In
cyrmenin B1 (2), the isopropyl terminus of B2 (3) is replaced
by an n-propyl unit (Figure 2). From a biosynthetic point of
view, these modifications arise from different starter units in
the polyketide synthesis of the side chain. As in several
other myxobacterial metabolites, the starter CoA esters are
derived by oxidative degradation of the amino acids leucine,
valine, and norvaline.[13]
Figure 3. Natural and synthetic β-methoxyacrylates
Figure 2. Cyrmenin A (1), B1 (2) and B2 (3)
Because of their relatively simple structure and low
mammalian toxicities, the strobilurins have been optimized
extensively by chemical synthesis and developed to com-
Conclusion
mercially important agricultural fungicides.[19] Interestingly,
during this process, the nitrogen-linked pharmacophore of
the cyrmenins has already been anticipated by synthetic
chemists (see, e.g., 6),[20] but it was not developed further.
With their two adjacent didehydroamino acids, the cyr-
menins represent a unique series of natural products. Al-
though the O-methyl-didehydroserine unit is not known to
exist in other natural products, its carbon-atom backbone
is identical to the well-known β-methoxyacrylate pharmac-
ophore of two related families of natural fungicides. The
strobilurins (e.g., A, 5) and oudemansins[14] were discovered
in 1976 by Steglich and Anke as new class of fungicides.
They have in common a β-methoxyacrylate unit that is
linked to the rest of the molecule by the α-carbon atom, and
they are all produced by higher fungi. The second family
is produced by myxobacteria and comprises myxothiazoles
Experimental Section
General: UV: Shimadzu UV/Vis scanning spectrometer UV-2102,
ethanol as solvent [Uvasol, Merck]. IR: Nicolet 20 DXB FTIR
spectrometer. NMR: Bruker DMX 600 (1H: 600.1 MHz; 13C:
150.9 MHz), ARX 400 (1H: 400.1 MHz; 13C: 100.6 MHz), AM 300
(1H: 300.1 MHz; 13C: 75.5 MHz) spectrometer; the internal stand-
ard was the signal of the solvent. Mass spectrometry (DCI): Finni-
(e.g., Z, 7),[15] melithiazoles[16] (ϭ cystothiazols[17]), and gan MAT 95 spectrometer (DCI with isobutane), resolution M/
haliangicin.[18] They are characterized by the presence of a
β-methoxyacrylamide or methyl ester unit, which is linked
∆M ϭ 1000; high-resolution data from peak matching (M/∆M ϭ
10000).
by the β-carbon atom to the rest of the molecule. Both the
Isolation of Cyrmenins: The adsorber resin was harvested by centri-
α- and β-linked acrylates were identified by Jagow et al.[4c]
fugation of the fermentation batch (300 L) of the Archangium ge-
to be inhibitors of mitochondrial respiration at the
phyra strain Ar 9944 grown in the presence of Amberlite XAD 16
cytochrome bc1 complex. Most remarkably, the cyrmenins (2 L). The collected cell mass and adsorber resin (7.48 kg) were
Eur. J. Org. Chem. 2004, 431Ϫ435
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
433

T. Leibold, F. Sasse, H. Reichenbach, G. Höfle
FULL PAPER
extracted with methanol (7 ϫ 2.5 L, total 17.5 L). The aqueous (s), 1496 (s) cmϪ1. DCI MS (ϩ, NH3): m/z (%) ϭ 410 (100) [M ϩ
phase (2.25 L) that remained after evaporation of the organic sol-
vent, was adjusted to pH ϭ 7 and extracted four times with ethyl
acetate (5 L in all). The organic layer was dried with magnesium
sulfate and the solvents were evaporated. To remove the lipophilic
components, the residue (80 g) was partitioned between methanol
containing 3% water (800 mL) and heptane (3 ϫ 800 mL). Concen-
tration of the polar layer yielded the raw product (53 g), which was
separated by silica gel chromatography [LiChroprep Si 100 silica
gel (540 g), 63Ϫ200 µm (Merck), 9 ϫ 60 cm; eluents: CH2Cl2 (4 L)
and CH2Cl2/diethyl ether (95:5; 6 L)]. After HPLC analysis, the
fractions containing cyrmenins (1Ϫ3) were combined and concen-
trated to dryness (residue 2.55 g). LH-20 chromatography with di-
chloromethane/methanol (4:1) as solvent removed the major part
of the impurities to yield the cyrmenin complex (1.40 g). Finally,
NH4]ϩ. HR-DCI MS: C21H36N3O5 [M ϩ NH4]ϩ: calcd. 410.2655;
found 410.2678. tR ϭ 4.32 min (CH3CN/5 m NH4OAc buffer,
63:37).
Synthesis of Reference Compounds
Synthesis of Methyl (Z)-2-Benzoylamino-3-methoxyacrylate (4) and
Methyl 2-Benzoylamino-3,3-dimethoxypropanoate: A solution of 4-
methoxymethylene-2-phenyl-4H-oxazol-5-one[7a] (50 mg, 0.25
mmol) and 4-DMAP (10 mg, 82 µmol, 0.33 equiv.) in MeOH (abs.;
2.5 mL) was stirred at room temp. for 2 h. The solvent was evapo-
rated in vacuo and the resulting oil was purified by chromatography
(silica gel; CH2Cl2/Et2O, 9:1) to yield 4 as colorless crystals (8.6 mg,
37 µmol, 15%) and the acetal as a colorless solid (51 mg,
0.19 mmol, 76%).
˚
RP-MPLC separation [3 portions, HD-Sil, 60 A, 20 µm (YMC); 6
1
4: M.p. 152Ϫ154 °C (benzene) [ref.[21] 155Ϫ156 °C (benzene)]. H
ϫ 50 cm; eluent: CH3CN/50 m NH4OAc buffer; detection: UV
absorption at 277 nm] of this mixture furnished cyrmenin A [1;
440 mg (corresponding to 1.47 mg/mL of fermentation broth)], cyr-
menin B1 [2; 492 mg (corresponding to 1.64 mg/mL of fermen-
tation broth)], and cyrmenin B2 [3; 60 mg (corresponding to
0.20 mg/mL of fermentation broth)] as colorless oils.
NMR (CDCl3, 300.1 MHz): δ ϭ 3.72 (s, 3 H, 1ЈЈ-H), 3.86 (s, 3 H,
4-H), 7.17 (br. s, 1 H, NH), 7.31 (s, 1 H, 3-H), 7.41 (m, 2-H, 4Ј-H),
7.48 (m, 1-H, 5Ј-H), 7.83 (m, 2 H, 3Ј-H) ppm. 13C NMR (CDCl3,
75.5 MHz): δ ϭ 51.8 (q, C-1ЈЈ), 62.1 (q, C-4), 107.5 (s, C-2), 127.5/
128.5 (2 d, C-3Ј/C-4Ј), 131.7 (d, C-5Ј), 134.0 (s, C-2Ј), 154.8 (d, C-
3), 165.5 (s, C-1Ј), 165.6 (s, C-1) ppm.
Cyrmenin A (1): UV (ethanol): λmax. (lg ε) ϭ 256 (4.28) nm. IR
(KBr): ν˜ ϭ 3377 (w), 2953 (m), 2926 (m), 2854 (w), 1720 (m), 1658
Methyl 2-Benzoylamino-3,3-dimethoxypropanoate: 1H NMR
(CDCl3, 400 MHz): δ ϭ 3.43 (s, 6 H, 4-H), 3.76 (s, 3 H, 1ЈЈ-H),
4.68 (d, J ϭ 3.4 Hz, 1 H, 3-H), 5.03 (dd, J ϭ 3.3, 8.4 Hz, 1 H, 2-
H), 6.85 (br. d, J ϭ 8.2 Hz, 1 H, NH), 7.44 (m, 3 H, 4Ј-H, 5Ј-H),
7.80 (m, 2 H, 3Ј-H) ppm. 13C NMR (CDCl3, 100.6 MHz): δ ϭ 52.5
(q, C-1ЈЈ), 54.4 (d, C-2), 55.7 (q, C-4), 103.8 (d, C-3), 127.2 (d, C-
3Ј), 128.5 /131.8 (2 d, C-4Ј/C-5Ј), 133.7 (s, C-2Ј), 167.5 (s, C-1Ј),
169.7 (s, C-1) ppm.
(s), 1496 (s) cmϪ1 1H NMR (400.1 MHz, CDCl3): δ ϭ 0.85 (d,
.
J ϭ 6.6 Hz, 6 H, 18-H3/19-H3), 1.14 (m, 2 H, 16-H), 1.27 (m, 4 H,
14-H/15-H), 1.40 (m, 2 H, 13-H), 1.50 (m, J ϭ 6.6 Hz, 1 H, 17-H),
1.99 (s, 3 H, 8-CH3), 2.28 (m, J ϭ 1.6, 7.2 Hz, 2 H, 12-H), 3.76 (s,
3 H, 1-OCH3), 3.91 (s, 3 H, 2Ј-OCH3), 5.39 (br. s, 1 H, 5Ј-Hb), 5.80
(dt, J ϭ 7.2, 10.7 Hz, 1 H, 11-H), 6.24 (m, J ϭ 1.6, 10.7, 11.8 Hz,
1 H, 10-H), 6.67 (br. s, 1 H, 5Ј-Ha), 7.08 (br. s, 1 H, 3-H), 7.30 (br.
d, J ϭ 11.7 Hz, 1 H, 9-H), 7.33 (s, 1 H, 2Ј-H), 8.50 (br. s, 1 H, 6-
H) ppm. 13C NMR (100.6 MHz, CDCl3): δ ϭ 167.7 (s, C-7), 165.0
(s, C-1), 162.6 (s, C-4), 155.0 (d, C-2Ј), 139.8 (d, C-11), 134.2 (s, C-
5), 129.8 (d, C-9), 129.5 (d, C-8), 123.4 (d, C-10), 106.7 (s, C-2),
102.2 (t, C-5Ј), 62.4 (q, 2Ј-OCH3), 52.0 (q, 1-OCH3), 38.9 (t, C-16),
29.5 (2 t, C-13 / C-14), 28.2 (t, C-12), 27.9 (d, C-17), 27.3 (t, C-15),
22.6 (q, C-18), 12.5 (q, 8-CH3) ppm. DCI MS (ϩ, NH3): m/z (%) ϭ
424 (100) [M ϩ NH4]ϩ. HR-DCI MS: C22H38N3O5: calcd.
424.2811 [M ϩ NH4]ϩ; found 424.2821. tR ϭ 6.29 min (CH3CN/
5 m NH4OAc buffer, 63:37).
Photoisomerization of Methyl (Z)-2-Benzoylamino-3-methoxyacry-
late (4): A solution of methyl (Z)-2-benzoylamino-3-methoxyacry-
late (4) (20 mg, 85 µmol) and benzophenone (2.0 mg, 11 µmol) in
degassed MeOH (abs.; 2.0 mL) was irradiated in a quartz vial for
3 ϫ 30 min at room temp. using a high-pressure mercury vapor
lamp (Philips HPK, 125 W). After the equilibrium was reached, at
a (Z)/(E) ratio of ca. 4:1, the solvent was evaporated in vacuo and
the resulting oil was purified by preparative HPLC (VP, 250/21 Nu-
cleodur 100Ϫ10 C18 EC; CH3CN/50 m NH4OAc buffer, 1:9). A
mixture of (Z) isomer 4 and (E) isomer 4Ј (19 mg, 93%) was iso-
lated.
Cyrmenin B1 (2): UV (ethanol): λmax. (lg ε) ϭ 258.0 (4.22) nm. IR
(KBr): ν˜ ϭ 3380 (w), 2927 (m), 2854 (w), 1719 (m), 1658 (s), 1496
1
4Ј: H NMR (CDCl3, 300.1 MHz): δ ϭ 3.83 (s, 3 H, 1ЈЈ-H), 3.89
1
(s) cmϪ1. H NMR (400.1 MHz, CDCl3): δ ϭ 0.87 (t, J ϭ 7.1 Hz,
(s, 3 H, 4-H), 7.41 (m, 2-H, 4Ј-H), 7.48 (m, 1-H, 5Ј-H), 7.79 (m, 2
H, 3Ј-H), 8.08 (s, 1 H, 3-H) ppm. 13C NMR (CDCl3, 75.5 MHz):
δ ϭ 52.3 (q, C-1ЈЈ), 62.8 (q, C-4), 108.7 (s, C-2), 126.9/128.7 (2 d,
C-3Ј/C-4Ј), 131.6 (d, C-5Ј), 134.4 (s, C-2Ј), 155.3 (d, C-3), 164.6 (s,
C-1Ј), 165.6 (s, C-1) ppm.
3 H, 18-H), 1.25 (m, 8 H, 14-H/15-H/16-H/17-H), 1.37 (m, 2 H,
13-H), 1.99 (d, J ϭ 1.4 Hz, 3 H, 8-CH3), 2.28 (dq, J ϭ 1.7, 7.4 Hz,
2 H, 12-H), 3.76 (s, 3 H, 1-OCH3), 3.91 (s, 3 H, 2Ј-OCH3), 5.39 (t,
J ϭ 1.6 Hz, 1 H, 5Ј-Hb), 5.80 (m, J ϭ 0.8, 7.6, 11.3 Hz, 1 H, 11-
H), 6.24 (m, J ϭ 1.7, 11.1, 11.4 Hz, 1 H, 10-H), 6.66 (d, J ϭ 1.7 Hz,
1 H, 5Ј-Ha), 7.08 (br. s, 1 H, 3-H), 7.30 (m, J ϭ 1.4, 1.5, 11.8 Hz,
1 H, 9-H), 7.32 (s, 1 H, 2Ј-H), 8.49 (br. s, 1 H, 6-H) ppm. 13C
NMR (100.6 MHz, CDCl3): δ ϭ 167.8 (s, C-7), 165.2 (s, C-1), 162.8
(s, C-4), 155.4 (d, C-2Ј), 139.8 (d, C-11), 134.1 (s, C-5), 129.8 (d,
C-9), 129.6 (d, C-8), 123.4 (d, C-10), 106.7 (s, C-2), 102.4 (t, C-5Ј),
62.3 (q, 2Ј-OCH3), 51.9 (q, 1-OCH3), 31.8 (t, C-16), 29.5 (t, C-13),
29.2 (t, C-14), 29.1 (t, C-15), 28.1 (t, C-12), 22.6 (t, C-17), 14.0 (q,
C-18), 12.4 (q, 8-CH3) ppm. DCI MS (ϩ, NH3): m/z (%) ϭ 393
(1) [M ϩ H]ϩ, 410 (100) [M ϩ NH4]ϩ. HR-DCI MS: C21H36N3O5
[M ϩ NH4]ϩ: calcd. 410.2655; found 410.2701. tR ϭ 4.74 min
(CH3CN/5 m NH4OAc buffer, 63:37).
Acknowledgments
We thank B. Grek and D. Geier for their skillful assistance in the
isolation work, A. Roß, H. Schüler, and R. Krützfeld and co-work-
ers for their contributions in the large-scale fermentation and
downstream processing, Dr. V. Wray, B. Jaschok-Kentner, and C.
Kakoschke for measuring NMR spectra, and R. Christ for measur-
ing mass spectra.
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www.eurjoc.org Eur. J. Org. Chem. 2004, 431Ϫ435

Cyrmenins, Novel Antifungal Peptides
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Received June 18, 2003
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