Cyclic lipodepsipeptides verlamelin A and B, isolated from entomopathogenic fungus Lecanicillium sp.

Article abstract of DOI:10.1038/ja.2014.22

Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomopathogenic fungus Lecanicillium sp. HF627. As the structural elucidation of verlamelin so far was only preliminary, we studied and determined the absolute structure of these two compounds to be cyclo(5S-hydroxytetradecanoic acid-D-alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-Val). This is the first study that precisely analyzed the structure of verlamelin.

Full text of DOI:10.1038/ja.2014.22

The Journal of Antibiotics (2014) 67, 459–463
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
www.nature.com/ja
&
ORIGINAL ARTICLE
Cyclic lipodepsipeptides verlamelin A and B, isolated
from entomopathogenic fungus Lecanicillium sp.
Kei-ichi Ishidoh1, Hiroshi Kinoshita1, Yasuhiro Igarashi2, Fumio Ihara3 and Takuya Nihira1,4
Verlamelin and its new derivative (verlamelin B) were isolated from fermentation broth of entomopathogenic fungus
Lecanicillium sp. HF627. As the structural elucidation of verlamelin so far was only preliminary, we studied and determined the
absolute structure of these two compounds to be cyclo(5S-hydroxytetradecanoic acid-D-alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-
Val). This is the first study that precisely analyzed the structure of verlamelin.
The Journal of Antibiotics (2014) 67, 459–463; doi:10.1038/ja.2014.22; published online 2 April 2014
Keywords: cyclic lipodepsipeptide; entomopathogenic fungi; Lecanicillium; Verticillium lecanii
INTRODUCTION chromatography, C18 column chromatography and preparative
Entomopathogenic fungi are fungal parasites of insects. During their reverse phase HPLCs.
infection to insects, they are known to evade insect immunities and
Both major compound 1 and minor compound 2 were obtained as
kill the host insects by secreting several bioactive substances.1–3 As the colorless solid. The molecular formula of 1 was identified as
high productivity has been well established by continuous discovery of C45H71N7O11 based on high-resolution fast atom bombardment MS
novel bioactive compounds,4–6 entomopathogenic fungi have been (HRFABMS) (observed m/z 886.5289 [M þ H]þ , calcd 886.5290 for
regarded as a source of bioactive compounds. Especially, those C45H72N7O11). The molecular formula of 2 was C44H69N7O11
belonging to the genus Lecanicillium (formerly Verticillium lecanii) (HRFABMS, observed m/z 872.5140 [M þ H]þ , calcd 872.5134 for
can be good source of novel compounds, as exemplified by the C44H70N7O11), suggesting that 2 is a one-methylene-shorter derivative
isolation of indolosesquiterpenes (lecanindoles A–D7), phenopicolinic of 1, considering the almost identical UV/Vis spectrum (lmax 224,
acid derivatives (vertilecanins8) and pregnanes.9,10
Here we would like to report isolation and structure determination
278 nm).
All the 13C signals and all the 1H signals were assigned using
of two compounds, verlamelin (verlamelin A, 1) and it new derivative DEPT135, HSQC, COSY and HMBC (Table 1). 13C NMR of the two
(verlamelin B, 2) from entomopathogenic fungus Lecanicillium sp. compounds detected eight carbonyl carbons in the range of dC
Although the planar structure of verlamelin has already been 170–174, and 1H NMR in DMSO-d6 revealed eight broad resonances,
reported, its structure determination was only preliminary, because which were not detectable in the measurement using methanol-d4,
these reports lacked detailed information on the signal assignments of suggesting that certain numbers of amide linkages were present in
1H/13C, the signal connectivity in HSQC/HMBC and the D/L these compounds. Overlapping methylene signals in the high mag-
configuration of the component amino acids.11–13 Furthermore, the netic field suggested that both compounds have an alkyl chain. Based
configuration of a hydroxyl group on the 5-hydroxytetradecanoic acid on these data, 1 and 2 were assumed to bear typical lipopeptide
moiety has not yet been determined. Therefore, in order to substructures. By COSY and HMBC correlations, the component
completely establish the structure of verlamelin, we conducted a amino acids were estimated to be valine (Val), alanine (Ala), tyrosine
detailed structure determination of 1 and 2.
(Tyr), threonine (Thr) (1) or serine (Ser) (2), glutamine (Gln) and
proline (Pro). The remaining substructure was deduced to be a
5-hydroxytetradecanoic acid moiety by comparison of the dc values of
C-2 to C-6 with those on cordycommunin, which has the
RESULTS AND DISCUSSIONS
Lecanicillium sp.
HF627 was cultivated under static conditions in liquid medium 5-hydroxytetradecanoic acid substructure.14 As dH of the
containing sucrose 8%, yeast extract 4%, CaCO3 0.5% and HP20 1%. oxymethine H-5 was detected relatively downfield (4.79 (1), 4.77
The whole broth was extracted with n-butanol. Compounds 1 and 2 (2)), the C-5 was judged to be involved in ester bonding. The
were purified by
a series of steps with silica gel column connecting sequence of components was finally determined as
1International Center for Biotechnology, Osaka University, Osaka, Japan; 2Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan; 3National Institute of
Fruit Tree Science, Ibaraki, Japan and 4Mahidol University–Osaka University Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol
University, Bangkok, Thailand
Correspondence: Professor T Nihira, Mahidol University–Osaka University Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol
University, 272 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand.
E-mail:nihira@icb.osaka-u.ac.jp
Received 19 November 2013; revised 26 February 2014; accepted 5 March 2014; published online 2 April 2014

Verlamelin A and B
K-i Ishidoh et al
460
Table 1 NMR spectroscopic data (400MHz, DMSO-d6) for verlamelin A (1) and verlamelin B (2)
Verlamelin A (1)
Verlamelin B (2)
Position
dC, mult.
dH, mult. (J in Hz)
Position
dC, mult.
dH, mult. (J in Hz)
L-Val
D-Tyr
L-Val
D-Tyr
1
2
3
4
4’
NH
170.8, qC
59.4, CH
29.2, CH
18.5, CH3
19, CH3
—
—
1
2
3
4
5
170.8, qC
59.3, CH
29.2, CH
18.5, CH3
19, CH3
—
—
3.84, t (7.3)
1.91–2.04,b
0.82, d (6.4)
0.84, d (6.4)
8.46, bd (6.9)
3.86, t (7.3)
1.97–2.02,i m
0.81, d (6.4)
0.84, d (6.4)
8.41, bd (6.9)
m
NH
1
2
3
4
5, 9
6, 8
7
7-OH
NH
172.7, qC
54.2, CH
38.0, CH2
127.5, qC
130.3, CH
114.8, CH
155.8, qC
—
—
4.61, m
2.75, m
—
7.04, d (8.7)
6.60, d (8.7)
—
1
2
3
172.5, qC
54.0, CH
38.1, CH2
127.5, qC
130.3, CH
114.8, CH
155.8, qC
—
—
4.64, m
2.73, m
—
7.02, d (8.2)
6.59, d(8.2)
—
4
5, 9
6, 8
7
7-OH
NH
9.09, bs
7.50, bd (9.6)
9.11, bs
7.55, bd (8.1)
—
—
L-Gln
L-Pro
L-Gln
L-Pro
1
2
3
4
171.0, qC
52.7, CH
26.8, CH2
31.8, CH2
173.7, qC
—
—
4.04, m
1
2
3
170.9, qC
52.6, CH
27.1, CH2
31.8, CH2
173.8, qC
—
—
4.09, m
1.71, m; 1.89, m
1.97–2.02,i m
6.74, bs; 7.21, bs
7.75, bd (8.2)
1.71, m; 1.91–2.04,b
m
1.91–2.04,b
6.74, bs; 7.21, bs
7.78, bd (8.2)
m
4
CONH2
NH
CONH2
NH
1
2
3
4
5
170.9, qC
60.2, CH
29.2, CH2
24.2, CH2
46.8, CH2
—
4.33, m
1
2
3
4
5
171.0, qC
60.1, CH
29.2, CH2
24.2, CH2
46.7, CH2
—
4.33, m
1.81,j m; 2.09, m
1.81,j m
1.83–1.84,d m; 2.11–2.16,c
m
1.83–1.84,d
m
3.48, m; 3.62, m
3.45, m; 3.54, m
D-Ala
D-Ala
D-Ser
1
2
3
NH
171.0, qC
46.9, CH
16.3, CH3
—
—
4.48, m
1.18, d (6.4)
7.80, bd (6.4)
1
2
3
170.6, qC
47.0, CH
16.2, CH3
—
—
4.48, m
1.15, d (6.4)
7.76, bd (6.4)
NH
D-allo-Thr
1
2
3
4
3-OH
NH
170.5, qC
58.3, CH
66.4, CH
20.0, CH3
—
—
1
2
3
3-OH
NH
170.6, qC
55.3, CH
61.3, CH2
—
—
4.27, m
3.64, m
5.27, bt (4.6)
7.63, bd (7.7)
4.20, dd (7.1, 8.7)
3.99, m
1.08, d (6.4)
5.27, bs
—
—
7.48, bd (9.2)
5(S)-hydroxytetradecanoic acid
172.2, qC
5(S)-hydroxytetradecanoic acid
172.6, qC
1
2
3
4
5
6
7
8
—
1
2
3
4
5
6
7
8
9
10
11
12
13
14
—
34.7, CH2
19.8, CH2
32.7, CH2
73.2, CH
1.91–2.04,b m; 2.11–2.16,c
m
34.3, CH2
19.8, CH2
32.6, CH2
73.2, CH
1.97–2.02,i m; 2.21, m
1.33–1.35,e
m
1.33–1.37,k
m
1.33–1.35,e m; 1.42–1.46,f m
4.79, m
1.33–1.37,k m; 1.42–1.44,l m
4.77, m
33.5, CH2
25.0, CH2
29.0,a CH2
28.9,a CH2
28.7,a CH2
28.7,a CH2
31.3, CH2
22.1, CH2
14.0, CH3
1.42–1.46,f m
33.6, CH2
24.9, CH2
29.0,h CH2
28.9,h CH2
28.7,h CH2
28.7,h CH2
31.3, CH2
22.1, CH2
14.0, CH3
1.42–1.44,l m
1.20–1.23,g
1.20–1.23,g
1.20–1.23,g
1.20–1.23,g
1.20–1.23,g
1.20–1.23,g
1.20–1.23,g
m
m
m
m
m
m
m
1.20–1.23,m
1.20–1.23,m
1.20–1.23,m
1.20–1.23,m
1.20–1.23,m
1.20–1.23,m
1.20–1.23,m
m
m
m
m
m
m
m
9
10
11
12
13
14
0.83, t (6.9)
0.83, t (6.9)
a,hCarbon chemical shifts may be interchanged.
b,c,d,e,f,g,i,j,k,l,mThe proton resonances are overlapped.
5-hydroxytetradecanoic acid, Thr (1) or Ser (2), Ala, Pro, Gln, Tyr hydrolysate was labeled by Marfey’s reagent (Na-(5-Fluoro-2,4-
and Val, based on HMBC and ROESY (1) and NOESY (2) dinitrophenyl)-L-alaninamide). The retention times of the labeled
correlation. HMBC correlation of the oxymethine on 5- products in C18 HPLC (29.3, 30.2, 34.5, 37.3, 40.4, 42.1, 62.2min for
hydroxytetradecanoic acid with C-1 on Val demonstrated the verlamelin A; and 26.0, 30.2, 34.6, 37.3, 40.5, 42.2, 62.2min for
formation of a cyclic structure (Figure 1). Thus, the compound 2 verlamelin B) indicated that the component amino acids were L-Gln,
was proposed to be a new analog of verlamelin (verlamelin B) in L-Pro, D-Ala, L-Val, D-Tyr and D-allo-Thr (verlamelin A) or D-Ser
which Ser was replaced with Thr.
To determine the absolute configurations in the peptide moiety of
(verlamelin B) (see ‘Experimental procedure’ for standards).
As for the absolute configuration of C-5 in the 5-hydroxytetrade-
the verlamelins, Marfey’s analysis was conducted.15 After degradation canoic acid moiety, a modified Mosher’s method was used.16,17 After
of verlamelin A and verlamelin B under acidic conditions, the the reactive phenolic hydroxyl group was protected by methyl iodide
The Journal of Antibiotics

Verlamelin A and B
K-i Ishidoh et al
461
HMBC
HMBC
NOESY
ROESY
verlamelin A (1)
verlamelin B (2)
Figure 1 Key HMBC, ROESY (1) and NOESY (2) correlation.
(R) - (S) (ppm)
+ 0.45
+ 0.26 - 0.31
+ 0.49
+ 0.57
+ 0.71
- 0.79
- 0.86
O
O
MαNP
(R) - (S) (ppm)
+ 0.28 - 0.29
+ 0.47
R = CH3 (1)
R = H (2)
+ 0.64
+ 0.85
- 0.84
O
O
Figure 3 Stereochemical structure of verlamelin A (1) and B (2), refined in
this study.
MαNP
The potency of both compounds was equal when tested against
Cochliobolus miyabeanus and Alternalia solani. However, the new
derivative of verlamelin, 2 was less active against Fusarium oxysporum,
Cladosporium cucumerinum and Ustilago maydis (potency of 2: 4, 0.5
Figure 2 Partial (R)—(S) values of derivatives of (1) and (2) synthesized by
a modified Mosher’s method.
and the macrolactone ring was cleaved by methanolysis, the reaction and 464mg per disc, respectively), compared with 1 (potency of 1: 2,
products (purified by C18 HPLC) showed m/z at 932.5 and 918.5, 0.25 and 16mg per disc, respectively), suggesting that the methyl
respectively, suggesting that methanolysis proceeded successfully. group on the first amino-acid residue connected to the fatty acid
Reaction of the product from 1 with (R)- or (S)-MaNP acid moiety should have an important role for antifungal activities against
(M.W.: 230.2) gave products (purified by C18 HPLC) of m/z at these fungi. As antifungal spectrum of 1 agreed well with that of the
1144.6, implying that the esterification with MaNP occurred at a verlamelin reported previously,13 1 might possess identical absolute
single position. Similar esterification reaction of the product from 2 configuration to that of the original verlamelin.
gave products of m/z 1342.7, suggesting that the two molecules of
By detailed structure determination, the absolute structures of 1
MaNP were incorporated. Subsequent partial hydrolysis with K2CO3 and 2 were identified for the first time; they were found to consist
resulted in the loss of one MaNP at the primary alcohol of the Ser of a cyclic lipodepsipeptide, cyclo(5S-hydroxytetradecanoic acid-D-
residue and yielded a product of m/z at 1112.6, corresponding to the alloThr/Ser-D-Ala-L-Pro-L-Gln-D-Tyr-L-Val) (Figure 3). The related
monoester with an MaNP. Each monoester with an MaNP was compounds,
W493
A/B,
cyclo(3S-hydroxy-4R-methyltetra-
1
analyzed by H NMR and COSY to observe the anisotropic effect as decanoic acid-D-alloThr-L-Ala-D-Ala-L-Gln-D-Tyr-L-Val/Ile), from
the result of shielding by the MaNP group. The Dd (dR-dS) values of Fusarium sp.,18 and cordycommunin, cyclo(5-hydroxytetradecanoic
the Mosher’s esters indicated the 5-S configuration (Figure 2). Based acid-D-alloThr-L-Ala-L-Ala-L-Gln-L-Tyr-L-Val), from Ophiocordyceps
on these results, the absolute structures of verlamelin A and verlamelin communis,14 have also similar partial structure to fengycin (plilastatin
B were definitively determined for the first time, as shown in Figure 3. A1, inhibitor of phospholipase A2), which was isolated from Bacillus
Based on the previous results of in vitro antifungal activity of subtilis as an antifungal lipopeptide compound containing a cyclic
verlamelin,11–13 we measured the antifungal activity of 1 and 2 by disc peptide moiety composed of L-Tyr-D-alloThr-L-Glu-D-Ala-L-Pro-L-
diffusion method (summary of results, Supplementary Figure S11). Gln-D-Tyr-L-Ile.19,20 Judging from the correlation of the structure
The Journal of Antibiotics

Verlamelin A and B
K-i Ishidoh et al
462
and the antifungal activity among these cyclic peptide compounds, Stereochemistry of amino acids constituting verlamelin A and B
One milligram of aliquots of 1 or 2 were hydrolyzed by heating at 115 1C for
8 h in 10ml of 6 M HCl. After cooling to room temperature, they
were completely dried in vacuo and dissolved in 150 ml of water. Marfey’s
reagent (300 ml of 10mgmlꢁ1 solution in acetone) (Na-(2,4-dinitro-5-fluor-
ophenyl)-L-alaninamide) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)
was added, followed by the addition of 70ml of 1 M NaHCO3. The reactions
proceeded at 371C for 1 h and were quenched by addition of 70ml of 1 M HCl.
The resulting mixture was dried in vacuo and then dissolved in 1 ml of DMSO
to be analyzed by HPLC. Marfey’s derivatives of amino acids as a standard were
prepared by reacting 50 mM of amino acids in the same manner as described
above. HPLC analysis was carried out on a Shiseido Capcell-Pak C18 column
(Ø4.6ꢀ 250 mm) with a linear gradient of CH3CN from 10% to 50% in 0.05%
aqueous TFA solution (60 min from 10% to 50%, 5 min at 50%, 3 min from
50% to 10%), at a flow rate of 1.0 mlminꢁ1, with detection at 340 nm. The
glutamine (Gln) residues of 1 and 2 should be converted by acid hydrolysis to
glutamic acid (Glu). Retention times (min) of Marfey’s derivatives used as
standards were as follows: L-Ser (25.6), D-Ser (26.0), L-Thr (27.0), D-Thr
(31.8), L-alloThr (27.5), D-alloThr (29.3), L-Glu (30.1), D-Glu (31.9), L-Pro
(34.3), D-Pro (36.0), L-Ala (33.2), D-Ala (37.2), L-Val (42.1), D-Val (48.1),
L-Tyr (57.9) and D-Tyr (40.4, 62.2).
D-tyrosine after L-glutamine might be critical for antifungal activities
because cordycommunin, which possesses L-Tyr instead of D-Tyr, did
not show activity against Candida albicans and Magnaporthe grisea.14
EXPERIMENTAL PROCEDURE
General experimental procedure
NMR spectra were recorded on a JEOL JMN-ECS400 (JEOL, Tokyo, Japan) at
1
400 MHz for H and 100MHz for 13C. The 1H and 13C chemical shifts were
referenced to the solvent signal (dH 2.49 and dC 39.5 in dimethyl sulfoxide
(DMSO)-d6, dH 3.31 in CD3OD). HRFABMS were recorded on a JEOL JMS-
700 spectrometer. Optical rotation was measured on a P-1020 polarimeter
(Jasco, Tokyo, Japan). The UV spectrum was recorded on a Hitachi U-3200
spectrophotometer (Hitachi, Tokyo, Japan).
Fungal material
The entomopathogenic fungus Lecanicillium sp. HF627 was isolated from a
chillie thrips cadaver. Fungal conidia that had developed on the surface of the
cadaver were transferred onto solid Sabouraud maltose yeast-extract (SMY)
medium (4% maltose, 1% yeast extract, 1% peptone) with 1.5% agar and
incubated at 251C for several days. After several rounds of single-colony
isolation, the isolated strain was identified as Verticillium lecanii (Lecanicillium
sp.) according to its morphology. The fungus is deposited at the culture
collection of Natural Institute of Fruit Tree Science.
(R)- and (S)-MaNP acid ester derivatives of 1 and 2
A modified Mosher’s method using (R)- and (S)-MaNP acid (Tokyo Chemical
Industry Co., Ltd.) was used to clarify the stereogenic centers of the
hydroxytetradecanoic acid moiety.16 To protect the hydroxyl group of the
Tyr residue in advance, methylation was carried out as follows. To 1 and 2
Fermentation and isolation
The seed culture was prepared by inoculating conidia of strain HF627 into dissolved in acetonitrile (10 mg per 500 ml) were added 50ml of 1,8-
50ml of SMY medium in a 500-ml baffled flask, followed by cultivation on a diazabicyclo[5.4.0]undec-7-ene and 50ml of CH3I. After incubation at 501C
rotatory shaker at 28 1C and 160 r.p.m. for 3 days. For the main cultivation, the
for 3 h and quenching by dilution with water, the reaction products were
seed culture (2ml) was inoculated into 100 ml of Medium #5 (8% sucrose, 4% extracted three times with an equal volume of ethyl acetate. The ethyl acetate
yeast extract, 0.5% CaCO3, 1% activated HP20 (Mitsubishi Chemical Co., layer was washed with 1 M HCl and brine, dried over anhydrous sodium
Tokyo, Japan) by methanol) in 500-ml Erlenmeyer flasks, followed by static sulfate, and concentrated in vacuo, yielding crude methyl ethers of 1 and 2.
incubation for 21 days at 251C. The culture broth (1liter) was mixed with n-
To cleave the ester linkage between hydroxytetradecanoic acid and Val
butanol (500ml) and stirred for 1 h, and the n-butanol layer was collected after residue, methanolysis was carried out as follows. Crude verlamelin methyl
centrifugation (3000r.p.m., 10 min), dried over anhydrous sodium sulfate and ethers were dissolved in 2 ml of 0.5 M methanolic sodium metoxide and stirred
concentrated in vacuo using a rotary evaporator, yielding 3 g of brown gum as for 5 h at room temperature. After quenching with 1 M HCl, extraction with
the crude extract. The crude extract was fractionated on a silica gel 60 (70–230 ethyl acetate (equal volume, three times) and evaporation in vacuo, the cleaved
mesh; Merck, Darmstadt, Germany) column (Ø30mm) by stepwise elution product and the uncleaved reactant were collected individually by reverse
with increasing ethyl acetate concentrations (hexane/ethyl acetate¼ 4:1, 2:1,
phase C18 HPLC using 85% methanol as eluent. The recovered reactant was
3:2, 1:1, 1:2, 1:4 and 0:10 v/v), followed by stepwise elution with increasing reused for the same reaction, and the product was obtained as described above.
methanol concentrations (ethyl acetate/methanol¼ 40:1, 20:1, 10:1, 8:1, 4:1, The methyl ethers of 1 and 2 yielded 6.2 and 4.2 mg of methanolysis products,
2:1, 1:1, and 0:10 v/v). Fractions containing verlamelins were detected by silica respectively.
gel TLC (ethyl acetate/methanol¼ 2:1, Rf 0.21), combined and evaporated in
vacuo until dryness. The residues were further fractionated with a Sep-Pak methanolysis product of
Silica 35cc Vac cartridge (10 g; Waters, Milford, MA, USA) by stepwise elution mixed with 2 mg of dimethyl amino pyridine, 3 mg of 1-ethyl-3-(3-dimethy-
laminopropyl) carbodiimide (EDC), 2ml of triethyl amine and 2.1 mg
(R)- and (S)-MaNP acid esters of 1 were prepared as follows. The
1
methyl ether (in 200 ml of CH2Cl2) was
with increasing methanol concentrations (ethyl acetate/methanol ¼ 2:1, 3:2,
1:1, 1:2, 1:4, and 0:10 v/v). Verlamelins in the Sep-Pak fractions were further of (R)-MaNP acid or 2.1 mg of (S)-MaNP acid. After standing at room
separated by preparative reverse phase HPLC with a Shiseido Capcell-Pak C18 temperature overnight and quenching by addition of water, the reaction
column (Ø10ꢀ 250 mm) (Shiseido Co., LTD., Tokyo, Japan) with isocratic
mixture was extracted three times with an equal volume of ethyl acetate.
After washing the ethyl acetate extract with 1 M HCl and brine, drying
elution using 80% methanol at a flow rate of 4 mlminꢁ1. The peaks of 2 and
1, detected at 11 and 12min by UV (210nm), respectively, were collected over anhydrous sodium sulfate and concentrating in vacuo, the (R)- and
individually and evaporated in vacuo to dryness, yielding 2.7 and 65.5mg of (S)-MaNP acid ester derivatives of (3a and 3b, respectively) were
colorless amorphous solid, respectively (chromatogram of HPLC, see purified by reverse phase HPLC on Shiseido Capcell-Pak C18
Supplementary Figure S1).
column (Ø10ꢀ 250 mm) with isocratic elution using 90% methanol as a
Additional quadruplicate experiments without the Sep-Pak Silica fractiona- mobile phase.
1
a
tion were carried out further to obtain 2. As a result, a total of 97.7mg of 2 was
obtained.
(R)- and (S)-MaNP acid ester derivatives of 2 (4a and 4b, respectively) were
synthesized and purified in a similar manner as those of 1.
Compound (1): a colorless solid; [a]23 þ 2.5 (c 0.01, MeOH); UV
Compund 3a: HRFABMS m/z 1144.6558 [Mþ H]þ (calcd for
D
1
(MeOH) lmax (log e) 224 (4.20), 278 (3.29) nm; HRFABMS m/z 886.5289 C61H90N7O14, 1144.6546). For H, see Supplementary Figures S6 and S7.
[Mþ H]þ (calcd for C45H72N7O11, 886.5290). For 1H, 13C, see Table 1 (see
Compund 3b: HRFABMS m/z 1144.6543 [Mþ H]þ (calcd for
1
also Supplementary Figures S2 and S3).
Compound (2): a colorless solid; [a]23 ꢁ68.5 (c 0.01, MeOH); UV
C61H90N7O14, 1144.6546). For H, see Supplementary Figures S6 and S8.
Compund 4a: HRFABMS m/z 1112.6274 [Mþ H]þ (calcd for
D
1
(MeOH) lmax (log e) 224 (4.12), 278 (3.26) nm; HRFABMS m/z 872.5140 C60H86N7O13, 1112.6283). For H, see Supplementary Figures S6 and S9.
[Mþ H]þ (calcd for C44H70N7O11, 872.5134). For 1H, 13C, see Table 1 (see
Compund 4b: HRFABMS m/z 1112.6284 [Mþ H]þ (calcd for
1
also Supplementary Figures S4 and S5).
C60H86N7O13, 1112.6283). For H, see Supplementary Figures S6 and S10.
The Journal of Antibiotics

Verlamelin A and B
K-i Ishidoh et al
463
Antifungal assay
7
8
Roll, D. M. et al. The Lecanindoles, nonsteroidal progestins from the terrestrial fungus
Verticillium lecanii 6144. J. Nat. Prod. 72, 1944–1948 (2009).
Soman, A. G., Gloer, J. B., Angawi, R. F., Wicklow, D. T. & Dowd, P. F. Vertilecanins:
new phenopicolinic acid analogues from Verticillium lecanii. J. Nat. Prod. 64,
189–192 (2001).
Claydon, N., Grove, J. F., Pople, M. & Begley, M. J. New metabolic products of
Verticillium lecanii. Part 1. 3b-Hydroxy-4, 4, 14a-trimethyl-5a-pregna-7, 9 (11)-diene-
20S-carboxylic acid and the isolation and characterisation of some minor metabolites.
J. Chem. Soc. Perkin Trans. 1, 497–502 (1984).
In vitro antifungal activity of 1 and 2 against six indicator strains (Fusarium
oxysporum subsp. cucumerinum NBRC 31224, Cladosporium cucumerinum
NBRC 6370, Aspergillus niger NBRC 105649, Alternaria solani NBRC 7516,
Cochliobolus miyabeanus NBRC 9633 and Ustilago maydis NBRC 6907) was
tested by disc diffusion method as follows. Paper discs loaded with 10 ml of
verlamelin solution in DMSO were placed on the potato dextrose agar (Difco,
Detroit, MI, USA) plate, which was homogeneously inoculated with conidia,
myceria or vegetative culture of the indicator strains in advance. Growth-
inhibitory effect was observed after incubation at 281C for 1–3 days,
depending on indicator strains. Nystatin (16mg per disc) and DMSO alone
were used as the positive and negative controls, respectively.
9
10 Grove, J. F. 23, 24, 25, 26, 27-Pentanorlanost-8-en-3b, 22-diol from Verticillium
lecanii. Phytochemistry 23, 1721–1723 (1984).
11 Onishi, J. C., Rowin, G. L. & Miller, J. E. Antibiotic A43F. United States Patent
4201771. May 6 (1980).
12 Kim, J. C. et al. Verlamelin, an antifungal compound produced by a mycoparasite
Acremonium strictum. Plant Pathol. J. 18, 102–105 (2002).
13 Rowin, G. L. et al. Verlamelin, a new antifungal agent. J. Antibiot. 39, 1772–1775
(1986).
14 Haritakun, R., Sappan, M., Suvannakad, R., Tasanathai, K.
& Isaka, M. An
antimycobacterial cyclodepsipeptide from the entomopathogenic fungus Ophiocordy-
ceps communis BCC 16475. J. Nat. Prod. 73, 75–78 (2010).
15 Bhushan, R. & Bruckner, H. Marfey’s regent for chiral amino acid analysis: a review.
Amino acids 27, 231–247 (2004).
1
2
3
Wang, B., Kang, Q., Lua, Y., Bai, L. & Wanga, C. Unveiling the biosynthetic puzzle of
destruxins in Metarhizium species. Proc. Natl Acad. Sci. USA 109, 1287–1292
(2012).
Xu, Y. et al. Biosynthesis of the cyclooligomer depsipeptide bassianolide, an
insecticidal virulence factor of Beauveria bassiana. Fungal Genet. Biol. 46,
353–364 (2009).
Xu, Y. et al. Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulence
factor of the entomopathogenic fungus Beauveria bassiana. Chem. Biol. 15, 898–907
(2008).
16 Kasai, Y. et al. MaNP acid,
a powerful chiral molecular tool for preparation of
enantiopure alcohols by resolution and determination of their absolute configurations
by the 1H NMR anisotropy method. Chirality 16, 569–585 (2004).
17 Kusumi, T., Yabuuchi, T., Takahashi, H. & Ooi, T. Chiral anisotropic reagents for
determining the absolute configulation of secondary alcohols and carboxylic acids. Yuki
Gosei Kagaku Kyokaishi 63, 1102–1114 (2005).
4
5
Azumi, M. et al. Aurovertins F–H from the entomopathogenic fungus Metarhizium
anisopliae. J. Nat. Prod. 71, 278–280 (2008).
Isaka, M., Palasarn, S., Tobwor, P., Boonruangprapa, T. & Tasanathai, K. Bioactive
anthraquinone dimers from the leafhopper pathogenic fungus Torrubiella sp. BCC
28517. J. Antibiot. 65, 571–574 (2012).
18 Nihei, K., Itoh, H., Hashimoto, K., Miyairi, K. & Okuno, T. Antifungal cyclodepsipep-
tides, W493 A and B, from Fusarium sp.: isolation and structural determination.
Biosci. Biotechnol. Biochem. 62, 858–863 (1998).
19 Honma, M. et al. Termination of the structural confusion between plipastatin A1 and
fengycin IX. Bioorg. Med. Chem. 20, 3793–3798 (2012).
6
Putri, S. P., Kinoshita, H., Ihara, F., Igarashi, Y. & Nihira, T. Farinomalein, a maleimide-
bearing compound from the entomopathogenic fungus Paecilomyces farinosus. J. Nat.
Prod. 72, 1544–1546 (2009).
20 Samel, S. A., Wagner, B., Marahiel, M. A. & Essen, L. O. The thioesterase domain of
the fengycin biosynthesis cluster: a structural base for the macrocyclization of a non-
ribosomal lipopeptide. J. Mol. Biol. 359, 876–889 (2006).
Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics