Asperelines A-F, peptaibols from the marine-derived fungus Trichoderma asperellum

Article abstract of DOI:10.1021/np900190w

Fermentation of the marine-derived fungus Trichoderma asperellum, collected from the sediment of the Antarctic Penguin Island, resulted in the isolation of six new peptaibols named asperelines A-F (1-6), which are characterized by an acetylated N-terminus and a C-terminus containing an uncommon prolinol residue. Structures were determined by extensive 1D and 2D NMR (¹H- ¹H COSY, HMQC, HMBC, NOESY) spectroscopic data analysis combined with ESIMS/MS fragmentation. The absolute configurations of the amino acid residues possessing a chiral R-carbon and of the prolinol residue were determined to be L and S, respectively, using a new method of ¹H NMR spectroscopic comparison of complexes formed between the chiral reagent Ru(D ₄-Por*)CO and amino acids derived from the peptaibols with those formed with reference standards.

Full text of DOI:10.1021/np900190w

1036
J. Nat. Prod. 2009, 72, 1036–1044
Asperelines A-F, Peptaibols from the Marine-Derived Fungus Trichoderma asperellum
Jinwei Ren,† Chunmei Xue,† Li Tian,‡ Minjuan Xu,† Jian Chen,§ Zhiwei Deng,⊥ Peter Proksch,| and Wenhan Lin*,†
State Key Laboratory of Natural and Biomimetic Drugs, Peking UniVersity, Beijing 100083, People’s Republic of China, Biological
Department, Qingdao UniVersity of Science & Technology, Qingdao 266042, People’s Republic of China, Analytical and Testing Center,
Beijing Normal UniVersity, Beijing, 100875, People’s Republic of China, Institut fu¨r Pharmazeutische Biologie, Heinrich-Heine-UniVersita¨t,
Geb. 26.23, UniVersita¨tsstrasse 1, D-40225 Du¨sseldorf, Germany, and Key Laboratory of Pesticide & Chemical Biology, Ministry of Education,
College of Chemistry, Central China Normal UniVersity, 152 Luoyu Road, Wuhan 430079, People’s Republic of China
ReceiVed March 26, 2009
Fermentation of the marine-derived fungus Trichoderma asperellum, collected from the sediment of the Antarctic Penguin
Island, resulted in the isolation of six new peptaibols named asperelines A-F (1-6), which are characterized by an
acetylated N-terminus and a C-terminus containing an uncommon prolinol residue. Structures were determined by extensive
1D and 2D NMR (1H-1H COSY, HMQC, HMBC, NOESY) spectroscopic data analysis combined with ESIMS/MS
fragmentation. The absolute configurations of the amino acid residues possessing a chiral R-carbon and of the prolinol
1
residue were determined to be L and S, respectively, using a new method of H NMR spectroscopic comparison of
complexes formed between the chiral reagent Ru(D -Por*)CO and amino acids derived from the peptaibols with those
4
formed with reference standards.
Peptaibols are a class of linear or cyclic peptides characterized
by chain lengths of 4-21 amino acid residues, which are usually
classified according to their chain length, as long-chain (17-21
residues), medium-chain (11-16 residues), and short-chain (4-10
residues) sequences.1-3 Their structural patterns feature an abun-
dance of R-aminoisobutyric acid residues (Aib), an N-acyl terminus
such as an acetyl group, and a C-terminus containing an amino
alcohol residue (phenylalaninol, leucinol, valinol, and others).
Generally, the trivial name of peptaibols represents a group of
peptides containing a number of Aib residues and 2-amino alcohols.
In addition, the unique R-dialkyl amino acid residues of peptaibols
such as Acc, Lva, and EtNva are also included in the peptaibol
family.1,4 Naturally occurring peptaibols are mixtures of isoforms,
and more than 850 sequences have been isolated from at least 23
genera of ascomycetous fungi or their anamorphs.1-8 These peptides
are regarded to be synthesized nonribosomally by large multidomain
enzymes.9,10 They possess diverse bioactivities, as antibacterials,
antifungals, and antiparasitics,11-13 and are used as biocontrol
agents against fungal phytopathogens.14-16
Trichoderma species (Hyphomycetes) have been studied exten-
sively as potential sources of biocontrol agents, enzymes, and
diverse bioactive peptaibols.17-21 T. asperellum is widely distrib-
uted in soil and is a mycoparasitic fungus used for antiphytopatho-
gens.22 It is highly effective against the rice diseases caused by
seedborne pathogens.23 Additionally, it is a rich source of peptai-
bols, and previous chemical examination led to the isolation of two
trichotoxins, 1704E and 1717A,24 and identification of seven
peptaibols by means of “peptaibiomics” using a HPLC-MSn
method,25 which are characterized by a valinol linked to the
C-terminus. Trichotoxins A-5026 and A-4027 are also produced by
a strain of T. asperellum, which was originally misidentified as T.
Viride. Lieckfeldt et al.28 assumed that the T. Viride biocontrol
strains reported to have high-temperature optima are actually T.
asperellum and that no true T. Viride strain has been found to
produce antibiotics. Thus, all trichotoxins reported to date are
considered to be produced by strains of T. asperellum. In the course
of investigation of new metabolites and their biofunctions from
extremophilic microorganisms, a T. asperellum strain was isolated
from the sediment of the Antarctic Penguin Island. Chemical
investigation of its fermentation broth led to the isolation of six
new peptaibols (1-6), all of them featuring a structurally unique
prolinol residue at the C-terminus and an acetylated N-terminus.
Results and Discussion
The chromatographic separation of the fermentation broth (80 L)
using Amberlite-XAD, Si gel, and a Sephadex LH-20 column in
combination with semipreparative HPLC resulted in the isolation
of six pure peptaibols (1-6).
Aspereline A (1) was isolated as colorless crystals. The molecular
formula was determined to be C45H80N10O11 on the basis of the
HRESIMS (m/z 937.6088 [M + H]+) and NMR data, indicating
1
11 degrees of unsaturation. The H NMR spectrum in DMSO-d6
displayed nine resonances in the downfield range between δH 7.10
and 8.59, which were assignable to the NH protons of amides. The
13C NMR spectrum contained 10 carbonyl signals between δC 177.4
and 171.3, confirming 1 to be a peptide. Chemical shift assignments
for each amino acid residue were based on the interpretation of
1H, 13C NMR, DEPT, and 2D NMR data including 1H-1H COSY,
HMQC, and HMBC, which revealed the presence of nine amino
acid residues, involving an isoleucine (Ile), a valine (Val), an alanine
(Ala), and six 2-aminoisobutyric acids (Aib-1 to Aib-6), along with
acetyl and prolinol residues. For instance, the spin systems of the
R-H and NH resonances of Val (δH 3.51/δH 7.76), Ile (δH 3.62/δH
7.10), and Ala (δH 4.00/δH 7.37) and the correlations from the
R-methine resonances to the side chains of the individual amino
acid residues were clearly observed in the COSY spectrum. Six
NH singlets were attributed to the Aib residues, which were proven
by the HMBC correlation between NH resonances and the
quaternary carbons resonating around δC 56, characteristic of the
R-C of Aib residues. In addition to an acetyl group represented by
a methyl singlet at δH 1.93 (3H, s) and its HMBC correlation to a
carbonyl carbon at δC 171.3 (s), a prolinol ring was recognized
from the COSY couplings of H2-5 (δH 3.38, 3.40)/H-1 (δH 3.95),
H-1/H2-2 (δH 1.69, 1.75), H2-2/H2-3 (δH 1.60, 1.75), and H2-3/H2-4
(δH 3.30, 3.59). The carbons corresponding to the protons in the
molecule were assigned by a HMQC spectrum. The sequence of
amino acid residues was established through the HMBC correlations
of the NH and R-H resonances to the amide carbonyl carbons
through 2JCH and 3JCH couplings (Table 1). The HMBC correlations
of the acetyl methyl protons at δH 1.93 (3H, s, H-1) and the NH at
* To whom correspondence should be addressed. Tel: (86)10-82806188.
Fax: (86)10-82802724. E-mail: whlin@bjmu.edu.cn.
† Peking University.
‡ Qingdao University of Science & Technology.
§ Central China Normal University.
⊥ Beijing Normal University.
| Heinrich-Heine-University.
10.1021/np900190w CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/10/2009

Peptaibols from the Fungus Trichoderma asperellum
Journal of Natural Products, 2009, Vol. 72, No. 6 1037
δH 8.59 (s) to the carbonyl carbon at δC 171.3 (s) indicated that
the N-terminus of 1 was acetylated. Subsequently, the interactions
of NH (δH 8.59, s) with δC 175.4 (CdO, Aib-1), NH (δH 8.41, s)
with CdO (Aib-1) and δC 177.4 (CdO, Aib-2), NH (δH 7.76, d)
and R-H (δH 3.51, m, Val) with CdO (Aib-2) and δC 172.8 (CdO,
Val), NH (δH 7.53, s) with CdO (Val) and δC 175.4 (CdO, Aib-
3), NH (δH 7.10, d) and R-H (δH 3.62, m, Ile) with CdO (Aib-3)
and δC 173.4 (CdO, Ile), NH (δH 7.84, s) with CdO (Ile) and δC
174.1 (CdO, Aib-4), NH (δH 7.80, s) with CdO (Aib-4) and δC
175.4 (CdO, Aib-5), NH (δH 7.37, d) and R-H (δH 4.00, m, Ala)
with CdO (Aib-5) and δC 172.3 (CdO, Ala), and NH (δH 7.53, s)
with CdO (Ala) and δC 171.3 (CdO, Aib-6) established the
sequence of Ac-Aib-Aib-Val-Aib-Ile-Aib-Aib-Ala-Aib. The prolinol
residue was deduced to form an amide at the C-terminus on the
basis of HMBC correlations from the methylene protons at δH 3.30
(1H, m) and 3.59 (1H, m) and the NH (Aib-6) at δH 7.53 (s) to the
carbonyl carbon at δC 171.3. Additionally, the negative ion ESIMS
displayed the deprotonated molecule at m/z 935 [M - H]-, which
was selected as a precursor ion for ESIMS/MS experiments.
Analysis of the resulting product ion spectrum indicated that the
MS cleavage process followed B-type fragmentation and that
fragment ions resulting from sequential loss of the N-terminal amino
acid residues were observed. For example, ESIMS2 of the precursor
ion produced B-type ions at m/z 893 [M - Ac]-, 808 [M - Ac -
Aib]-, 723 [M - Ac - 2Aib]-, 624 [M - Ac - 2Aib - Val]-,
539 [M - Ac - 3Aib - Val]-, 426 [M - Ac - 3Aib - Val -
Ile]-, 341 [M - Ac - 4Aib - Val - Ile]-, and 256 [M - Ac -
5Aib - Val - Ile]- (Figure 1). This result further supported the
sequence assignment by NMR spectroscopic data analysis. In order
to determine the absolute configurations of the individual amino
acid residues in the parent peptide, a new method for the 1H NMR
spectroscopic comparison of complexes formed between the chiral
reagent Ru(D -Por*)CO30,31 and amino acids derived from the
4
peptaibols with those formed with reference standards is established.
Acidic hydrolysis29 of 1 followed by derivatization with a chiral
reagent Ru(D -Por*)CO was performed (Figure 2). The configu-
4
ration of the individual amino acid residue was determined through
1
the comparison of the H NMR data of the complex between the
L/D standard amino acids and the corresponding residue derived
from 1, analyzed as a mixture. For instance, the complex with L-Ala
exhibited resonances at δ -3.00 (R-H), -4.50 (NHa), and -5.69
(NHb), whereas the corresponding protons for the D-Ala complex
are at δ -2.83 (R-H), -4.43 (NHa), and -5.70 (NHb). Thus, the
1H NMR signals observed for the Ala complex in the mixture of
amino acids being hydrolyzed from 1 (Figure 3) were consistent
with the L-form. On the basis of the same protocol, the configuration
of Ile in 1 was determined to be the L-form (Figure 3). In addition,
the analytical result revealed the L-configuration for Val residue,
whereas prolinol possesses the S-configuration (see Supporting
Information for additional figures).
The molecular formula of aspereline B (2) was established as
C44H78N10O11, based on the HRESIMS (m/z 945.5744 [M + Na]+)
and NMR data. The molecular weight of 2 was thus 14 amu lower
than that of 1. Analysis of 2D NMR (1H-1H COSY, HMQC, and
HMBC) spectroscopic data revealed that the structure of 2 contained
spin systems for five Aib, two Ala, one Val, and one Ile, as well as
an acetyl group and a prolinol subunit. The sequence of the linear
peptide was established on the basis of HMBC cross-peaks from
2
NH and R-H resonances to amide carbonyl carbons through JCH
3
and JCH correlations (Figure 4), which indicated that the larger
part of the linear sequence was the same as that of 1. The only
difference was found at the N-terminus, where the Aib residue of

1038 Journal of Natural Products, 2009, Vol. 72, No. 6
Ren et al.
Table 1. 1H and 13C NMR Data and COSY and HMBC Correlations of 1 (in DMSO-d6)a
residue
Ac
position
δC, mult.
δH (J in Hz)
COSY correlation
HMBC correlation
CdO
CH3
CdO
1
2
3
NH
CdO
1
2
3
NH
CdO
1
171.3, qC
23.1, CH3
175.4, qC
56.1, qC
26.2, CH3
23.2, CH3
1.93, s
CdO
Aib-1
Aib-2
Val
1.35, s
1.37, s
8.59, s
C-3, C-1, CdO (A-1)
C-1, C-2, CdO (A-1)
CdO (Ac), CdO (A-1), C-1
177.4, qC
56.2, qC
27.8, CH3
24.4, CH3
1.35, s
1.34, s
8.41, s
C-3, C-1, CdO (A-2)
C-1, C-2, CdO (A-2)
CdO (A-1), CdO (A-2), C-1
172.8, qC
62.8, CH
29.2, CH
20.0, CH3
19.1, CH3
3.51, m
2.17, m
0.98, d (6.5)
0.91, d (6.5)
7.76, d (6.0)
NH, H-2
H-1, H-3, H-4
H-2
H-2
H-1
CdO (A-2), CdO (V-1), C-2, C-3, C-4
CdO (V-1), C-3, C-4, C-1
C-4, C-2, C-1
C-3, C-2, C-1
CdO (V-1), CdO (A-2), C-1
2
3
4
NH
Aib-3
Ile
CdO
1
2
3
NH
CdO
1
2
3
175.4, qC
56.3, qC
27.1, CH3
23.4, CH3
1.35, s
1.37, s
7.53, s
C-3, C-1, CdO (A-3)
C-1, C-2, CdO (A-3)
CdO (A-3), CdO (V-1), C-1
173.4, qC
61.1, CH
35.1, CH
26.0, CH2
3.62, m
1.75, m
1.15, m
NH, H-2
H-1, H-3, H-5
H-2, H-4
CdO (A-3), CdO(I), C-2, C-5, C-3
1.55, m
4
5
NH
10.4, CH3
15.2, CH3
0.76, t (7.0)
0.82, d (6.5)
7.10, d (6.5)
H-3
H-2
H-1
C-2, C-3
C-1, C-2, C-3
C-1, CdO(I), CdO (A-3)
Aib-4
Aib-5
CdO
1
2
3
NH
CdO
1
174.1, qC
56.4, qC
26.0, CH3
22.9, CH3
1.35, s
1.37, s
7.84, s
C-3, C-1, CdO (A-4)
C-1, C-2, CdO (A-4)
CdO (A-4), CdO (I), C-1
175.4, qC
56.4, qC
2
3
27.5, CH3
23.5, CH3
1.35, s
1.33, s
7.80, s
C-3, C-1, CdO (A-5)
C-1, C-2, CdO (A-5)
CdO (A-5), CdO (A-4), C-1
NH
CdO
1
Ala
172.3, qC
49.6, CH
17.6, CH3
4.00, m
1.33, m
NH, H-2
H-1
CdO (A-5), CdO(Al), C-2
CdO (Al), C-1
2
NH
CdO
1
7.37, d (7.5)
H-1
CdO (A-5), CdO(Al), C-1, C-2
Aib-6
171.3, qC
56.3, qC
2
3
NH
1
2
25.1, CH3
22.9, CH3
1.37, s
1.37, s
7.53, s
C-3, C-1, CdO (A-6)
C-1, C-2, CdO (A-6)
CdO (A-5), CdO (A-6), C-1
CdO(A-6), C-5, C-2
Prolinol
60.2, CH2
26.3, CH2
3.95, m
1.69, m
1.75, m
1.60, m
1.75, m
3.30, m
3.59, m
3.38, m
3.40, dd (3.0, 10.5)
H-5, H-2
H-1, H-3
H-1, H-3
H-2, H-4
H-2, H-4
H-3, H-4b
H-3, H-4a
H-1, H-5b
H-1, H-5a
3
4
5
25.2, CH2
48.0, CH
61.5, CH2
C-1, CdO(A-6), C-3, C-2
C-1, CdO(A-6), C-3, C-2
C-1, C-2
C-1, C-2
a A ) Aib, I ) Ile, V ) Val, Al ) Ala.
Figure 1. (-) ESIMS/MS fragmentation of 1 by ionization of [M - H]-.
1
1 was replaced by an alanine in 2, as evident from the H-1H
at δH 1.32 (3H, d) and 8.66 (1H, d, J ) 7.5 Hz, NH) and the HMBC
interaction of the NH and the acetyl protons at δH 1.92 (3H, s) to
COSY correlation of δH 4.01 (1H, m, R-H) with the methyl protons

Peptaibols from the Fungus Trichoderma asperellum
Journal of Natural Products, 2009, Vol. 72, No. 6 1039
Figure 2. Route to prepare Ru(D4-Por)CO complexes of the amino acids derived from 1.
the carbonyl carbon at δC 171.5 (s). The assignment of the amino
acid sequence was further corroborated by ESIMS/MS fragmenta-
tion. The configurations of the amino acid residues and of the
prolinol unit were the same as for 1.
(1H, m, R-H), and 7.06 (1H, d, J ) 7.0 Hz, NH). Detailed HMBC
data analysis (Figure 4) confirmed that the second Val of 4 replaced
the Ile in 1. The negative ion ESIMS/MS revealed B-type fragments,
which confirmed the entire sequence of 4 to be Ac-Aib-Aib-Val-
Aib-Val-Aib-Aib-Ala-Aib-prolinol.
Aspereline C (3) had the same molecular formula as 2, as
determined by HRESIMS data. A comparison of the NMR
spectroscopic data (Table 2) revealed that 3 possesses the same
amino acid residues as 2. In addition, the signals of an acetyl group
and a prolinol residue were also observed from its 1H and 13C NMR
spectra. 2D NMR data (Figure 4) indicated that the amino acid
residues corresponding to both the N- and C-termini were Aib
residues as in the case of 1, which were connected to the acetyl
and prolinol residues, respectively. The NH resonance at δH 7.38
(1H, s, NH) belonging to the Aib adjacent to the C-terminus
interacted with the R-H of an alanine at δH 4.07 (1H, m, Ala-2) in
the NOESY spectrum and clarified the location of the alanine near
the C-terminus. The COSY correlations of δH 3.93 (1H, m, R-H)/
δH 7.79 (1H, d, J ) 4.5 Hz, NH) and R-H/Me (δH 1.31, d) confirmed
the presence of a second Ala residue (Ala-1). The HMBC
correlation of the protons at δH 4.07, 3.93, and 7.42 (d, NH-Ala-2)
to the amide carbonyl carbon at δC 172.6 (s) allowed the
determination of an Ala-Ala dipeptide linkage. The remaining
sequence of the residues was identical to that of 1 on the basis of
the 2D NMR data analysis and the comparison of the NMR data
with those reported for 1. The ESIMS/MS spectra of protonated
and deprotonated 3 (Figure 5) exhibited the successive fragment
ions as generated by B-type fragmentation from the N-terminus
and X-type fragmentation from the C-terminus, respectively. These
findings provided additional evidence for assigning the amino acid
sequence as Aib-Aib-Val-Aib-Ile-Aib-Ala-Ala-Aib. The configura-
tions of the amino acid residues were in agreement with those of 1.
HRESIMS data indicated that aspereline D (4) had the same
molecular formula as 2 and 3. Analysis of 1D and 2D NMR
spectroscopic data disclosed the presence of six Aib, two Val, and
one Ala, along with an acetyl group and a prolinol residue. The
sequence of the amino acid residues was established by 2D NMR
(NOESY and HMBC) and ESIMS/MS techniques, which revealed
the structure of 4 to be closely related to that of 1. The only
difference found was the presence of an additional Val residue of
4 instead of the Ile residue of 1, which was evident from the COSY
coupling system of the proton signals at δH 2.16 (1H, m, H-2),
0.87 (3H, d, J ) 6.5 Hz, H-3), 0.86 (3H, d, J ) 6.5 Hz, H-4), 3.36
The HRESIMS data provided the molecular formula of aspereline
E (5) as C45H80N10O12, according to the protonated molecular ion
1
at m/z 953.6031 [M + H]+ and NMR data. The H and 13C NMR
data of 5 (Table 2) were closely related to those of 1, except for
the presence of a serine residue noted by the 1H-1H COSY
correlations from δH 4.05 (1H, ddd) to δH 3.69 (1H, dd) and 3.81
(1H, dd) and also the NH at δH 7.43 (1H, d, J ) 8.0 Hz). This
residue was deduced to be connected to the Aib at the C-terminus,
replacing an Ala of 1, on the basis of the HMBC correlations from
δH 4.05 and 7.49 (1H, s, NH-Aib) to δC 169.5 (s) and from the
latter NH to δC 56.5 (s, R-C, Aib) and 171.3 (s, Aib). The negative
ESIMS/MS spectrum revealed that an additional early fragmentation
was caused by the loss of 31 amu, which was generated from
cleavage of the CH2OH unit of Ser, while the remaining fragments
originated by a cleavage mechanism similar to that for other linear
peptides. Moreover, the positive ion ESIMS/MS spectrum followed
the regular cleavage pathway through X-type fragmentation. The
configurations of the amino acid residues were determined using
the same protocol described for 1, which resulted in the L-
configuration for Val, Ala, Ile, and Ser.
Aspereline F (6) had the molecular formula C46H82N10O11, as
determined by the HRESIMS and NMR data. The 1H and 13C NMR
data (Table 2) indicated that it is a linear peptide, closely related
to the other asperelines. The 1H NMR spectrum presented four R-H
protons at δ 3.73 (1H, dd, J ) 6.0, 7.0 Hz), 3.51 (1H, dd, J ) 6.5,
7.0 Hz), 3.60 (1H, m), and 4.00 (1H, m), which were ascribed to
two Val, one Ile, and one Ala, as proven by the COSY and HMBC
correlations. The remaining residues were Aib units. Thus, this
peptide contained nine amino acids apart from an acetyl and a
prolinol residue. The amino acid sequence was unambiguously
established through HMBC correlations from the NH and R-H
protons to the carbonyl carbons, which defined the sequence of 6
to be Aib-Val-Val-Aib-Ile-Aib-Aib-Ala-Aib. The N- and C-termini
are substituted by acetyl and prolinol residues, respectively, as in
the case of 1-5. The negative ion ESIMS/MS data supported the
structure assignment. The configurations of the chiral amino acid

1040 Journal of Natural Products, 2009, Vol. 72, No. 6
Ren et al.
Figure 3. 1H NMR spectroscopic comparison of the Ru(D -Por*)CO complexes between L-Ala and L-Ile in the mixture derived from 1 and
4
the reference standards.
residues and prolinol were assigned as L and S using the same
method as described for 1.
In addition to the 1D and 2D NMR spectra, ESIMS/MS
effectively elucidated the peptaibol sequences. The experimental
results indicated that the negative ion ESIMS/MS of the deproto-
nated precursor ion always produces sequential fragments from the
N-terminus via the B-type pathway, whereas the amino acid residues
are fragmented sequentially from the C-terminus through an X-type
route in positive ion ESIMS/MS (Figure 6). In the case of the
peptaibol containing the Ser residue, prior cleavage occurred at Ser
by loss of a CH2OH residue in the negative ion ESIMS/MS. Thus,
the combination of the ESIMS/MS spectra, recorded by deproto-
nated and protonated ionization, allowed the determination of the
entire sequences of the peptaibols. This method also can be used
to determine the individual peptides from a peptide-containing
mixture through online HPLC-ESIMS/MS techniques.
Compounds 1-6 were tested against fungi and bacteria, but they
showed only weak inhibitory activity toward the early blight
pathogen Alternaria solani, the rice blast Pyricularia oryzae, and
the bacteria Staphylococcus aureus and Escherichia coli with IC50
> 100 µg/mL and IC90 > 500 µg/mL.
Herein, a new group of peptaibols displaying nine amino acid
residues is described, which enriches the diverse peptaibol family.
The (S)-prolinol residue is reported here as a novel, C-terminal
constituent of peptaibiotics for the first time. In addition, this is
the first report on the production of peptaibols by a psychrophilic
fungus isolated from an Antarctic habitat. Recently, Bru¨ckner and
his co-workers1 hypothesized that extremophilic fungi, including
those from Antarctic habitats, are capable of producing peptaibiotics.
The results presented here serve as a first proof for this hypothesis.
In the 1H NMR spectra, peptaibols are easily recognized by the
broad signals integrated around δH 1.34-1.37 arising from the

Peptaibols from the Fungus Trichoderma asperellum
Journal of Natural Products, 2009, Vol. 72, No. 6 1041
Figure 4. HMBC and NOESY correlations of asperelines B-F (2-6).
NMR and 2D NMR spectra were recorded on Bruker Avance-500 and
Varian INOVA-500 NMR spectrometers using TMS as an internal
standard. The chemical shifts are given in δ (ppm) and coupling
constants in Hz. ESIMS spectra were measured on a Quttra Premier
XE tandem mass spectrometer (Micromass, UK), while HRESIMS
spectra were measured on a LTQ Obitrap X1 Thermo Scientific and a
FT-MS-Bruker APEX IV (7.0T). Silica gel (GF254) for TLC and H-Sil
gel (200-300 mesh) for column chromatography (CC) were purchased
from Qingdao Marine Chemical Company, Qingdao, China. Semi-
preparative HPLC was performed on an Alltech-HPLC (USA) using a
Kromasil column (ODS, 10 µm, 10 × 250 mm). The chemical reagents
used for chromatography were purchased from Beijing Chemical Works
Co. Ltd. (Beijing).
multiple methyl groups of Aib’s, which distinguish these com-
pounds from other peptides not having Aib residues.
The organometallic compound Ru(D -Por*)CO served as a new
4
type of sensitive chiral NMR shift reagent to determine the L/D
configurations of amino acids at the microgram level. It is achieved
by observing the resonances of amino acids exclusively below 0
ppm upon binding the amino group to the ruthenium porphyrin in
an axial orientation to form a stable complex. The significant upfield
shifts of the NH2 and R-H resonances are induced by the ring-
current effect generated by the Ru(D -Por*)CO planar geometry.
4
The resonances of the protons of L- and D-amino acids are shifted
upfield to different extents. The chiral reagent Ru(D -Por*)CO
4
Fungal Strain and Identification. Trichoderma asperellum was
collected from the sediment of the Antarctic Penguin Island. The strain
(Y19-07, accession number HTTM-Z00002) was isolated and identified
by Prof. T. Li of the First Institute of Oceanography, SOA, and was
deposited in SOA and the State Key Laboratory of Natural and
Biomimetic Drugs, Peking University. The strain sequence is accessed
under GenBank DQ767600.
DNA Extractions. The strain Y19-07 was cultured in liquid medium
(potato broth in seawater 20%, peptone 0.2%, yeast extract 0.1%,
glucose 3%, NaCl 2%, MgCl2 ·6H2O 0.15%, KCl 0.02%, FePO4
0.001%, pH 6.5) at 15 °C for 2 days. The mycelium, DNA extraction
buffer (500 µL), NaCl (5M, 25 µL), and glass beads (250-300 µL)
were mixed thoroughly in a microcentrifuge tube. The suspension was
centrifuged for 2 min at 10 000 rpm. The supernatant was then
transferred into a new microcentrifuge tube, and a phenol-chloroform-
isoamyl alcohol (25:24:1, 500 µL) mixture was added. The suspension
was centrifuged for 5 min at 10 000 rpm. To a microcentrifuge tube
with the supernatant, the 2-propanol (i-PrOH) (100 µL, 70%) was added
at -20 °C for 1 h. After centrifuging for 3 min (10 000 rpm), the pellet
was resuspended in 70% EtOH (800 µL) to centrifuge for 5 min at
10 000 rpm. The air-dried supernatant was suspended in 500 µL of TE
buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA) and mixed with EDTA-
Sark (50 µL) and proteinase K (20 mg/mL, 2.5 µL) to incubate for 30
induced a greater upfield shift for the R-H than for the alkyl side
chain protons, and the R-H resonances of L- and D-amino acids are
baseline-resolved. Thus, the chemical shift differences of the R-H
in association with other signals within the amino acid residue are
employed to distinguish the L- and D-configurations. The advantage
of this method is that it can discriminate the configurations of
constituent amino acids in a peptide in a mixture of hydrolyzed
products without chromatographic separation.
In recent years, large numbers of peptaibol sequences have been
elucidated, due to the development of modern spectroscopic
techniques, including various 2D NMR methods and ESIMS/MS
spectrometric techniques, which have been frequently used for the
complete sequencing of peptaibols. The optimization of the
production of peptides, which are important for biotechnological
and pharmacological studies, requires further investigation.
Experimental Section
General Experimental Procedures. Melting points were measured
on a XT4A microscopic melting-point detector. Optical rotations were
recorded on a Perkin-Elmer 341 LC polarimeter. UV spectra were
measured on a Shimadzu UV-210A spectrophotometer. 1H and 13C

1042 Journal of Natural Products, 2009, Vol. 72, No. 6
Table 2. 1H and 13C NMR Data of 2-6 in DMSO-d6
Ren et al.
2
3
4
5
6
no.
Ac
CdO
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
171.5
22.9
Ala-1
174.4
51.2
171.4
23. 5 1.93, s
Aib-1
175.6
56.2
171.6
23.4
Aib-1
175.9
56.3
171.6
23.7
Aib-1
173.8
56.2
171.3
23.6
Aib-1
176.2
56.3
CH3
1.92, s
1.93, s
1.93, s
1.94, s
CdO
1
4.01, m
2
3
NH
17.6
1.32, d (7.0)
26.3
23.4
1.33, s
1.35, s
8.87, s
26.2
23.0
1.34, s
1.34, s
9.25, s
26.2
26.3
1.36, s
1.36, s
9.18, s
26.8
23.1
1.38, s
1.35, s
8.58, s
8.66, d (7.5)
Aib-1
176.9
56.2
27..5 1.40, s
23.2
Aib-2
177.4
56.2
27.8
24.4
Aib-2
177.6
56.3
27.1
23.4
Aib-2
177.6
56.2
27.1
27.7
Val-1
172.6
61.8
29.0
19.2
19.7
1
2
3
4
3.73, dd (6.0,7.0)
2.14, m
0.91, d (6.5)
0.96, d (6.5)
8.05, d (6.0)
1.40, s
1.36, s
1.33, s
1.33, s
1.36, s
1.36, s
1.35, s
NH
7.87, s
8.60, s
8.84, s
8.79, brs
Val
Val
Val-1
172.9
62.9
29.4
19.2
20.1
Val
Val-2
173.9
62.9
29.2
19.5
20.0
CdO
1
2
3
4
NH
172.4
62.5
29.2
19.1
20.1
173.0
62.9
29.2
19.2
20.1
172.9
62.9
29.2
3.55, dd (6.5,7.0)
2.10, m
0.89, d (7.0)
0.96, d (7.0)
7.67, d (6.5)
3.48, m
2.17, m
0.98, d (6.5)
0.91, d (6.5)
7.73, d (7.5)
3.46, m
2.16, m
3.47, dd (6.0,7.0)
2.18, m
0.99, d (6.5)
0.91, d (6.5)
7.75, d (6.0)
3.51, dd (6.5,7.0)
2.12, m
0.99, d (6.5) 20.1
0.98, d (6.5) 19.1
7.78, d (8.0)
0.92, d (6.5)
0.97, d (6.5)
7.62, d (6.5)
Aib-2
175.5
56.3
26.9
23.6
Aib-3
175.9
56.2
27.1
23.4
Aib-3
175.5
56.3
27.8
25.1
Aib-3
Aib-2
175.3
56.2
27.5
25.1
CdO
1
2
3
176.1
56.4
27.8
26.3
1.41, s
1.35, s
7.77, s
1.43, s
1.40, s
7.54, s
1.34, s
1.34, s
7.55, s
1.44, s
1.36, s
7.59, s
1.36, s
1.35, s
8.07, s
NH
Ileu
Ileu
Val-2
173.4
62.9
29.1
20.2
19.1
Ileu
Ileu
CdO
1
2
3
4
5
NH
173.5
61.2
35.2
26.3
10.7
15.3
173.5
61.4
34.9
26.1
10.4
15.2
174.7
61.3
34.9
173.3
61.3
35.7
26.2
11.2
15.5
3.60, dd (7.0,8.5)
1.80, m
1.15, m 1.55, m
0.78, t (7.0)
0.82, d (6.5)
7.40, d (8.5)
3.61, dd (6.5,7.2)
1.87, m
1.16, m 1.55, m
0.77, t (7.0)
0.83, d (7.2)
7.21, d (6.5)
3.36, m
2.16, m
3.60, dd (6.5,7.0)
1.88, m
1.15, m 1.59, m
0.78, t (7.5)
0.82, d (6.5)
7.27, d (6.5)
3.60, m
1.85, m
0.87, d (6.5) 26.0
0.86, d (6.5) 10.4
15.1
1.33, m 1.14, m
0.80, t (6.5)
0.85, d (7.0)
7.14, d (6.5)
7.06, d (7.0)
Aib-3
175.5
62.5
25.0
23.2
Aib-4
175.9
56.2
26.5
22.9
Aib-4
175.4
56.3
23.2
27.5
Aib-4
Aib-3
175.5
56.3
26.9
23.2
CdO
1
2
3
175.8
56.4
25.2
26.0
1.36, s
1.34, s
7.79, s
1.45, s
1.35, s
7.86, s
1.34, s
1.33, s
7.87, s
1.36, s
1.32, s
7.99, s
1.36, s
1.35, s
7.60, s
NH
Aib-4
174.2
56.3
Ala-1
172.6
51.1
Aib-5
174.1
56.3
Aib-5
175.3
56.7
Aib-4
174.2
56.2
CdO
1
3.93, m
2
3
NH
23.1
26.3
1.35, s
1.32, s
7.86, s
17.0
1.31, d (7.0)
26.3
24.3
1.34, s
1.33, s
7.83, s
27.7
27.1
1.43, s
1.43, s
7.91, s
26.4
24.4
1.36, s
1.35, s
7.72, s
7.79, d (4.5)
Ala-2
172.3
49.6
Ala-2
172.0
49.6
Ala
Ser
169.5
57.2
Ala
172.3
49.6
CdO
1
2
172.2
49.6
17.6
4.00, m
1.32, d (7.0)
4.07, m
1.32, s
4.00, m
4.05, ddd (3.0,8.0,8.5)
4.00, m
1.32, m
17.0
17.6
1.34, d (7.0) 62.1
3.69, dd (3.0,11.5) 3.81, 17.6
dd (8.5,11.5)
7.43, d (8.0)
Aib-5
NH
7.37, d (7.0)
7.42, d (9.0)
7.38, d (7.5)
7.42, d (7.5)
Aib-5
171.3
56.2
26.8
23.6
Aib-5
171.2
56.2
25.9
22.8
Aib-6
171.3
56.3
25.9
22.7
Aib-6
CdO
1
2
3
171.3
56.5
25.2
25.9
171.2
56.4
25.9
23.4
1.36, s
1.34, s
7.54, s
1.35, s
1.35, s
7.38, s
1.37, s
1.37, s
7.53, s
1.38, s
1.38, s
7.49, s
1.36, s
1.35, s
7.52, s
NH
prolinol
1
2
60.2
26.3
3.96, m
1.58, m
1.74, m
1.41, m
1.75, m
3.28, m
3.59, m
60.2
26.3
3.94, m
1.69, m
1.75, m
1.60, m
1.75, m
3.26, m
3.57, m
60.2
26.2
3.49, m
1.44, m
1.72, m
1.52, m
1.77, m
3.58, m
3.25, m
3.35, m
3.40, m
60.2
26.3
3.94, m
1.67, m
1.74, m
1.60, m
1.75, m
3.27, m
3.59, m
3.35, dd (7.5,10.5)
3.40, dd (3.0,10.5)
60.2
26.3
3.96, m
1.67, m
1.74, m
1.60, m
1.74, m
3.27, m
3.59, m
3.39, dd (7.5,10.5)
3.52, dd (3.0,10.5)
3
4
5
25.2
48.0
61.4
25.2
48.0
25.1
48.0
25.6
48.0
61.4
25.2
48.0
61.5
3.35, dd (6.5,10.5) 61.4
3.40, dd (3.0,10.5)
3.35, dd (5.0,10.5) 61.4
3.40, dd (3.4,10.5)
min at 37 °C. The incubated solution was diluted by NaCl (5 M, 17
µL) and then centrifuged for 5 min (10 000 rpm). Afterward, EtOH
(70%, 800 µL) was added gently to centrifuge for 5 min at 10 000
rpm. The supernatant was air-dried and finally resuspended in TE buffer
(40 µL).
PCR Amplifications. Each PCR mixture (50 µL) contained MgC12
(3 µL, 25 mmol/L), DNA polymerase buffer (5 µL, 10 × Taq), dNTP
(1.0 µL, 5 mM/L), primer (1.0 µL, each 10 µM), Taq DNA polymerase
(0.25 unit), diluted genomic DNA (0.5 µL), and deionized water (38
µL). Primers used for PCR amplifications were ITS1 and ITS4 (5-

Peptaibols from the Fungus Trichoderma asperellum
Journal of Natural Products, 2009, Vol. 72, No. 6 1043
Figure 5. (+) and (-) ESIMS/MS fragmentation of 3.
for 10 min, readjusting pH to 7.3, adding agar (20 g), autoclaving at
121 °C for 20 min, cooling to 50 °C, and adding warm (50 °C) sterile
artificial seawater (ASW) (750 mL). ASW consists of NaCl (28.13 g),
KCl (0.77 g), CaCl2 ·2H2O (1.60 g), MgCl2 ·6H2O (4.80 g), NaHCO3
(0.11 g), MgSO4 ·7H2O (3.50 g), and distilled water (1000 mL). Pieces
of mycelium were cut into small segments and aseptically transferred
to a 250 mL Erlenmeyer flask containing 100 mL of culture media
(potato broth in seawater 20%, peptone 0.2%, yeast extract 0.1%,
glucose 3%, NaCl 2%, MgCl2 ·6H2O 0.15%, KCl 0.02%, FePO4
0.001%, pH 6.5). The flasks were incubated at 15 °C on a rotary shaker,
at 150 rpm, for 12 days.
Extraction and Isolation. The culture broth (80 L) was centrifuged
at 4000 rpm. The supernatant was adsorbed onto Amberlite-XAD
(macroporous resin), then the column was eluted with H2O, 5% EtOH,
and 95% EtOH, successively. The 95% EtOH fraction was collected and
evaporated in vacuo to give a residue, which was partitioned between H2O
and EtOAc. The organic layer was concentrated in vacuo to give the EtOAc
fraction. The EtOAc fraction (14.4 g) was subjected to vacuum liquid
chromatography (160-200 mesh Si gel) and eluted with a gradient of
petroleum ether (PE)-acetone (5:1, 3:1, 2:1, 1:1) to obtain seven fractions
(FA-FG). FC (1.9 g) was fractionated on a Sephadex LH-20 column
Figure 6. Cleavage pathway of (+)- and (-)-ESIMS/MS fragmen-
tation of peptaibols.
TCCGTAGGTGAACCTGCGG-3/5-TCCTCCGCTTATTGATATGC-
3). Amplifications were performed using the following parameters: a
5 min step at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min
at 51 °C, and 50 s at 72 °C, and then extended to a final 10 min at 72
°C. The genomic DNA was verified by 1% agarose gel electrophoresis.
Culture Conditions. The initial cultures were maintained on the
artificial seawater agar by dissolving beef extract (10 g) and peptone
(10 g) by heating in tap water (250 mL) to adjust pH to 7.8 and boiling

1044 Journal of Natural Products, 2009, Vol. 72, No. 6
Ren et al.
eluting with MeOH to yield four subfractions (SF1-SF4). SF1 (300 mg)
mainly contained peptides as determined by 1H NMR spectroscopy, and
it was subsequently separated on semipreparative HPLC (YMC, 10 µm,
10 × 250 mm) with a mobile phase of MeOH-H2O (70:30) to obtain 1
(79 mg, tR 73.9 min), 6 (15 mg, tR 85.2 min), a mixture of 2 and 3 (43
mg), and a mixture of 4 and 5 (32 mg). The mixture of 2 and 3 was then
separated by semipreparative HPLC (YMC, 10 µm, 10 × 250 mm) using
CH3CN-H2O (4:6) as the mobile phase to obtain 2 (7.0 mg, tR 31.5 min)
and 3 (8.0 mg, tR 46.5 min), while the mixture of 4 and 5 was separated
by the same protocol as for compounds 2 and 3 using CH3CN-H2O (38:
62) as the mobile phase to yield 4 (12 mg, tR 71.5 min) and 5 (15 mg, tR
87.5 min).
H5-5), -1.60 (1H, m, H-4a), -3.44 (1H, m, H-4b), -3.22 (1H, m,
R-H), -4.70 (1H, m, NH).
Acknowledgment. This work was supported by grants from NSFC
(No. 30672607), the National Hi-Tech Projects (2006AA09Z446,
2006DFA31100, 2007AA09Z448, 2006AA09Z405, 2007AA09Z435),
China Uni-PhD Base Project (20060001149), and International Coop-
eration Projects of BMBF-MOST.
Supporting Information Available: 1D and 2D NMR (COSY,
HMQC, HMBC, NOESY) and ESIMS/MS spectra of compounds 1-6
1
and the H NMR spectra of Ru(D -Por*)CO complexes of reference
4
Aspereline A (1): colorless needles (MeOH); mp 296-298 °C;
standards and the amino acids derived from the peptaibols. This material
is available free of charge via the Internet at http://pubs.acs.org.
[R]20 -42 (c 0.1, MeOH); 1H and 13C NMR data, see Table 1;
D
(-)ESIMS/MS m/z 935 [M - H]-, 893, 808, 723, 624, 539, 426;
(+)ESIMS/MD m/z 937 [M + H]+, 835, 751, 680, 595, 510, 397, 312;
HRESIMS m/z 937.6088 [M + H]+(calcd for C45H81N10O11, 937.6081).
References and Notes
Aspereline B (2): colorless needles (MeOH); mp 273-275 °C; [R]20
D
(1) Bru¨ckner, H.; Becker, D.; Gams, W.; Degenkolb, T. Chem. BiodiVersity
2009, 6, 38–56.
1
-5.0 (c 0.1, MeOH); H and 13C NMR data, see Table 2; (-)ESIMS
m/z 921 [M - H]-, 879, 808, 723, 624, 539, 426; (+)ESIMS m/z 945
[M + Na]+, 815, 731, 660, 575, 490, 377, 292; HRESIMS m/z 945.5744
[M + Na]+ (calcd for C44H78N10O11Na, 945.5743).
(2) Degenkolb, T.; Gams, W.; Bru¨ckner, H. Chem. BiodiVersity 2008, 5,
693–706.
(3) Degenkolb, T.; Bru¨ckner, H. Chem. BiodiVersity 2008, 5, 1817–1843.
(4) Toniolo, H.; Bru¨ckner, H. Peptaibiotics-Fungal Peptides Containing
R-Dialkyl R-Amino Acids, Verlag Helvetica Chimica Acta, Zu¨rich, Wiley-
VCH: Weinheim, 2009.
Aspereline C (3): colorless needles (MeOH); mp 284-285 °C;
[R]20 -22 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2;
D
(-)ESIMS/MS m/z 921 [M - H]-, 879, 794, 709, 610, 525, 412, 327;
(+)ESIMS/MS m/z 945 [M + Na]+, 815, 731, 660, 589, 504, 391,
306; HRESIMS m/z 945.5746 [M + Na]+ (calcd. for C44H78N10O11Na,
945.5743).
(5) Degenkolb, T.; Kirschbaum, J.; Bru¨ckner, H. Chem. BiodiVersity 2007,
4, 1052–1067.
(6) Ishiyama, D.; Satou, T.; Senda, H.; Fujimaki, T.; Honda, R.; Kanazawa,
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Aspereline D (4): colorless needles (MeOH); mp 301-303 °C;
[R]20 -10.5 (c 0.1, MeOH); 1H and 13C NMR data, see Table 2;
D
(-)ESIMS/MS m/z 921 [M - H]-, 879, 794, 709, 610, 525, 426;
(+)ESIMS/MS m/z 945 [M + Na]+, 815, 731, 660, 575, 490, 391,
306, 211; HRESIMS m/z 945.5740 [M
+
Na]+ (calcd for
C44H78N10O11Na, 945.5743).
Aspereline E (5): colorless needles (MeOH); mp 295-297 °C; [R]20
D
1
-12 (c 0.1, MeOH); H and 13C NMR data, see Table 2; (-)ESIMS/
MS m/z 951, 922, 880, 794, 709, 610, 525, 412; (+)ESIMS/MS m/z
976 [M + Na]+, 847, 761, 674, 589, 504, 391, 306; HRESIMS m/z
953.6031 [M + H]+(calcd for C45H81N10O12, 953.6035).
Aspereline F (6): colorless needles (MeOH); mp 281-283 °C; [R]20
D
1
-12 (c 0.1, MeOH); H and 13C NMR data, see Table 2; (-)ESIMS/
MS m/z 949, 907, 822, 723, 624, 539, 426; (+)ESIMS/MS m/z 973
[M + Na]+, 844, 759, 688, 603, 518, 405, 321; HRESIMS m/z 973.6047
[M + Na]+ (calcd for C46H82N10O11Na, 973.6042).
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Ru((+)-D -Por*)CO preparation. The chiral reagent Ru((+)-D -
4
4
Por*)CO was synthesized as reported.29,30
Configuration Determination. Compound 1 (0.2 mg) was dissolved
in 6 N HCl (2 mL) and heated at 105 °C for 24 h in a sealed ampule. The
resulting solution was cooled to rt and concentrated in vacuo to remove
H2O and HCl. Then, 500 µL of SOCl2-CH3OH (v/v ) 1:10) was added
and heated at 75 °C in a H2O bath for 1 h. The dried sample was mixed
with 40 µL of saturated Na2CO3 to basify the aqueous solution. Then a
0.6 mL solution of 2.0 mg of Ru((+)-D -Por*)CO in CDCl3 was added to
4
extract the amino acids. The CDCl3 layer was quickly passed through a 2
× 5 mm silica gel column and then collected in a NMR tube. A 1H NMR
spectrum was recorded at 600 MHz. The standards L/D Ala, L/D Ile, L/D
Val, L/D Ser, and S/R prolinols were purchased from Sigma, and the same
protocol was used as for 1 to record the 1H NMR spectrum of the individual
complexed amino acids. The complexed amino acid residues from 2-6
were treated similarly to 1.
L-Ala-Ru((+)-D -Por*)CO: 1H NMR δ (CDCl3) -2.00 (3H, d, Me),
4
-3.00 (1H, m, R-H), -4.46 (1H, m, NHa), -5.66 (1H, m, NHb).
1
L-Ile-Ru((+)-D -Por*)CO: H NMR δ (CDCl3) -0.21 (3H, t, J )
4
7.0 Hz, H-4), -0.75 (1H, m, H-3a), -1.10 (1H, m, H-3b), -0.90 (1H,
m, H-2), -1.50 (3H, d, J ) 6.5 Hz, H-5), -2.92 (1H, m, R-H), -4.30
(1H, m, NHa), -5.60 (1H, m, NHb).
L-Val-Ru((+)-D -Por*)CO: 1H NMR δ (CDCl3) -0.90 (1H, m,
4
H-2), -1.10 (3H, d, J ) 6.5 Hz, H-3), -1.36 (3H, d, J ) 6.5 Hz,
H-4), -3.07 (1H, m, R-H), -4.30 (1H, m, NHa), -5.61 (1H, m, NHb).
1
L-Ser- Ru((+)-D -Por*)CO: H NMR δ (CDCl3) -0.87 (1H, m,
4
H-2b), -0.81 (1H, m, H-2b), -2.63 (1H, m, R-H), -4.02 (1H, m, NHa),
(31) Zhang, R.; Yu, W.; Wong, K.; Che, C. J. Org. Chem. 2001, 66,
8145–8153.
-4.18 (1H, m, NHb).
1
S-Prolinol-Ru((+)-D -Por*)CO. H NMR δ (CDCl3) -0.77 (2H,
m, H2-2), -1.42 to -1.52 (2H, m, H2-3), -1.52 to -1.55 (2H, m,
4
NP900190W