Screening the Biosphere: The Fungicolous Fungus Trichoderma phellinicola, a Prolific Source of Hypophellins, New 17-, 18-, 19-, and 20-Residue Peptaibiotics

Abstract

To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola) growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non-ribosomal antibiotic polypeptides, peptaibiotics, which contain the non-proteinogenic marker amino acid, α-aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and, consequently, released into the host. By means of liquid chromatography coupled to electrospray high-resolution time-of-flight mass spectrometry, we detected ten 20-residue peptaibols in the specimen. Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of an agar plate culture of T. phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably, this strain could be identified as a potent producer of 39 new 17-, 18-, and 19-residue peptaibiotics, which display the same building scheme as the 20-residue peptaibols found in the specimen. Two of the 19-residue peptaibols are tentatively assigned to carry tyrosinol, a novel C-terminal residue, as deduced from high-resolution tandem mass-spectrometry data. For the new peptaibiotics produced by T. phellinicola, the name 'hypophellin(s)', based on the teleomorph name, is introduced.

Full text

Screening the Biosphere: The Fungicolous Fungus Trichoderma phellinicola,
a Prolific Source of Hypophellins, New 17-, 18-, 19-, and 20-Residue
Peptaibiotics
1
)
by Christian Rene´ Rçhricha)2), Anita Iversenb)2), Walter Michael Jaklitschc), Hermann Voglmayrc),
Andreas Vilcinskasa)d), Kristian Fog Nielsenb), Ulf Thrane b),Hans von Dçhrene), Hans Brcknerf)3),
and Thomas Degenkolb*b)d)
a) Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) , Bioresources Project Group,
Winchesterstrasse 2, D-35394 Giessen (C. R. R.: phone : þ49-641-99-37617,
e-mail: christian.roehrich@ime.fraunhofer.de; A. V.: phone : þ49-641-99-39500, fax: þ49-641-4808-581,
e-mail: andreas.vilcinskas@ime.fraunhofer.de)
b) Department of Systems Biology, Center for Microbial Biotechnology, Technical University of
Denmark (DTU), Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby (A. I.: phone: þ45-45252725,
e-mail: aive@bio.dtu.dk; K. F. N.: phone: þ45-45252602, fax: þ45-45884922, e-mail: kfn@bio.dtu.dk;
U. T.: phone: þ45-45252630, fax: 45-45884148, e-mail: ut@bio.dtu.dk)
c) Department of Systematic and Evolutionary Botany, Faculty Centre of Biodiversity,
University of Vienna, Rennweg 14, A-1030 Vienna (W. M. J.: phone: þ43-1-4277-54055,
e-mail: walter.jaklitsch@univie.ac.at; H. V.: phone : þ43-4277-54050,
e-mail: hermann.voglmayr@univie.ac.at)
d) Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of
Applied Entomology, Institute of Phytopathology and Applied Zoology (IPAZ) , University of Giessen
(JLU), Heinrich-Buff-Ring 26–32, D-35392 Gießen (phone: þ49-641-99-37601;
e-mail: thomas.degenkolb@ernaehrung.uni-giessen.de)
e) Biochemistry and Molecular Biology OE 2, Institute of Chemistry, Technical University of Berlin,
Franklinstraße 29, D-10587 Berlin (phone: þ49-30-314-22697; fax: þ49-30-314-24783;
e-mail: doehren@chem.tu-berlin.de)
f) Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ) , Department of
Food Sciences, Institute of Nutritional Science, University of Giessen, Heinrich-Buff-Ring 26– 32,
D-35392 Gießen (phone : þ49-711-349919; e-mail: hans.brueckner@ernaehrung.uni-giessen.de)
To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we
screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola)
growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non-
ribosomal antibiotic polypeptides, peptaibiotics, which contain the non-proteinogenic marker amino
acid, a-aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and,
consequently, released into the host. By means of liquid chromatography coupled to electrospray high-
resolution time-of-flight mass spectrometry, we detected ten 20-residue peptaibols in the specimen.
Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of
an agar plate culture of T.phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably,
this strain could be identified as a potent producer of 39 new 17-, 18-, and19-residue peptaibiotics, which
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 787
 2013 Verlag Helvetica Chimica Acta AG, Zrich
1) The term residue covers both a-amino acids and the C-terminal b-amino alcohol.
2) These authors contributed equally to this work.
3) Visiting Professor at Department of Food Sciences and Nutrition, College of Food Sciences and
Agriculture, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.

display the same building scheme as the 20-residue peptaibols found in the specimen. Two of the 19-
residue peptaibols are tentatively assigned to carry tyrosinol, a novel C-terminal residue, as deduced from
high-resolution tandem mass-spectrometry data. For the new peptaibiotics produced by T. phellinicola,
the name hypophellin(s), based on the teleomorph name, is introduced.
1. Introduction. – 1.1. Fungi as a Prolific Source of Bioactive Natural Products. The
current estimate of the total number of fungal species ranges between 1.0 and 1.5
million [1], whereas the number of those validly described should now exceed only
98,000 [2]. Of the 33,500 bioactive microbial metabolites known to date, the fungal
kingdom contributes ca. 15,600. Approximately 10,000 of them were shown to display
anti-infective, antitumor, and/or antiviral activities. Microbial-derived drugs on the
market comprise ca. 400– 500 active pharmaceutical agents [ 3], including therapeuti-
cally relevant antibiotics of fungal origin such as b-lactams, fusidic acid, and
griseofulvin, as well as the two immunosuppressants mycophenolic acid and cyclo-
sporine A [4].
Given that less than 1% of microorganisms visible under the microscope have been
cultivated under laboratory conditions so far, microbial diversity provides an
enormous, yet underestimated potential for future drug discovery [5] and in the
search for new agricultural antibiotics [6] .
1.2. The Potential of Trichoderma Species as Biological Control Agents (BCAs).
Species of the ubiquitous fungal genus Trichoderma and its Hypocrea teleomorphs have
attracted considerable interest in the past two decades because of the pivotal role of
their secondary metabolites in the antagonistic activities of biocontrol species [7 –9] .
Most of them occur as opportunistic, plant (endo)symbionts [10], some of which exhibit
pronounced antimicrobial activity towards economically important plant pathogens.
Recent examples include:
the hyperparasite Trichoderma stromaticum (syn. Hypocrea stromatica), the
active agent of Tricovab a commercial formulation against Crinipellis (syn.
Moniliophthora)perniciosa, the Witches broom pathogen of cocoa (Theobroma
cacao) [11] [12];
T.paucisporum and T.theobromicola, displaying in vitro-activities against frosty
pod rot of cocoa, Moniliophthora roreri [13] ;
T.martiale, which, in small-scale in situ field trials, proved highly effective against
black pod rot of cocoa caused by Phytophthora palmivora [14] .
The mode of action of phytoprotective Trichoderma species is considered rather
complex. Depending on the species or even strains investigated, the following
mechanisms may contribute to the antagonistic potential towards plant pathogenic
fungi:
i) Competition for nutrients and/or space, ii) growth promotion of plants, especially
colonization of roots, resulting in improved root and plant growth, iii) induction of
localized and systemic resistance responses in plants, iv) mycoparasitism, v) increase of
uptake and concentration of nutrients by the plant, including the production of
siderophores, and vi) production of volatile and non-volatile antibiotics [10] .
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)788

1.3. Peptaibiotics – Non-Ribosomally Biosynthesized Fungal Peptide Antibiotics
Containing a,a-Dialkyl-a-amino Acids. During the past two decades, peptaibiotics
have regained particular interest because of their unique bioactivities, resulting from
their amphipathicity and helical conformations [15] . These are attributed to the
presence of high proportions of peptide-bound a-aminoisobutyric acid (Aib),
frequently accompanied by d- and/or l-isovaline (Iva) [16], and, in a few sequences,
l-a-ethylnorvaline (EtNva), or 1-aminocyclopropane-1-carboxylic acid (Acc) [17].
The presence of these a,a-dialkyl-a-amino acids (Fig. 1,a) has been confirmed in acidic
hydrolysates of more than 30 genera of fungi [18].
Peptaibiotics are defined as non-ribosomally biosynthesised, linear or cyclic
polypeptide antibiotics of exclusively fungal origin which i) have a molecular weight
between 500 and 2,200 Da, thus containing 4 –21 residues; ii) show a high content of the
marker Aib, as well as further a,a-dialkylamino acids; iii) are characterized by the
presence of other non-proteinogenic amino acids and/or lipoamino acids; iv) possess an
acylated N-terminus, and v) in the case of linear peptides, have a C-terminal residue
that, in most of them, consists of a free or O-acetylated, amide-bonded b-amino
alcohol. The C-terminus might also be an amine, amide, sugar alcohol, 2,5-
diketopiperazine, a heterocyclic residue, or an amino acid with free carboxy terminus.
The majority of Aib-containing peptides carry a C-terminal residue representing a b-
amino alcohol. Only this group is referred to as peptaibols sensu stricto, whereas for the
others the comprehensive name peptaibiotics is used [17] .
1.4. Detection of Peptaibiotics in T. phellinicola Growing on Its Natural Host. The
genus Trichoderma, which currently consists of ca. 200 validly described species the
number of which increases continually [19– 28], is generally recognized as the most
prolific source of peptaibiotics [17]. However, reports on the detection of peptaibiotics
in samples collected in the natural habitat of the producer(s) are rare. Most of the ca.
Fig. 1. a) Structures and configurations of a,a-dialkylamino acids found in peptaibiotics.b)Building
scheme of subfamily-1 (SF1) peptaibiotics, produced by Hypocrea phellinicola. Variable positions are
underlined. Minor sequence variations are parenthesized. Deletions of certain amino acid positions are
highlighted in different shades: C-terminal deletions are highlighted in dark, deletions of Gln in medium,
and deletions of [Aib/Ala]6in light gray. a) Deleted in 17-, 18-, and 19-residues hypophellins. b) Deleted
in the 17-residue sequence 29.c) Deleted in 18-residue sequences 11,12, and 28, and in the 17-residue
sequence 29.d) Detected with DTU maXis gradient only. e) Detected with JLU micrOTOF-Q II gradient
only.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 789

1,000 individual sequences of peptaibiotics known to date have been sequenced in
extracts of fungal cultures grown under artificial laboratory conditions.
The first example of peptaibiotics isolated from natural specimens were hypelcins A
and B obtained from ca. 2 kg of dried, crushed stromata of Hypocrea peltata [29 – 31]. In
1997 and 1999, three reports were published on the isolation of peptaibiotics from
fruiting bodies of Scleroderma texense,Tylopilus neofelleus, and Boletus sp., respec-
tively; all being members of the Boletales [32– 34]. However, in 2002, Kiet et al. [35]
isolated chrysospermins A– D from the Vietnamese species Xerocomus langbianensis
(Boletaceae, Boletales) and attributed the detection of these four 19-residue peptaibols
[36] to an unrecognized infection of X.langbianensis with Sepedonium sp. This
phenomenon was later commented on by Degenkolb et al. [37] [38]. Finally, Neuhof
et al. [39] corroborated the assumption of Kiet et al. [ 35] by analyzing four fruiting
bodies of members of the order Boletales infected by Sepedonium chrysospermum and
S.microspermum, respectively. Notably, all samples were screened positive for
peptaibiotics of the chrysospermin type. In 2006, Lehr et al. [40] demonstrated that
16-residue peptaibols, the antiamoebins, were solely responsible for antibiosis in
herbivore dung naturally colonized by or artificially inoculated with Stilbella fimetaria
(syn. S.erythrocephala).
1.5. Bioactivities of Peptaibiotics from Trichoderma.Peptaibiotics are thus assumed
to play a key role in the infection process of a host by a fungicolous species because of
their unique ability of forming voltage-gated ion channels. This phenomenon is best
described by the dipole flip-flop gating model in planar lipid bilayers [41]. Their well-
documented membrane activity, however, may also account for other striking
bioactivities, such as neurolepsy [42], inhibition of amyloid b-peptide formation [43],
inhibition of HIV-1 integrase [44], suppression of tumor cells, targeted calcium-
mediated apoptosis, and autophagy in human hepatocellular carcinoma cells [45], as
well as induction of defence responses and systemic resistance in tobacco against
tobacco mosaic virus [46] and programmed cell death in fungal plant pathogens [47].
1.6. Choice of the Model Organism. Trichoderma phellinicola, a recently described
polyporicolous species, which specifically occurs on effused basidiomes of Phellinus
spp., was chosen as a model organism. Specimens of H.phellinicola have so far been
recorded from Austria, Denmark, Germany [20], and the Czech Republic (see Exper.
Part). This species is possibly specific for Phellinus ferruginosus [20].
To confirm the above hypothesis of peptaibiotic production under in vivo
conditions, a specimen of Trichoderma phellinicola growing on its host Phellinus
ferruginosus, was screened for peptaibiotics. For comparison, the ex-type culture of T.
phellinicola, CBS 119283 (¼C.P.K. 2137), was investigated. Both morphs were
analyzed using a peptaibiomics approach as described in [48 – 50].
2. Results. – 2.1. General Considerations. All 17-, 18-, 19-, and 20-residue sequences
discussed below were obtained from Trichoderma phellinicola [20]. The name
hypophellins (HPHs), which covers the entirety of long-chain peptaibiotics ( >17
residues) produced by this species, is proposed. We base this name on the teleomorph
name Hypocrea phellinicola, which used to be the valid name of the holomorph in dual
nomenclature [20]. The introduction of a new name for peptaibiotics from a
phylogenetically well-defined species is more favorable than earlier names for many
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)790

of the 19- and 20-residue peptaibiotics mentioned below, viz. suzukacillins, trichocel-
lins, trichokonins, and longibrachins, which were produced by phylogenetically
undefined Trichoderma species with thus highly questionable names. The latter issue
is further complicated by the fact that many of the peptaibiotic-producing Trichoderma
strains reported in the literature have never been deposited in a public culture
collection, or deposition was terminated [51] .
Hypophellins are numbered consecutively with Arabic numbers as follows: i)
sequences produced by the specimen; ii) sequences produced by the culture CBS
119283 grown and analyzed at JLU; iii) sequences produced by the culture CBS 119283
grown and analysed at DTU.
2.2. Peptaibiotic Pattern of the Teleomorph. Notably, the teleomorph of Trichoderma
phellinicola proved to be a prolific source of ten 20-residue peptaibols, compounds 1–
10, displaying the characteristic building scheme of subfamily 1 (SF1), one of the nine
peptaibol subfamilies (Fig. 1,b, and Tables 1 and 2), as introduced by Chugh and
Wallace [52]
4
).
One Gln residue is found in position 7, and another one towards or at the C-
terminus in position 18, whereas position 19 is either occupied by a third Gln or a Glu
residue. A highly conserved Pro residue is located in position 14 of the peptide chain.
All sequences have a Gly residue in position 11 and terminate in Pheol. At least seven,
at most nine, residues are occupied by Aib. Variable amino acid residues are located in
positions 2, 6, 17, and 18 (Fig. 1,b).
Most of the peptaibols sequenced resemble previously described compounds
(Fig. 1,b,Table 1, and Fig. 2,a) such as longibrachins A and B [53], trichobrachins II
[57], trichoaureocins [54], trichokonins [55] [62][63], and suzukacillins A [60].
2.3. Peptaibiotic Pattern of the Culture.2.3.1. General Considerations. As observed
before [20], ascospores of T.phellinicola are unstable and die rapidly after collecting.
This might have been the reason why no agar culture could be obtained from our
specimen. As a substitute, the ex-type culture of T. phellinicola CBS 119283 (¼C.P.K.
2137) was provided, and its peptaibiotic pattern was analyzed. Except for the two
lipopeptaibols 48 and 49, the remaining compounds 11–47 represent the characteristic
building scheme of SF1, resembling the previously described 20-residue peptaibols
suzukacillins A, trichosporins B, and trichocellins A [60][61] [64 –67] .
2.3.2. micrOTOF-Q II Screening. In contrast to the specimen analyzed, the ex-type
plate culture grown and analyzed at the Justus Liebig University of Giessen (JLU)
produced two new 18- and fifteen new 19-residue peptaibols, compounds 11–27, which
lacked the [Ala/Aib]6residue of the 20-residue peptaibols found in the specimen
(Tables 3 and 4, and Fig. 2,b). The two truncated 18-residue sequences, compounds 11
and 12, terminated in free Gln. Sequences 14 and 16 –27 carry a C-terminal Pheol. For
compounds 13 and 15, a C-terminal tyrosinol residue (abbreviated as Tyrol) was
tentatively deduced from HR-ESI-MS/MS data (Tables 3 and 4,Fig. 3).
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 791
4) These subfamilies were introduced at a time when the total number of peptaibiotics described did
not exceed 200 sequences. As of October 2012, ca. 1,000 individual sequences are known, which also
exhibit new building schemes and constituents. Consequently, there is an urgent need to reconsider
this classification.

2.3.3. maXis Screening. All SF1 peptaibiotics, compounds 12,14,19,28–47,ofthe
ex-type plate culture grown and analyzed at DTU (Tables 5 and 6, and Fig. 2,c) exhibit
the characteristic deletion of the Ala/Aib residue in position 6. However, different
positional isomers and homologues were found, e.g., the 17-residue deletion sequence
29, lacking the C-terminal dipeptide [Gln18Pheol19]. In compound 31, a Ser-residue
was found in position 3, whereas compound 30 exhibited a Gly residue in position 4.
Overall, the structural diversity of peptaibiotics produced by the two cultures was much
higher as compared to the specimen: variable amino acid residues were in positions 2, 3,
4, 5, 6, 17, 18, and 20 (Fig. 1,b).
2.4. Lipopeptaibols as Trace Components in the Plate Cultures. Two lipopeptaibols,
compounds 48 and 49, were produced as trace components in the DTU plate culture.
Compound 49 probably represents trichogin A IV [68] [69] or a positional isomer
thereof. The new positionally isomeric compound 48, named lipophellin 1, is
characterized by the deletion of [Gly]5of compound 49 (Tables 5 and 6, and Fig. 2,c).
3. Discussion. – 3.1. Hypophellins, Novel Long-Chain Peptaibiotics from T.
phellinicola.The most notable result of this investigation is, indeed, the unequivocal
confirmation of peptaibiotic biosynthesis in the natural habitat of T. phellinicola
growing on its host Phellinus ferruginosus, commonly known as the Rusty Porecrust.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)792
Table 1. Sequences of 20-Residue Peptaibiotics Detected in the Specimen of Hypocrea phellinicola
No. tR[min] [MþH]þResidue
12a)3 4 5 67891011
137.8–38.1 1937.1209 Ac Aib Ala Aib Ala Aib Ala Gln Aib Vxx Aib Gly
237.8–38.1 1938.1068 Ac Aib Ala Aib Ala Aib Ala Gln Aib Vxx Aib Gly
339.1–39.3 1951.1358 Ac Aib Ala Aib Ala Aib Ala Gln Aib Vxx Aib Gly
439.8–40.0 1952.1192 Ac Aib Ala Aib Ala Aib Ala Gln Aib Vxx Aib Gly
540.2–40.4 1951.1416 Ac Aib Ala Aib Ala Aib Aib Gln Aib Vxx Aib Gly
641.0– 41.2 1952.1258 Ac Aib Ala Aib Ala Aib Aib Gln Aib Vxx Aib Gly
741.3– 41.7 1965.1615 Ac Aib Ala Aib Ala Aib Aib Gln Aib Vxx Aib Gly
842.3–42.5 1966.1354 Ac Aib Ala Aib Ala Aib Aib Gln Aib Vxx Aib Gly
943.0–43.2 1979.1718 Ac Aib Aib Aib Ala Aib Aib Gln Aib Vxx Aib Gly
10 44.0–44.3 1980.1636 Ac Aib Aib Aib Ala Aib Aib Gln Aib Vxx Aib Gly
a) Variable residues are underlined in the table header. Minor sequence variants are underlined in the

We here describe for the first time the in vivo detection of non-ribosomal peptide
antibiotics
5
), which may significantly contribute to the complex interaction of a
fungicolous ascomycete growing on its basidiomycetous host.
3.2. The Peptaibiome of the Specimen. The teleomorph produced a microheteroge-
neous mixture of ten 20-residue HPHs, four of which, 6,8,9, and 10, are new (Table 1).
Compared to smaller sequences consisting of less than 17 residues, long-chain
peptaibiotics display a higher membrane-pore-formation activity by several orders of
magnitude [71].
Depending on the individual sequence, seven to nine Aib residues are present,
which strongly promote the formation of helical structures. i.e., a-or310-helices, and
even mixed forms [72 –74], which is due to the steric constraints imposed by the
geminal Me groups of the Ca-atom [75]. All of them exhibit the structurally important
features, which are required for the formation of transmembrane ion channels in
artificial lipid bilayer membranes, as compiled by Duclohier [76], and Duclohier and
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 793
(micrOTOF-Q II screening)
Compound identical or positionally
isomeric with
Ref.
12 13 14 15 16 17 18 19 20
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol Longibrachin A I [53]
Trichoaureocin 3 [54]
Trichokonin VI (¼gliodeliquescin A) [55] [56]
Trichobrachins II-5, II-6 [57]
Trichobrachin IIb A [58] [59]
Lxx Aib Pro Vxx Aib Aib Glu Gln Pheol Longibrachin B II [53]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol Trichokonin VII [55]
Trichoaureocin 4 [54]
Suzukacillin A-10a [60]
Trichobrachins II-7, II-8, II-9 [57]
Trichobrachin IIb B [58] [59]
Lxx Aib Pro Vxx Aib Vxx Glu Gln Pheol Longibrachin B III [53]
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol Trichokonin VIII (¼trichosporin B-IVc) [55] [61]
Trichoaureocin V [54]
Trichobrachin IIb C [58] [59]
Lxx Aib Pro Vxx Aib Aib Glu Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol Longibrachin A IV [53]
Trichoaureocin VI [54]
Trichobrachin IIb D [58] [59]
Lxx Aib Pro Vxx Aib Vxx Glu Gln Pheol New (longibrachin IV: [ Gln]18 ![Glu]18)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (homolog of 7)
Lxx Aib Pro Vxx Aib Vxx Glu Gln Pheol New (homolog of 8)
sequences. This applies to Tables 1,3, and 5.
5) Hypophellins were simultaneously detected in an LC/MS/MS screening of 15 specimens belonging
to nine Hypocrea species, which have been collected in their natural habitat. Recently, a manuscript
on the in vivo detection of hypopulvins, novel peptaibiotics from the polyporicolous fungus H.
pulvinata, has been published. The results therein corroborate that peptaibiotics are produced by a
fungicolous fungus during infection of its natural hosts [70].

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)794
Table 2. Diagnostic Fragment Ions of 20-Residue Peptaibiotics Detected in the Specimen of Hypocrea phellinicola (micrOTOF-Q II screening)
Diagnostic fragment ions Peaks [m/z]a)
12345678910
tR[min] 37.8–38.1 38.6 – 38.7 39.1 – 39.3 39.8 –40.0 40.2 –40.4 41.0 – 41.2 41.3– 41.7 42.3–42.5 43.0 – 43.2 44.0 – 44.3
[MþNa]þ1959.1047 1960.0872 1962.1376 n.d. 1973.1212 1974.1064 1987.1372 1988.1245 2001.1535 2002.1445
[MþH]þ1937.1209 1938.1036 1951.1358 1952.1192 1951.1416 1952.1258 1965.1615 1966.1354 1979.1718 1980.1636
a1100.0808 100.0808 100.0808 100.0806 100.0809 100.0805 100.0808 100.0807 n.d. n.d.
a2171.1181 171.1181 171.1197 171.1195 171.1188 171.1185 171.1191 171.1200 185.1315 185.1311
a3256.1657 256.1657 256.1662 256.1663 256.1666 256.1665 256.1669 256.1671 270.1811 270.1808
a4327.2121 327.2121 327.2155 327.2142 327.1992 327.2097 327.2102 327.2116 341.2238 341.2180
a5n.d. n.d. 412.2739 412.2739 412.2572 412.2720 412.2776 412.2766 n.d. n.d.
b1128.0758 128.0758 128.0762 128.0757 128.0763 128.0758 128.0765 128.0760 128.0758 128.0748
b2199.1102 199.1102 199.1109 199.1107 199.1111 199.1111 199.1113 199.1115 213.1251 213.1251
b3284.1604 284.1604 284.1615 284.1614 284.1618 284.1615 284.1623 284.1625 298.1845 298.1802
b4355.1982 355.1982 355.1973 355.1972 355.1981 355.1976 355.1986 355.1988 369.2109 369.2144
b5440.2479 440.2479 440.2494 440.2492 440.2502 440.1497 440.2508 440.2510 454.2669 454.2699
b6511.2839 511.2839 511.2850 511.2852 525.3019 525.3015 525.3023 525.3026 539.3231 539.3231
b7639.3431 639.3431 639.3455 639.3451 653.3661 653.3626 653.3690 653.3681 667.3870 667.3881
b8724.3937 724.3937 724.3961 724.3957 738.4118 738.4109 738.4130 738.4132 752.4381 752.4367
b9823.4750 823.4590 823.4611 823.4601 837.4777 837.4772 837.4790 837.4795 851.5058 851.5067
b10 908.5298 908.5095 908.5131 908.5129 922.5302 922.5290 922.5314 922.5317 936.5594 936.5602
b11 965.5490 965.5311 965.5470 965.5316 979.5501 979.5476 979.5508 979.5506 993.5822 993.5819
b12 1078.6366 1078.6151 1078.6340 1078.6138 1092.6478 1092.6289 1092.6325 1092.6326 1106.6642 1106.6710
b13 1163.6824 1163.6642 1163.6810 1163.6662 1177.6994 1177.6816 1177.6853 1177.6859 1191.7215 1191.7196
y7774.4598 775.4614 788.4742 789.4595 774.4586 775.4436 788.4750 789.4596 788.4750 789.4596
y7–H
2O 756.4445 757.4491 n.d. 771.4507 n.d. 757.4308 n.d. 771.4468 n.d. 771.4468
y7– AA (20) 623.3556 624.3581 637.3711 638.3573 623.3563 624.3414 637.3722 638.3576 637.3722 638.3576
y7– AA (20-19) 495.2979 496.2999 509.3124 510.2961 495.2955 496.2793 509.3120 510.2962 509.3120 510.2962
y7– AA (20-18) 367.2385 367.2350 381.2515 381.2517 367.2364 367.2358 381.2519 381.2514 381.2519 381.2514
y7– AA (20-17) 282.1900 282.1831 282.1850 282.1820 282.1853 282.1838 282.1839 282.1839 282.1839 282.1839
a) n.d., Not detected.

Wro
´blewski [77]. A multitude of bioactivities has been described for 20-residue
peptaibols of similar structure, which are compiled in Table 7.
3.3. The Peptaibiome of the Ex-Type Plate Culture. In contrast to what has been
observed for the specimen, 20-residue peptaibols could not be detected. Instead, fifteen
19-residue peptaibols were detected in the micrOTOF-Q II screening and another
eighteen in the maXis screening. Although sequences of 11 –47 still exhibit the
characteristic building scheme of SF1, they are distinguished from the 20-residue
peptaibols of the teleomorph specimen by a deletion of the Aib/Ala residue in position
6(DAla/Aib6) of the peptide chain. This deletion, however, is predicted not to
negatively influence the bioactivity of these long-chain peptaibols, as all important
structural features are still present, which comply with the requirements for the
formation of transmembrane ion channels in artificial lipid bilayer membranes
[76][77]. The three 18-residue sequences, 11,12, and 28, exhibit a deletion of the C-
terminal amino alcohol, whereas the dipeptide [Gln18Pheol19] is deleted in 29, a 17-
residue sequence. Truncated versions of SF1 peptaibols lacking the C-terminal amino
alcohol or even the adjacent Gln residue have been reported before.
The ten 19-residue peptaibiotics, trichobrachins I (TB I) , lacking the C-terminal
Pheol residue, as well as the two 18-residue trichobrachins II-1 and -2 (TB II), which
exhibit a deletion of the C-terminal dipeptide [Gln19Pheol20], were shown to originate
from 20-residue trichobrachins II (TB II) by enzymatic degradation [57]. Two minor
desPheol compounds F30, representing 1.3% of the alamethicin (ALM) mixture
investigated, have been detected by non-aqueous capillary electrophoresis (NACE)
coupled to electrospray mass spectrometry [94].
3.4. l-Phenylalaninol as Constituent of Natural Products. C-Terminal l-Pheol is
commonly found in peptaibiotics [17] [18] but has also been infrequently reported as a
constituent of other plant and fungal secondary metabolites such as N-benzoyl-l-
phenylalaninol from Catharanthus pusillus [95] and Diospyros quaesita [96], O-acetyl-
N-(N’-benzoyl-l-phenylalanyl)-l-phenylalaninol from Euphorbia fischeriana and E.
kansui [97], and N-benzoyl-O-[N’-benzoyl-l-phenylalanyl]-l-phenylalaninol from
Penicillium arenicola (syn. P.canadense)[98].
3.5. l-Tyrosinol as a Constituent of Natural Products. To the best of our knowledge,
neither d- nor l-tyrosinol
6
) has ever been reported as constituent of either linear or
cyclic peptides of microbial origin, including peptaibiotics. However, l-tyrosinol is a
cryptic building block of the following natural products:
farinosone C, an amide from Paecilomyces farinosus RCEF 0101 [99];
cordyceamides A and B from a liquid culture of Cordyceps sinensis [100] ;
preoxazinin-7, the linear precursor [101] , and cyclic oxazinins from the digestive
glands of Mytilus galioprovincialis [102] [103].
3.6. The Lifestyle of Trichoderma phellinicola: Findings and Thoughts. Taken these
findings together, we dare predict a mycoparasitic lifestyle of the host-specific
polyporicolous Trichoderma phellinicola:
It has been demonstrated by in vitro studies that chitinases and b-1,3-glucanases act
synergistically with peptaibiotics in inhibiting spore germination and hyphal elongation
of Botrytis cinerea. Parallel formation of hydrolytic enzymes and 19-residue antifungal
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 795
6) C-Terminal b-amino alcohols with the d-configuration have not yet been reported for peptaibiotics.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)796
Fig. 2. Base-peak chromatograms (BPCs) of a) the H. phellinicola specimen screened with the
micrOTOF-Q II,b) the H. phellinicola ex-type plate culture screened with the micrOTOF-Q II, and
c) the H. phellinicola specimen screened with the maXis. †, co-eluting peptaibiotics, not sequenced;
‡, non-peptaibiotic metabolite.

trichorzianins A and B by the potent mycoparasite Trichoderma atroviride
7
)is
triggered in the presence of cell walls of plant-pathogenic fungi [106]. Trichorzianins
have previously been shown to form voltage-gated ion channels in planar lipid bilayers
[107] and to modify the membrane permeability of liposomes, and they are active
against Rhizoctonia solani and Phythophthora cactorum [108]. Based on these findings,
a model of how peptaibiotics such as trichorzianins and hydrolases interact synergisti-
cally was proposed.
First, the host cell wall is digested enzymatically; thereafter, peptaibiotics will
penetrate the cell membrane to form ion channels. Cell leakage reduces the ability of
the host to effectively repair its cell wall. Eventually, inhibition of chitin and b-glucan
synthesis further amplifies the destructive effect of chitinases and b-1,3-glucanases
[108]. These mechanisms, however, may also account for the recently published
induction of programmed cell death in plant fungal pathogens [47] caused by the 20-
residue peptaibol trichokonin VI (¼gliodeliquescin A [56])
8
), from T. koningii,T.
pseudokoningii, and T. deliquescens (syn. Gliocladium deliquescens) [20]. The presence
of peptaibiotics was also shown to play a role in the induction of plant defence
responses [110].
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 797
Fig. 2 (cont. )
7) The trichorzianin-producing strain ATCC 36042 ( ¼CBS 391.92) was originally identified as T.
harzianum [104] but later shown to belong to T.atroviride [105]. The high degree of
misidentification of Trichoderma species prior to introduction of phylogenetic analysis is still
regarded a major problem, unless authors describe how their cultures were identified [17].
8) Gliodeliquescin A has been isolated from Gliocladium deliquescens NRRL 1086 [109] and not from
NRRL 3091 [56]. According to phylogenetic data (18S-rRNA, and ITS 1 and 2), G.deliquescens
NRRL 1086 (¼CBS 228.48¼ATCC 10097) was re-identified as G.viride (www.straininfo.net/
strains/260309).

3.7. Remarks on Non-Ribosomal Biosynthesis and Module Skipping by T.
phellinicola. The exclusive production of 20-residue peptaibols by the T. phellinicola
teleomorph indicates the presence of a 20-module NRPS. As the culture CBS 119283
has been shown to produce 17-, 18-, and 19-residue peptaibiotics only, it is likely to
contain a 19-module NRPS, lacking the 6th module activating Ala or Aib. In addition,
modules 3 and 4 show differing substrate specificities, as compared to the teleomorph,
thus permitting the incorporation of Ala or Ser in position 3 and of Gly, Ala, or Ser in
position 4, respectively. These findings indicate substantial variations in the sequences
of the SF1-type peptaibol synthetases of both strains. As has been discussed in the case
of SF4-type peptaibols, genes involved in secondary-metabolite products show a much
broader sequential variety than housekeeping genes [50]. We here, indeed, find
evidence for a significant structural variation within a large gene.
Experimental Part
Chemicals. All solvents used, MeCN (99.9%), MeOH (99.9%) , CH2Cl2(99.8%) , and HCOOH
(98%), were of LC/MS grade from Sigma-Aldrich (D-Steinheim). Water was purified by a Merck-
Millipore Milli-Q Synthesis A10 system (D-Schwalbach/Ts.) .
Origin of Specimen. The teleomorphic specimen of Trichoderma phellinicola growing on its host
Phellinus ferruginosus was collected in the Na
´rodn park Podyj (Czech Republic, Moravia), near
Hardegg at the bridge across the River Thaya, just across the border between Austria and the Czech
Republic.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)798
Table 3. Sequences of 18- and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS 119283) of
No. tR[min] [MþH]þResidue
12 3 4 5 6 7 8 9 10 11
11 30.9–31.1 1747.0135 Ac Aib Ala Aib Ala Ala – Gln Aib Lxx Aib Gly
12 31.8– 32.0 1761.0324 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
13 32.2–32.6 1896.0995 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
14 32.5–32.7 1910.1131 Ac Aib Ala Aib Ser Aib – Gln Aib Lxx Aib Gly
15 32.8–33.1 1910.1140 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
16 35.1–35.3 1896.1035 Ac Aib Ala Ala Ser Aib – Gln Aib Lxx Aib Gly
17 37.0–37.2 1866.0928 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
18 37.7–37.9 1880.1095 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
19 38.3–38.4 1880.1136 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
20 38.8–39.2 1894.1331 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
21 39.8–40.1 1895.1278 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
22 40.6–40.9 1908.1474 Ac Aib Ala Aib Ala Vxx – Gln Aib Lxx Aib Gly
23 41.5– 41.6 1909.1391 Ac Aib Ala Aib Ala Vxx – Gln Aib Lxx Aib Gly
24 42.1–42.3 1922.1601 Ac Aib [255] Ala Aib – Gln Aib Lxx Aib Gly
25 43.4–43.6 1936.1738 Ac Aib [269] Ala Aib – Gln Aib Lxx Aib Gly
26 44.2–44.4 1936.1750 Ac Aib Vxx Aib Ala Vxx – Gln Aib Lxx Aib Gly
27 45.0–45.6 1950.1894 Ac Aib Lxx Aib Ala Vxx – Gln Aib Lxx Aib Gly

Origin of Trichoderma phellinicola CBS 119283 (ex-type). All details concerning this new species
were given by Jaklitsch [20].
Extraction of Specimens. The teleomorph was extracted with CH2Cl2/MeOH 1:1 (v/v), the solvent
was evaporated in vacuo (Rotavapor R-215,Bchi, D-Essen), and the extract was cleaned up over Sep-
Pak Classic C18 cartridges (Waters, D-Eschborn) as described by Krause et al. [48].
Cultivation and Extraction of Pure Cultures. Cultures of the specimen were grown on potato dextrose
agar (PDA; Becton Dickinson, D-Heidelberg) at 238for 6 d. These subcultures were used for inoculation
of the main cultures. After 10 d of cultivation at 238in the dark, main cultures were extracted as described
for the teleomorph.
LC/MS Analysis. Two QTOF systems, both from Bruker Daltonic (D-Bremen) controlled by HyStar
v. 3.2 were used. Both instruments were equipped with an orthogonal ESI source and coupled to a Dionex
UltiMate 3000 UHPLC (Dionex, D-Idstein) .
System 1: high-resolution micrOTOF Q-II mass spectrometer. For separation, an Acclaim 120 C8,
3mm, 2.1150 mm, column ( Dionex, D-Idstein) at a flow rate of 0.25 ml/min1and a temp. of 358was
used. Eluent Aconsisted of H2Oþ0.1% HCOOH and eluent Bof 95% MeCN þ0.1% HCOOH.
Subsamples of 10 ml were injected. The column was held at 80% A/20% Bfor 5 min, then a gradient from
20% Bto 100% over 55 min was applied. Thereafter, the column was held at 100% Bfor 15 min, returned
to the start conditions in 1 min, and finally equilibrated for 14 min.
Samples were screened for peptaibiotics in the positive-ion mode using the following three-step
routine procedure: first a full scan was recorded from m/z50 to 3000. In System 1, this was followed by
CID measurements from m/z50 to 2000, recorded at energy of 150 eV. Finally, results of CID-MS were
verified by MS/MS experiments on selected precursor ions. For precursors of m/z<1000, a collision
energy of 30 eV was applied, precursor ions in the m/zrange from 1000 to 1500 were fragmented at a
collision energy of 35 eV and precursor ions of m/z>1500 at a collision energy of 40 eV. The isolation
width for MS/MS experiments was set to 1 Da.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 799
Hypocrea phellinicola (micrOTOF-Q II screening)
Compound identical or positionally isomeric with Ref.
12 13 14 15 16 17 18 19 20
Lxx Aib Pro Vxx Aib Vxx Gln Gln New (trichocellin A-VI – [Aib]5– Pheol) [67]
Lxx Aib Pro Vxx Aib Vxx Gln Gln New (trichocellin A-VI – [Ala]6– Pheol) [67]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Tyrol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Tyrol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New (trichosporin B IIIa – [Aib]6) [64] [61]
New (trichobrachin IIb A – [Ala]6) [58][59]
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New (suzukacillin A-11a – [Ala]6) [60]
New (trichosporin B-VIa – [Aib]6) [61]
New (trichosporin B-VIIa – [Aib]6) [66]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (trichosporin B-IVb – [Aib]6,
trichosporin B-VIb – [Aib]6)
[61]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (suzukacillin A-10b – [Ala]6) [60]
Lxx Aib Pro Vxx Aib Vxx Glu Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Glu Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol –
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol –
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New

System 2: The maXis 3G QTOF mass spectrometer operated at a resolution of 40,000 FWHM. An
Acquity BEH300 C18,1.7mm, 2.1150 mm, column ( Waters, D-Eschborn) was used for separation, using
H2Oþ0.1% HCOOH (eluent A) and 100% MeCN þ0.1% HCOOH (eluent B). The flow rate was set to
0.3 ml/min and the temp. to 408. The gradient started with 90% A/10% Band was changed to 50% A/50%
Bat 7 min, then to 30% A/70 % Bat 25 min, then raised to 100% Bat 38 min, and held at 100% Buntil
41 min before setting to starting conditions from time 42 min to 46 min. Three ml were injected. MS were
scanned in the m/zrange of 100–2,000. Auto MS with precursor ion-dependent collision energy
optimization was used for fragmentation in the range of 10–65 eV.
Data interpretation was performed using the DataAnalysis v. 4.0 software (Bruker Daltonic,D-
Bremen). Use of high-resolution (HR)ESI-MS allowed the unequivocal sequencing of fragment-ion
series according to the Roepstorff/FohlmanBiemann nomenclature. In cases where the isomeric amino
acids (Leu/Ile and Val/Iva, resp.) or the corresponding amino alcohols (Leuol/Ileol) with the same
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)800
Table 4. Diagnostic Fragment Ions of 18- and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS
Diagnostic fragment Peaks [m/z]a)
ions 11 12 13 14 15 16 17 18
tR[min] 30.9–31.1 31.8–32.0 32.2 – 32.6 32.5 –32.7 32.8 –33.1 35.1–35.3 37.0 – 37.2 37.7 – 37.9
[MþNa]þ1768.9850 1783.0115 1918.0846 1932.0976 1932.1017 1918.0877 1888.0616 1902.0891
[MþH]þ1747.0135 1761.0324 1896.0995 1910.1131 1910.1140 1896.1035 1866.0928 1880.1095
a1100.0718 n.d. n.d. n.d. n.d. 100.0720 100.0720 n.d.
a3256.1647 256.1624 242.1508 256.1641 256.1707 242.1511 242.1506 256.1675
a4n.d. n.d. n.d. n.d. 327.1979 n.d. n.d. 327.2046
a5n.d. n.d. n.d. n.d. n.d. n.d. 398.2312 n.d.
b1128.0687 128.0658 128.0709 128.0708 128.0835 128.0715 128.0719 128.0721
b2199.1075 199.1076 199.1109 199.1110 199.1161 199.1118 199.1115 199.1081
b3284.1611 284.1617 270.1453 284.1622 284.1637 270.1434 270.1471 284.1634
b4355.1972 355.1977 341.1846 371.1863 355.2023 357.1765 341.1815 355.1988
b4–H
2O n.d. n.d. n.d. 353.1758 n.d. 339.1676 n.d. n.d.
b5426.2340 440.2546 426.2354 456.2441 440.2494 442.2277 426.2314 440.2546
b6554.2840 568.3175 554.2840 584.3226 568.3175 570.2870 554.2989 568.3023
b7639.3523 653.3691 639.3539 669.3625 653.3679 655.3443 639.3530 653.3685
b8752.4400 766.4531 752.4386 782.4408 766.4563 768.4296 752.4353 766.4519
b9837.4860 851.5024 837.4880 867.4961 851.5028 853.4825 837.4896 851.5066
b10 894.5048 908.5271 894.5061 924.5223 908.5250 910.5022 894.5076 908.5242
b11 1007.5856 1021.6063 1007.5967 1037.6039 1027.6073 1023.5862 1007.5917 1021.6085
b12 1092.6441 1106.6573 1092.6442 1122.6523 1106.6575 1108.6413 1092.6474 1106.6629
b12 –H
2O n.d. n.d. n.d. n.d. n.d. 1090.6265 1074.6077 1088.6332
y6655.3841 655.3841 – – – – – –
y6–AA (18) 509.3130 509.3130 – – – – – –
y6–AA (18-17) 381.2540 381.2540 – – – – – –
y6–AA (18-16) 282.1709 282.1709 – – – – – –
y7– – 804.4624 788.4706 804.4669 788.4697 774.4592 774.4593
y7–H
2O – – 786.4472 770.4510 n.d. 770.4510 756.4383 756.4383
y7–AA (19) – – 637.3680 637.3708 637.3725 637.3705 623.3566 623.3559
y7–AA (19-18) – – 509.3068 509.3140 509.3085 509.3103 495.2961 495.2962
y7–AA (19-17) – – 381.2489 381.2515 381.2545 381.2513 367.2370 367.2373
y7–AA (19-16) – – n.d. n.d. 282.1814 282.1814 282.1815 282.1815
a) n.d., Not detected.

elemental composition could not be distinguished, the abbreviations Lxx, Vxx, and Lxxol were used
instead [48–50] .
This study was supported by the Hessian Ministry for Science and Art by a grant from theLOEWE-
Schwerpunkt program Insect Biotechnology to A. V. DTU acknowledges the grant from the Danish
Research Council (FI 2136-08-0023) for the maXis QTOF system, and MYCORED ( EC KBBE-2007-
222690-2) for supporting A. I. Support by the Austrian Science Fund (FWF; project P22081-B17) is
acknowledged by W. M. J. The authors are indebted to Prof. Dr. Hartmut Laatsch (Institute of Organic
and Biomolecular Chemistry, University of Gçttingen, Germany) for his valuable comments on the
occurrence of tyrosinol as a constituent of natural products.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 801
119283) of Hypocrea phellinicola (micrOTOF-Q II screening)
19 20 21 22 23 24 25 26 27
38.3–38.4 38.8 – 39.2 39.8 – 40.1 40.6 –40.9 41.5 – 41.6 42.1 – 42.3 43.4–43.6 44.2 – 44.4 45.0 –45.6
1902.0921 1916.1081 1917.1085 1930.1235 1931.1236 1944.1425 1958.1599 1958.1548 1972.1635
1880.1136 1894.1331 1895.1278 1908.1474 1909.1391 1922.1601 1936.1738 1936.1750 1950.1894
100.0721 100.0721 100.0747 100.0722 100.0722 100.0722 n.d. n.d. n.d.
242.1514 256.1682 256.1682 256.1677 256.1649 n.d. n.d. n.d. n.d.
313.1832 327.2048 327.2049 327.2042 327.2050 n.d. n.d. n.d. n.d.
n.d. 412.2533 412.2564 426.2817 n.d. n.d. n.d. n.d. n.d.
128.0722 128.0724 128.0718 128.0720 128.0708 128.0712 128.0672 128.0701 128.0684
199.1121 199.1081 199.1118 199.1083 199.1141 [255] [269] 227.1404 241.1564
270.1476 284.1608 284.1608 284.1641 284.1631 312.1955 326.2055
341.1814 355.1988 355.1973 355.1972 355.1972 383.2306 397.2427 383.2297 397.2477
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
426.2314 440.2531 440.2513 454.2685 454.2686 468.2836 482.3001 482.2988 496.3148
554.2989 568.3022 568.3119 582.3249 582.3249 596.3361 610.3531 610.3608 624.3766
639.3513 653.3673 653.3654 667.3841 667.3836 681.3976 695.4131 695.4110 709.4286
752.4346 766.4505 766.4489 780.4662 780.4659 794.4802 808.4949 808.4934 822.5109
837.4888 851.5044 851.5023 865.5205 865.5199 879.5335 893.5492 893.5457 907.5631
894.5075 908.5234 908.5216 922.5386 922.5395 936.5517 950.5713 950.5659 964.5813
1007.5920 1021.6065 1021.6039 1035.6228 1035.6231 1049.6347 1063.6526 1063.6516 1077.6661
1092.6463 1106.6606 1106.6578 1120.6786 1120.6785 1134.6898 1148.7069 1148.7051 1162.7188
1074.6284 1088.6331 1088.6424 1102.6441 1102.6440 1116.6595 1130.6997 1130.7031 1144.7051
––––– – –––
––––– – –––
––––– – –––
––––– – –––
788.4710 788.4710 789.4647 788.4718 789.4597 788.4710 788.4705 788.4678 788.4668
770.4509 770.4509 771.4475 770.4508 771.4390 770.4507 770.4507 770.1538 770.1538
637.3707 637.3707 638.3638 637.3705 638.3574 637.3721 637.3678 637.3649 637.3676
509.3096 509.3096 510.3014 509.3108 510.2964 509.3105 509.3113 509.3093 509.3082
381.2513 381.2513 381.2483 381.2524 381.2520 381.2505 381.2508 381.2506 381.2492
n.d. n.d. 282.1837 282.1813 282.1813 282.1813 282.1920 282.1781 282.1917

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)802
Table 5. Sequences of 10-, 11-, 17-, 18-, and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS
No. tR[min] [MþH]þResidue
12
3 4 5 6 7 8 9 10 11
28 10.8 1747.0131 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
12 11.2 1761.0273 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
14 12.2 1911.1213 Ac Aib Ala Aib Ser Aib – Gln Aib Lxx Aib Gly
29 12.6 1632.9708 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
30 13.0 1880.1000 Ac Aib Ala Aib Gly Aib – Gln Aib Lxx Aib Gly
31 13.2 1882.0784 Ac Aib Ala Ser Ala Aib – Gln Aib Lxx Aib Gly
19 13.5 1880.1008 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
32 14.1 1896.0964 Ac Aib Ala Ser Ala Aib – Gln Aib Lxx Aib Gly
33 14.9 1880.1035 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
34 15.5 1866.0863 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
35 15.9 1880.1012 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
36 16.2 1867.0706 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
37 16.4 1880.1007 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
38 16.7 n.d. Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
39 16.8 1880.1009 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
40 17.0 n.d. Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
41 17.2 1880.0997 Ac Aib Ala Ala Ala Aib – Gln Aib Lxx Aib Gly
42 17.5 1894.1210 Ac Aib Ala Aib Ala Vxx – Gln Aib Lxx Aib Gly
43 17.7 1895.1007 Ac Aib Ala Aib Ala Aib – Gln Aib Lxx Aib Gly
44 18.0 1894.1177 Ac Aib Ala Ala Ala Vxx – Gln Aib Lxx Aib Gly
45 18.6 1908.1341 Ac Aib Ala Aib Ala Vxx – Gln Aib Lxx Aib Gly
46 20.0 1922.1467 [227]a) Aib Ala Aib – Gln Aib Lxx Aib Gly
47 21.5 1936.1660 [241] b) Aib Ala Aib – Gln Aib Lxx Aib Gly
48 22.0 1009.7031 Occ) Aib Gly Lxx Aib – Gly Lxx Aib Gly Lxx Lxxol
49 22.1–22.2 1066.7242 Oc Aib Gly Lxx Aib Gly Gly Lxx Aib Gly Lxx Lxxol
a) The N-terminal sequence of compound 46, which is represented by a mass difference of 227 Da, could
241 Da, could not be assigned. c) Oc, Tentatively assigned as n-octanoyl residue.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 803
119283) of Hypocrea phellinicola (maXis screening)
Compound identical or positionally
isomeric with
Ref.
12 13 14 15 16 17 18 19 20
Lxx Aib Pro Vxx Aib Aib Gln Gln New (trichocellin A-V – [Ala]6– Pheol) [67]
Lxx Aib Pro Vxx Aib Vxx Gln Gln New (trichocellin A-VI – [ Ala]6– Pheol) [67]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln New (12 – [Gln]18)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (17: [Ala]4![Gly]4)
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (trichosporin B-IVb – [Aib]6,
trichosporin B-VIb – [Aib]6)
[61]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (positional isomer of 19,37, and 41)
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New ( positional isomer of 17)
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New (trichosporin B-VIa – [ Aib]6,
trichosporin B-VIIb – [Aib]6)
[61][66]
Lxx Aib Pro Vxx Aib Aib Glu Gln Pheol New (35 : [ Gln]17 ![ Glu]17)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (positional isomer of 19,33, and 41)
Lxx Aib Pro Vxx Aib Aib Glu Gln Pheol New (39 : [ Gln]17 ![ Glu]17)
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New ( positional isomer of 35)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (trichosporin B-VIIa – [Aib]6) [66]
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (positional isomer of 19,33, and 37)
Lxx Aib Pro Vxx Aib Aib Gln Gln Pheol New
Lxx Aib Pro Vxx Aib Vxx Glu Gln Pheol New (positional isomer of 40)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (positional isomer of 45)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol New (positional isomer of 44)
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol –
Lxx Aib Pro Vxx Aib Vxx Gln Gln Pheol –
New
Trichogin A IV [68]
Sequence 13 or 14 from Trichoderma cf.
strigosum CBS 119777
[49]
Partial sequence 4 from Hypocrea citrina
CBS 853.70
[48]
Partial sequence 4 from Hypocrea vinosa
CBS 247.63
[48]
not be assigned. b) The N-terminal sequence of compound 47, which is represented by a mass difference of

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)804
Table 6. Diagnostic Fragment Ions of 10-, 11-, 17-, 18-, and 19-Residue Peptaibiotics Detected in the Ex-
Diagnostic fragment ions Peaks [m/z]a)
28 12 14 28 30 31
tR[min] 10.8 11.2 12.2 12.6 13.0 13.2
[MþH]þ1747.0131 1761.0273 1911.1213 1632.9708 1880.1000 1882.0784
b1n.d. n.d. n.d. n.d. n.d. n.d.
b2n.d. 199.1093 n.d. 199.1087 199.1087 199.1123
b3284.1601 284.1607 284.1613 284.1609 284.1604 286.1389
b4355.1989 355.1980 371.1938 355.1975 341.1819 357.1760
b4–H
2O n.d. n.d. 438.2353 n.d. 412.2541 424.2167
b5440.2512 440.2509 456.2470 440.2506 426.2347 442.2296
b6568.3097 568.3098 584.3039 568.3096 554.2926 570.2869
b7653.3615 653.3626 669.3571 653.3619 639.3458 655.3404
b8766.4456 766.4471 782.4415 766.4461 752.4294 768.4257
b9851.5003 851.4996 867.4953 851.4987 837.4826 853.4789
b10 908.5192 908.5208 924.5190 908.5199 894.5026 910.4971
b11 1021.6077 1021.6046 1038.5981 1021.6053 1007.5901 1023.5860
b12 1106.6578 1106.6578 1122.6537 1106.6590 1092.6412 1108.6356
b12 –H
2O n.d. n.d. n.d. n.d. n.d. n.d.
y5– – – 527.3191 – –
y5–AA (17) – – – 381.2497 – –
y5–AA (17-16) – – – 282.1814 – –
y5–AA (17-15) – – – 197.1287 – –
y6641.3626 655.3768 – – – –
y6–AA (18) 495.2923 509.3095 – – – –
y6–AA (18-17) 367.2353 381.2500 – – – –
y6–AA (18-16) 282.1812 282.1816 – – – –
y6–AA (18-15) 197.1274 197.1288 – – – –
y7– – 788.4676 – 788.4676 774.4501
y7–H
2O – – 637.3673 – 637.3673 623.3515
y7–AA (19) – – 509.3117 – 509.3117 495.2926
y7–AA (19-18) – – 381.2509 – 381.2509 367.2344
y7–AA (19-17) – – 282.1814 – 282.1814 282.1813
y7–AA (19-16) – – 197.1284 – 197.1284 197.1270
a) n.d., Not detected.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 805
Type Culture (CBS 119283) of Hypocrea phellinicola (maXis screening)
19 32 33 34 35 36
13.5 14.1 14.9 15.5 15.9 16.2
1880.1008 1896.0964 1880.1035 1866.0863 1880.1012 1867.0706
n.d. n.d. n.d. 128.0697 n.d. n.d.
199.1123 199.1123 199.1123 199.1074 199.1078 199.1078
270.1449 286.1389 270.1449 270.1449 284.1605 270.1438
341.1826 357.1760 341.1826 341.1819 355.1975 341.1816
n.d. 424.2191 408.2242 408.2280 422.2402 n.d.
426.2349 442.2296 426.2349 426.2349 440.2506 426.2354
554.2934 570.2869 554.2934 554.2933 568.3087 554.2932
639.3463 655.3404 639.3463 639.3465 653.3621 639.3461
752.4301 768.4257 752.4301 752.4303 766.4461 752.4295
837.4813 853.4789 837.4813 837.4833 851.4992 837.4824
894.5075 910.4971 894.5075 894.5044 908.5203 894.5037
1007.5825 1023.5860 1007.5825 1007.5891 1021.6041 1007.5911
1092.6420 1108.6370 1092.6440 1092.6432 1106.6582 1092.6413
n.d. n.d. n.d. n.d. n.d. n.d.
––––––
––––––
––––––
––––––
––––––
––––––
––––––
––––––
––––––
788.4661 788.4667 788.4668 774.4504 774.4503 775.4366
637.3669 637.3683 637.3683 623.3499 623.3499 624.3356
509.3092 509.3078 509.3078 495.2931 495.2931 496.2769
381.2498 381.2500 381.2500 367.2337 367.2337 367.2337
282.1806 282.1815 282.1815 282.1812 282.1812 282.1814
197.1288 197.1286 197.1286 197.1284 197.1284 197.1287

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)806
Table 6 (cont.)
Diagnostic fragment ions Peaks [m/z]a)
37 38 39 40 41 42
tR[min] 16.4 16.7 16.8 17.0 17.2 17.5
[MþH]þ1880.1007 n.d. 1880.1009 n.d. 1880.0997 1894.1210
b1n.d. n.d. 128.0701 128.0713 n.d. n.d.
b2199.1075 199.1075 199.1077 199.1075 199.1075 199.1080
b3270.1444 284.1603 284.1602 284.1599 270.1444 284.1604
b4341.1819 355.1974 355.1975 355.1973 341.1819 355.1974
b4–H
2O n.d. n.d. 422.2399 n.d. n.d. 436.2493
b5426.2350 440.2504 440.2499 440.2504 426.2350 454.2659
b6554.2935 568.3091 568.3080 568.3086 554.2935 582.3240
b7639.3462 653.3619 653.3613 653.3615 639.3462 667.3770
b8752.4307 766.4459 766.4450 766.4452 752.4307 780.4612
b9837.4843 851.4983 851.4983 851.4987 837.4843 865.5140
b10 894.5019 908.5197 908.5205 908.5230 894.5019 922.5363
b11 1007.5901 1021.6066 1021.6041 1021.6054 1007.5901 1035.6190
b12 1092.6420 1106.6569 1106.6577 1106.6577 1092.6420 1120.6761
b12 –H
2O n.d. n.d. n.d. 1088.6517 n.d. 1103.6621
y5––––––
y5–AA(17) ––––––
y5–AA (17-16) – – – – – –
y5–AA (17-15) – – – – – –
y6––––––
y6–AA(18) ––––––
y6–AA (18-17) – – – – – –
y6–AA (18-16) – – – – – –
y6–AA (18-15) – – – – – –
y7788.4660 775.4348 774.4505 788.4664 788.4664 774.4522
y7–H
2O 637.3704 624.3348 623.3515 637.3670 637.3670 623.3499
y7–AA (19) 509.3084 469.2766 495.2929 509.3079 509.3079 495.2931
y7–AA (19-18) 381.2504 367.2338 367.2338 381.2493 381.2493 367.2337
y7–AA (19-17) 282.1806 282.1808 282.1808 282.1807 282.1807 282.1812
y7–AA (19-16) 197.1288 197.1283 197.1274 197.1282 197.1282 197.1284
a) n.d., Not detected.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 807
43 44 45 46 47 48 49
17.7 18.0 18.6 20.0 21.5 22.0 22.1–22.2
1895.1007 1894.1177 1908.1341 1922.1467 1936.1660 1009.7031 1066.7242
n.d. 128.0684 128.0684 n.d. n.d. n.d. n.d.
199.1084 199.1074 199.1080 227.1386 241.1536 212.1663 212.1644
284.1606 270.1440 284.1604 312.1916 326.2076 269.1858 269.1850
355.1969 341.1818 355.1974 383.2288 397.2443 382.2698 382.2695
n.d. 422.2401 436.2550 n.d. n.d. – –
440.2499 440.2501 454.2659 468.2807 482.2975 467.3234 467.3230
568.3077 568.3087 582.3240 596.3410 610.3540 524.3442 524.3428
653.3609 653.3614 667.3770 681.3925 695.4084 637.4289 581.3654
766.4466 766.4453 780.4612 794.4774 808.4926 722.4814 694.4498
851.4985 851.4983 865.5140 879.5284 893.5450 779.5027 779.5029
908.5184 908.5202 922.5363 936.5518 950.5672 892.5860 836.5243
1021.6039 1021.6067 1035.6190 1049.6372 1063.6524 – 949.6064
1106.6577 1106.6590 1120.6744 1134.6878 1148.7083 – –
1088.6389 n.d. 1102.6586 n.d. n.d. – –
–––––––
–––––––
–––––––
–––––––
–––––––
–––––––
–––––––
–––––––
–––––––
789.4503 788.4660 788.4670 788.4660 788.4650 – –
638.3516 637.3677 637.3670 637.3677 637.3678 – –
510.2927 509.3076 509.3079 509.3076 509.3077 – –
381.2498 381.2495 381.2493 381.2495 381.2492 – –
282.1814 282.1807 282.1807 282.1807 282.1814 – –
197.1292 197.1284 197.1282 197.1284 197.1277 – –

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)808
Fig. 3. Sequencing of compounds 13 and 15 containing a new C-terminal residue with a peak at m/z804.46,
tentatively assigned as tyrosinol (Tyrol)

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Table 7. Biological Activities of Selected 20-Residue Peptaibols Structurally Closely Related to
Hypophellins
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Longibrachins Ion-channel formation in BLM, antimycoplasmic [53]
Suzukacillins Antibacterial, antifungal [78]
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[46]
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Trichosporins B Uncoupling of the respiratory activity of rat liver mitochondria [64][84]
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medullary chromaffin cells
[85 – 87]
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Paracelsins Antibacterial (gþ) [89]
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[90]
Mosquitocidal (larvae of Culex pipiens) [91]
Toxic against aquatic invertebrates (Daphnia magna,Artemisia salina) [92] [93]
Ion-channel formation in BLM [71]
Antifungal [93]

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