Thermoactinoamide A, an Antibiotic Lipophilic Cyclopeptide from the Icelandic Thermophilic Bacterium Thermoactinomyces vulgaris

Article abstract of DOI:10.1021/acs.jnatprod.7b00560

The thermophilic bacterium Thermoactinomyces vulgaris strain ISCAR 2354, isolated from a coastal hydrothermal vent in Iceland, was shown to contain thermoactinoamide A (1), a new cyclic hexapeptide composed of mixed d and l amino acids, along with five mi

Full text of DOI:10.1021/acs.jnatprod.7b00560

Article
pubs.acs.org/jnp
Thermoactinoamide A, an Antibiotic Lipophilic Cyclopeptide from
the Icelandic Thermophilic Bacterium Thermoactinomyces vulgaris
Roberta Teta,† Viggo Thor Marteinsson,‡,⊥ Arlette Longeon,§ Alexandra M. Klonowski,‡ Rene Groben,‡
́
́
́
Marie-Lise Bourguet-Kondracki,§ Valeria Costantino,† and Alfonso Mangoni*,†
†The NeaNat Group, Dipartimento di Farmacia, Universita
80131 Napoli, Italy
̀
degli Studi di Napoli Federico II, Via D. Montesano 49,
‡Matis ohf., Vinlandsleid 12, 113 Reykjavik, Iceland
§Laboratoire Molec
́ ́
ules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Museum National d’Histoire
Naturelle, 57 Rue Cuvier (C.P. 54), 75005 Paris, France
⊥Faculty of Food Science and Nutrition, University of Iceland, Saemundargata 2, 101 Reykjavik, Iceland
S
* Supporting Information
ABSTRACT: The thermophilic bacterium Thermoactinomyces vulgaris
strain ISCAR 2354, isolated from a coastal hydrothermal vent in Iceland,
was shown to contain thermoactinoamide A (1), a new cyclic hexa-
peptide composed of mixed D and L amino acids, along with five minor
analogues (2−6). The structure of 1 was determined by one- and two-
dimensional NMR spectroscopy, high-resolution tandem mass spectrom-
etry, and advanced Marfey’s analysis of 1 and of the products of its partial
hydrolysis. Thermoactinoamide A inhibited the growth of Staphylococcus
aureus ATCC 6538 with an MIC value of 35 μM. On the basis of liter-
ature data and this work, cyclic hexapeptides with mixed D/L config-
urations, one aromatic amino acid residue, and a prevalence of lipophilic
residues can be seen as a starting point to define a new, easily accessible
scaffold in the search for new antibiotic agents.
are actually produced by microorganisms associated with
he global increase in the incidence of drug-resistant bacteria
Tdictates novel inputs and efforts in antibacterial drug dis- them.7,8
Submarine thermal vents are another example of such a
covery. Multidrug-resistant bacteria are estimated to kill 23 000
people in the US and 25 000 in Europe per year.1 At the same
time, the development of new antibiotics is declining, and cross-
resistance between newly introduced and currently used
antibiotics is not uncommon. Therefore, not only new molecules
but new scaffolds and new targets are needed.
When it comes to the discovery of new scaffolds and new
targets, natural products have always played a major role. This is
particularly true for antibiotics, where only three of the 20 classes
of approved antibiotics are not based on the scaffold of a natural
product (sulfa drugs, quinolones, and oxazolidinones).2 In this
respect, it has been observed that microorganisms living in
specialized ecological niches may offer more chances to discover
new scaffolds than widespread microorganisms.3
Examples of bacteria living in specialized ecological niches
include the bacterial communities associated with many marine
sponges. They are abundant, highly sponge-specific, and dis-
tinctly different from those in the surrounding water, so much so
that marine sponges have been considered as natural microbial
fermenters;4 moreover, the distinctness of sponge-associated
bacterial communities translates into a similar distinctness of
their biosynthetic genes.5,6 Not surprisingly, there is increasing
evidence that many of the metabolites found in sponges
specialized ecological niche, which in addition is virtually
unexplored with respect to natural products discovery. Hydro-
thermal vents are inhabited by thermophilic bacteria, which have
the ability to grow at very high temperatures. From the Icelandic
Strain Collection and Records (ISCAR) a total of 150 bacterial
strains that were originally isolated from various Icelandic marine
environments were screened for biological activity. One of those
strains showed antimicrobial activity and was chosen for further
analysis. The isolate, ISCAR 2354, originated from a coastal hot
spring on the Snæfellsness peninsula in West Iceland (64°57′52″
N, 23°5′21.53″ W). According to 16S rRNA gene sequencing
(GenBank accession number M372920), the strain belongs to
the species Thermoactinomyces vulgaris (Thermoactinomyceta-
ceae, Bacillales, Firmicutes), showing 100% identity to other
isolates of the species. From strain ISCAR 2354, a new anti-
bacterial cyclic hexapeptide with mixed D and L amino acids,
namely, thermoactinoamide A (1), was isolated, along with five
minor analogues (thermoactinoamides B−F, 2−6), whose
structures were partly elucidated by mass spectrometry. In this
Received: June 30, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00560
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paper, we wish to report the isolation, structure elucidation, and
antibacterial activity of thermoactinoamide A (1).
use with the Orbitrap high-resolution MS detector,10 and further
modified on account of the Ile residue present in 1. In fact, in our
experience, a C18 HPLC column cannot resolve the FDAA
derivatives of D/L-Ile from those of D/L-allo-Ile. However, the use
of a C3 column has been proposed to overcome difficulties with
reversed-phase separations of this compound class.11 We found
that a pentafluorophenyl (PFP) bonded-phase column can
provide baseline separation of six isobaric Leu, Ile, and allo-Ile
derivatives and is suited for use with an MS-selective detector
(Figure 1A). A small amount of compound 1 (12 μg) was hydro-
lyzed by treating it with 6 N HCl/AcOH (1:1) at 120 °C for 12 h.
The hydrolysate was reacted with 100 μL of 1% 1-fluoro-2,4-
dinitrophenyl-5-D-alaninamide (D-FDAA), the resulting D-FDAA
derivatives of Tyr, Val, Leu, and Ile were analyzed by HPLC-ESI-
HRMS, and their retention times were compared with authentic
standards prepared by reaction of L- and D-FDAA with L-Tyr,
L-Val, L-Leu, L- Ile, and D-allo-Ile. Analysis of the resulting chro-
matograms (Figure 1) revealed that the peptide 1 contains
2 × L-Leu, 1 × D-Leu, 1 × D-allo-Ile, 1 × L-Val, and 1 × D-Tyr.
The location of the D-Leu residue in compound 1 could not be
determined from the above data. Therefore, compound 1 was
subjected to partial hydrolysis, which produced a complex
mixture of linear peptides ranging from two to six amino acids.
HPLC separation of the mixture yielded two pure peptides
(7 and 8). The peptide 7 showed an [M + H]+ ion in the ESIMS
spectrum at m/z 620.4018, corresponding to the molecular
formula C32H54N5O7, in accordance with a linear pentapeptide
originating from the loss of one leucine or isoleucine residue
from compound 1. The sequence of the peptide was determined
as H-Leu(III)-Ile-Tyr-Val-Leu(I)-OH by analysis of the frag-
mentation pattern observed in its MS/MS spectrum (Figure S4),
showing that the lost amino acid residue was Leu(II) (aa-4 in
structure 1). Hydrolysis and Marfey’s analysis of this fragment as
described above revealed the presence of D-allo-Ile and L-Leu, but
not D-Leu. This showed that the lost amino acid residue, i.e.,
Leu(II), was indeed D-Leu.
RESULTS AND DISCUSSION
■
The thermophilic bacterium T. vulgaris strain ISCAR 2354 was
grown at 60 °C in 166 medium9 with 1% NaCl for 24 h.
An organic extract of the culture was derived through sonication
of the cells, followed by two extractions with H2O-saturated
BuOH and concentration of the organic phase. The organic
extract was preliminarily analyzed by liquid chromatography
electrospray high-resolution mass spectrometry (LC-ESI-HRMS)
and was shown to contain six related compounds, which according
to their MS/MS fragmentations appeared to be cyclic peptides.
The most abundant compound (1) was isolated and analyzed by
MS, MS/MS, and NMR spectroscopy.
The ESI-HRMS spectrum of compound 1 showed, in addition
to the [M + H]+ ion at m/z 715.4752 (base peak), an [M + Na]+
ion at m/z 737.4570, a doubly charged [M + Ca]2+ ion at
m/z 377.2147, and an [M + Ca + HCOO]+ ion at m/z 799.4277,
all within 1 ppm of the calculated mass for the molecular formula
C38H63N6O7. The fragmentation pattern observed in the tandem
mass (MS/MS) spectrum of compound 1 was clearly suggestive
of a peptide, with fragments originating from loss of one valine,
one leucine or isoleucine, and one tyrosine residue. Consid-
eration of the molecular formula led to the conclusion that the
peptide 1 contains one tyrosine, one valine, and four leucine/
isoleucine residues and that the peptide is cyclic. The overall
fragmentation pattern (Figure S3) indicated that the tyrosine
residue is flanked by a valine and a leucine/isoleucine residue.
A complete set of 2D NMR spectra of compound 1 was
recorded. The proton spectrum contained six distinct amide NH
signals (thus showing that no N-methylated residue is present in
the molecule) and six distinct α-proton signals (Table 1).
Analysis of the TOCSY spectrum (Figure S2) showed cor-
relation of all the protons of the six side chains with the cor-
responding α-proton and/or amide NH signals (Table 1). This
information, combined with the analysis of the COSY spectrum,
led to the identification of a tyrosine, a valine, an isoleucine, and
three leucine residues. The sequence of the six amino acids in the
peptide was determined on the basis of the NOESY spectrum,
which contained a clear correlation peak between each α-proton
and the amide NH proton of the subsequent amino acid residue
in the sequence (Table 1). The amino acid sequence determined
in this way was cyclo(Tyr-Val-Leu(I)-Leu(II)-Leu(III)-Ile).
The D/L configuration of the six amino acid residues was
determined using an advanced Marfey’s method, adapted to the
The peptide 8 showed an [M + H]+ ion in the ESIMS
spectrum at m/z 521.3333, corresponding to the molecular
formula C27H44N4O6, in accordance with a linear tetrapeptide
originating from the loss of one valine and one leucine or
isoleucine residue from compound 1. There is only one such
tetrapeptide that can be obtained from compound 1, i.e.,
H-Leu(II)-Leu(III)-Ile-Tyr-OH, and this sequence was confirmed
by the MS/MS fragmentation pattern (Figure S5). Hydrolysis and
Marfey analysis of this fragment showed the presence of both
B
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Table 1. NMR Data of Thermoactinoamide A (1) (1H 700 MHz, 13C 175 MHz, CD3SOCD3)
a
b
amino acid
D-Tyr
pos.
δC, type
δH, mult (J in Hz)
7.49, d (5.7)
NOESY
Ile-NH, Ile-2, Ile-3
HMBC
Ile-1
NH
1
171.2, C
54.8, CH
37.6, CH2
2
4.64, ddd (8.6, 5.7, 5.4)
2.92, dd (13.6, 5.4)
2.75, dd (13.6, 8.6)
Val-NH, Tyr-5/9
Tyr-5/9
Tyr-1
Tyr-1
Tyr-1
3
a
b
Tyr-5/9
4
126.6, C
5/9
6/8
7
129.9, CH
114.7, CH
155.8, C
6.91,d (8.3)
6.63, d (8.3)
Tyr-2, Tyr-3a, Tyr-3b
L-Val
NH
1
7.91, d (6.2)
Leu(I)-NH, Tyr-2
Leu(I)-NH
Tyr-1
170.5, C
2
60.0, CH
29.0, CH
17.4, CH3
18.6, CH3
3.62, t (5.8)
1.86, octet (6.6)
0.68, d (6.8)
0.66, d (6.8)
7.75, d (8.1)
Tyr-1, Val-1
3
4
5
L-Leu(I)
NH
1
Val-NH, Val-2, Leu(II)-NH
Leu(II)-NH
Val-1
170.7, C
50.6, CH
39.2, CH2
2
4.23, ddd (10.5, 8.1, 5.2)
1.59, m
Leu(I)-1
3
a
b
1.54, m
4
24.0, CH
22.8, CH3
20.9, CH3
1.46, m
5
0.85, d (6.8)
0.78, d (6.8)
7.53, d (8.1)
6
D-Leu(II)
L-Leu(III)
D-allo-Ile
NH
1
Leu(I)-NH, Leu(I)-2, Leu(III)-NH
Leu(III)-NH
Leu(I)-1
Leu(II)-1
171.4, C
50.5, CH
40.8, CH2
2
4.33, q (7.4)
1.48, m
3
a
b
1.48, m
4
24.2, CH
22.5, CH3
21.8, CH3
1.47, m
5
0.87, d (6.8)
0.82, d (6.8)
8.13, d (7.1)
6
NH
1
Leu(II)-NH, Leu(II)-2, Ile-NH
Ile-NH
Leu(II)-1
Leu(III)-1
171.7, C
51.1, CH
39.3, CH2
2
4.34, q (7.1)
1.46, m
3
a
b
1.36, m
4
24.0, CH
22.1, CH3
22.0, CH3
1.50, m
5
0.89, d (6.8)
0.83, d (6.8)
8.26, d (9.0)
6
NH
1
Leu-III-NH, Leu(III)-2, Tyr-NH
Leu(III)-1
170.8, C
2
55.8, CH
35.9, CH
14.3, CH3
25.5, CH2
4.25, dd (9.0, 5.0)
1.97, m
Tyr-NH
Tyr-NH
Leu(III)-1, Ile-1
3
4
0.78, d (7.0)
1.23, m
5
a
b
1.11, m
6
11.2, CH3
0.81, t (7.3)
a
b
Mixing time 300 ms. Correlations also present in the TOCSY spectrum are not shown. Selected HMBC correlations from proton stated to the
indicated carbon.
L-Leu and D-Leu in the peptide, further confirming the location of
the D-Leu residue.
the corresponding amino acids are the same as in 1 (see also
Figures S6−S10).
The minor cyclic hexapeptides thermoactinoamides B−F
(2−6) were present in much smaller amounts in the extract of
T. vulgaris. They were not isolated, but only partially char-
acterized by analysis of the respective MS/MS spectra. Putative
structures for compounds 2−6 are shown in Table 2. They were
obtained by analogy with compound 1, under the assumptions
that (i) differences between compounds 2−6 and compound 1
are confined to a single amino acid and (ii) the configurations of
Several hexapeptides have been isolated from natural sources,
but most of them (such as the iron-binding ferrichromes or
the cyanobacterial oxazole- or thiazole-containing peptides)
are unrelated to the thermoactinoamides. A few more similar
peptides (i.e., peptides made of unmodified, mostly lipophilic
proteogenic amino acids with mixed D/L configurations) have
been isolated from actinomycetes (it must be noted that, in spite
of the name, the genus Thermoactinomyces is phylogenetically far
C
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Figure 1. Advanced Marfey’s analysis of compound 1 using a pentafluorophenyl (PFP) bonded-phase column. (A) Extracted-ion chromatograms at m/z
384.1415 of the D-FDAA derivatives from the hydrolysis of 1 and of D- and L-FDAA derivatives L-Leu, L-Ile, and D-allo-Ile. (B) Extracted-ion
chromatograms at m/z 434.1306 of the D-FDAA derivatives from the hydrolysis of 1 and of D- and L-FDAA derivatives L-Tyr. (C) Extracted-ion
chromatograms at m/z 370.1357 of the D-FDAA derivatives from the hydrolysis of 1 and of D- and L-FDAA derivatives L-Val.
Table 2. Putative Structures of the Minor Cyclic Hexapeptides 2−6 from T. vulgaris
name
molecular formula
aa-1
aa-2
L-Val
aa-3
aa-4
aa-5
aa-6
1
2
3
4
5
6
thermoactinoamide A
thermoactinoamide B
thermoactinoamide C
thermoactinoamide D
thermoactinoamide E
thermoactinoamide F
C38H62N6O7
C37H60N6O7
C39H64N6O7
C41H60N6O8
C35H64N6O6
C38H62N6O6
D-Tyr
D-Tyr
D-Tyr
D-Tyr
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
D-Leu
L-Leu
L-Leu
L-Leu
L-Tyr
L-Leu
L-Leu
D-allo-Ile
D-Val
L-Val
L-Leu/L-Ile
L-Val
D-allo-Ile
D-allo-Ile
D-allo-Ile
D-allo-Ile
D-Leu/D-allo-Ile
L-Val
D-Phe
L-Val
from Actinomycetales).12 Among them are phepropeptins A−D
from Streptomyces sp.,13 desotamides from Streptomyces scopulir-
idis SCSIO ZJ46,14 wollamides from Streptomyces scopuliridis
SCSIO ZJ46,15 and nocardiamides A and B from the Nocardiopsis
sp. CNX03716 (see Figure S1 for the structures of some of these
compounds). In particular, nocardiamides appear very similar
to thermoactinoamide A (1) in amino acid composition, but
show a different pattern of D/L configurations for the amino acid
residues, suggesting a different, possibly unrelated biosynthetic
origin.
Antibacterial Activity of Thermoactinoamide A (1). The
cyclic hexapeptide 1 was tested against a panel of three Gram-
positive (Staphylococcus aureus ATCC 6538, Enterococcus faecalis
ATCC 29212, Bacillus cereus ATCC 14579) and three Gram-
negative (Escherichia coli ATCC 8739, Pseudomonas aeruginosa
ATCC 13388, Klebsiella pneumoniae ATCC 11296) bacterial
strains. It exhibited a significant and selective growth inhibi-
tion against S. aureus with a minimum inhibitory concentration
(MIC) value of 35 μM. It was inactive against the other strains up
to 140 μM.
Thermoactinoamide A (1), which revealed a significant and
selective activity against S. aureus ATCC 6538, may be a pro-
mising antibacterial compound. A comparably narrow antibiotic
activity against S. aureus ATCC 29213 with an MIC value of
23 μM was previously reported for the related cyclic hexapep-
tides desotamides A and B.14 While it is probably premature to
draw conclusions about the exact structural requirements for the
antibiotic activity of cyclic hexapeptides, the common structural
features of thermoactinoamides and desotamides (cyclic hexa-
peptides with mixed D/L configurations, one aromatic amino
acid residue, prevalence of lipophilic residues) can be seen as a
starting point to define a new, easily accessible scaffold for
antibiotic drugs. Future research to develop thermoactinoamides
along these lines will involve evaluation of their activity against
MRSA strains, definition of their structure−activity relationship,
and identification of their mechanism of action.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a Jasco P-2000 polarimeter at the sodium D line.
Electronic circular dichroism (ECD) spectra were recorded using a
Jasco-715 spectropolarimeter. NMR spectra were determined on Varian
Unity Inova spectrometers at 700 MHz; chemical shifts were referenced
to the residual solvent signal (CD3SOCD3: δH 2.50, δC 39.52). For an
accurate measurement of the coupling constants, the one-dimensional
1H NMR spectra were transformed at 64 K points (digital resolution:
0.09 Hz). Through-space 1H connectivities were evidenced using a NOESY
experiment with a mixing time of 300 ms. The HSQC spectra were
optimized for 1JCH = 145 Hz, and the HMBCexperiments for 2,3JCH = 8 Hz.
High-resolution ESIMS and HR-ESI-HPLC experiments were performed
on a Thermo LTQ Orbitrap XL mass spectrometer coupled to a Thermo
U3000 HPLC system. High-performance liquid chromatography (HPLC)
separations were achieved on an Agilent 1260 Infinity Quaternary LC
apparatus equipped with a diode-array detector .
Collection, Cultivation, and Extraction. The thermophilic
bacterial strain Thermoactinomyces vulgaris ISCAR 2354, used for the
isolation and characterization of thermoactinoamide A, originated
from a coastal hot spring in Icelandic marine waters (64°57′52″ N,
23°5′21.53″ W). A second strain of the same species (ISCAR 2850) that
was isolated from a deep sea hydrothermal vent sediment core at 400 m
depth (66°36′51″ N, 17°39′09″ W), was also found to contain thermo-
actinoamide A. Both strains were identified based on their 16S rRNA
gene sequences (GenBank accession numbers M372920 and M372921,
respectively) with 100% identity to other T. vulgaris sequences in
GenBank. However, phylogenetic analysis based on 16S rDNA
sequences of Thermoactinomyces species suggested that the genus
D
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should be taxonomically re-evaluated using other useful taxonomic
markers.12
preculture was assessed by measuring the optical density (OD) at
620 nm and was adjusted by dilution in order to obtain a suspension of
0.03 OD. An aliquot of 200 μL of the bacterial suspension was dis-
tributed in each well containing 2-fold serial dilutions of the peptide.
The 96-well microplates were incubated at 34 °C overnight with shaking
(450 rpm). The optical density of the wells was measured at 620 nm with
a microplate reader, and MICs, performed in triplicate, were defined as
the lowest concentration of drug that completely inhibits bacterial
growth. MIC values of positive controls: ampicillin [S. aureus (0.11 μM),
E. faecalis (4.59 μM)], gentamicin [B. cereus (17.2 μM), P. aeruginosa
(10.5 μM)], and chloramphenicol [E. coli (19.3 μM), K. pneumoniae
(3.17 μM)].
The strains were grown at 60 °C in 250 mL culture flasks in medium
166 containing 1% NaCl for 24 h.9 The bacterial culture was sonicated
for 3 min using a Branson Sonifier 250 with the duty cycle on constant-
output control on 4 and the output at 20 to break up the cells. The whole
culture fluid was transferred to a 1 L centrifuge flask and mixed with
100 mL of H2O-saturated BuOH with 10% MeOH before centrifugation
(10 min, 10000g, 4 °C). The upper, organic phase was transferred into a
new flask, and the BuOH extraction steps were repeated. The organic
extract was filtered through a 0.45 μm PTFE filter and then concentrated
on a rotary evaporator at 40 °C to obtain an extract for the analyses.
Isolation of Thermoactinoamide A. The organic extract of
ISCAR 2354 (739 mg) was analyzed by LC-ESI-HRMS on a Thermo
LTQ Orbitrap XL using a 5 μm Kinetex C18 column (50 × 2.10 mm)
maintained at 25 °C and a gradient system [eluent A: 0.1% HCOOH in
H2O; eluent B: CH3CN; gradient program: 30% B 5 min, 30% → 99% B
over 17 min; flow rate 200 μL min−1]. The extract was shown to contain
six related compounds (compounds 1−6), which accounted for most of
the peaks in the total ion current chromatogram. An MS/MS spectrum
was recorded for each compound. Analysis of the fragmentation pattern
suggested them to be cyclic peptides. The most abundant compound, 1,
was isolated by reversed-phase HPLC using a 10 μm Kinetex C18
column (250 × 10 mm) [eluent A: 0.1% HCOOH in H2O; eluent B:
MeOH; gradient program: 60% B 5 min, 60% → 100% B over 17 min,
100% B 13 min; flow rate 5 mL min−1, wavelength 280 nm], thus
obtaining pure thermoactinoamide A (1, 1.2 mg, tR 18.7 min).
Thermoactinoamide A (1): colorless, amorphous solid; [α]25D +80
(c 0.5, MeOH); ECD (412 μM, MeOH) λmax (Δε) 280 (+0.5); 1H and
13C NMR data, Table 1; HRESIMS (positive ion mode, 0.1% HCOOH
in MeOH) m/z 715.4752 [M + H]+ (calcd for C38H62N6O7, 737.4753),
m/z 737.4570 [M + Na]+ (calcd for C38H62N6O7Na+, 737.4572),
377.2147 [M + Ca]2+ (calcd for C38H62N6O7Ca2+, 377.2147), 799.4277
[M + Ca + HCOO]+ (calcd for C39H63N6O9Ca+, 799.4277).
Advanced Marfey’s Analysis. Compound 1 (12 μg) was
hydrolyzed with 6 N HCl/AcOH (1:1) at 120 °C for 12 h. The
residual HCl fumes were removed under N2 stream. The hydrolysate of
1 was dissolved in TEA/acetone (2:3, 100 μL), and the solution was
treated with 100 μL of 1% 1-fluoro-2,4-dinitrophenyl-5-D-alaninamide
(D-FDAA) in CH3CN/acetone (1:2). The vial was heated at 50 °C for
1.5 h. The mixture was dried, and the resulting D-FDAA derivatives
of Tyr, Val, Leu, and Ile were redissolved in MeOH (100 μL) for
subsequent analysis. Authentic standards of L-Tyr, L-Val, L-Leu, L-Ile, and
D-allo-Ile were treated with L-FDAA and D-FDAA as described above
and yielded the L-FDAA and D-FDAA standards. Marfey’s derivatives of
1 were analyzed by HPLC-ESI-HRMS, and their retention times were
compared with those from the authentic standard derivatives. A 2.6 μm
Kinetex PFP column (100 × 4.6 mm) maintained at 25 °C was eluted at
200 μL min−1 with 0.1% HCOOH in H2O and MeOH. The gradient
program was as follows: 60% MeOH 5 min, 60% → 100% MeOH over
30 min, 100% MeOH 15 min. Mass spectra were acquired in positive ion
detection mode, and the data were analyzed using the Xcalibur suite of
programs.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.7b00560.
■
S
Structures of some known cyclic hexapeptides from
actinomycetes, 1D sections of the TOCSY spectrum of
1, MS/MS spectra of 1−8, and copies of the MS and one-
and two-dimensional NMR spectra of 1 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-081678532. Fax: +39-081678552. E-mail: alfonso.
mangoni@unina.it.
■
ORCID
Valeria Costantino: 0000-0001-8723-9505
Alfonso Mangoni: 0000-0003-3910-6518
Notes
The authors declare no competing financial interest.
A patent application about these results has been filed to the
Icelandic patent office, Patent Number 9079.
ACKNOWLEDGMENTS
We acknowledge the financial support of the European Union
Seventh Framework Programme (BlueGenics, FP7-KBBE-2012-6)
■
̀
under grant agreement no. 311848 and by Universita degli Studi
di Napoli Federico II under the STAR project SeaLEADS.
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