Svetamycins A-G, Unusual Piperazic Acid-Containing Peptides from Streptomyces sp.

Article abstract of DOI:10.1021/acs.joc.7b00228

Seven new halogenated peptides termed svetamycins A-G (1-7) have been isolated from laboratory cultures of a Streptomyces sp. Svetamycins A-D, F, and G are cyclic depsipeptides, whereas svetamycin E is a linear analogue of svetamycin C. Their structures were determined using extensive spectroscopic analysis, and their stereochemical configuration was established by a combination of NMR data, quantum mechanical calculations, and chemical derivatizations. Svetamycins are characterized by the presence of a hydroxyl acetic acid and five amino acids including a rare 4,5-dihydroxy-2,3,4,5-tetrahydropyridazine-3-carboxylic acid, a γ-halogenated piperazic acid, and a novel δ-methylated piperazic acid in svetamycins B-C, E, and G. Moreover, isotope-labeled substrate feeding experiments demonstrated ornithine as the precursor of piperazic acid and that methylation at the δ position of the piperazyl scaffold is S-adenosyl-l-methionine (SAM)-dependent. Svetamycin G, the most potent antimicrobial of this suite of compounds, inhibited the growth of Mycobacterium smegmatis with an MIC₈₀ value of 2 μg/mL.

Full text of DOI:10.1021/acs.joc.7b00228

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Article
Svetamycins A-G, Unusual Piperazic Acid-
Containing Peptides from Streptomyces sp.
Denis Dardic, Gianluigi Lauro, Giuseppe Bifulco, Patricia Laboudie, Peyman
Sakhaii, Armin Bauer, Andreas Vilcinskas, Peter E. Hammann, and Alberto Plaza
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 10 May 2017
Downloaded from http://pubs.acs.org on May 11, 2017
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The Journal of Organic Chemistry is published by the American Chemical Society.
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Svetamycins AꢀG, Unusual Piperazic Acidꢀ
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Containing Peptides from Streptomyces sp.
Denis Dardić,† Gianluigi Lauro ‡ Giuseppe Bifulco,‡ Patricia Laboudie,ǁ Peyman Sakhaii,§ Armin
Bauer,⊥ Andreas Vilcinskas,† Peter E. Hammann,⊥ Alberto Plaza*,†,±
†
SanofiꢀFraunhofer Natural Product Center of Excellence, Fraunhofer IME, Industriepark
Höchst Bldg. G878, 65926, Frankfurt am Main, Germany
‡
Dipartimento di Farmacia, Università di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano
(SA), Italy
ǁ
Infectious Diseases Therapeutic Area, Sanofi R&D, Campus Mérieux, 1541 avenue Marcel
Mérieux, XNord 315, 69280 Marcy L’Etoile, France
§
NMR Laboratory, Chemistry & Biotechnology Development Frankfurt Chemistry, Sanofiꢀ
Aventis Deutschland GmbH, Industriepark Hoechst, Bldg. G849, 65926, Frankfurt am Main,
Germany.
⊥ R&D Infectious Diseases Therapeutic Area, SanofiꢀAventis Deutschland GmbH, Industriepark
Höchst Bldg. G878, 65926, Frankfurt am Main, Germany
*Eꢀmail: Alberto.Plaza@merckgroup.com
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ABSTRACT
Seven new halogenated peptides termed svetamycins AꢀG (1ꢀ7) have been isolated from
laboratory cultures of a Streptomyces sp. Svetamycins AꢀD, F, and G are cyclic depsipeptides
whereas svetamycin E is a linear analogue of svetamycin C. Their structures were determined
using extensive spectroscopic analysis, and their stereochemical configuration was established
by a combination of NMR data, quantum mechanical calculations, and chemical derivatizations.
Svetamycins are characterized by the presence of a hydroxyl acetic acid and five amino acids
including a rare 4,5ꢀdihydroxyꢀ2,3,4,5ꢀtetrahydropyridazineꢀ3ꢀcarboxylic acid, a γꢀhalogenated
piperazic acid, and a novel δꢀmethylated piperazic acid in svetamycins BꢀC, E, and G. Moreover,
isotopeꢀlabeled substrate feeding experiments demonstrated ornithine as the precursor of
piperazic acid and that methylation at the δ position of the piperazyl scaffold is SꢀadenosylꢀLꢀ
methionine (SAM)ꢀdependent. Svetamycin G, the most potent antimicrobial of this suite of
compounds, inhibited the growth of Mycobacterium smegmatis with an MIC80 value of 2 ꢀg/mL.
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INTRODUCTION
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The emergence of resistance to antibiotics is unavoidable due to the natural selection for
resistant pathogens that takes place with each generation of newly discovered antibiotics.1
Therefore, there is a continuous need to identify new lead antimicrobials with improved activities
and/or novel mechanisms of action. Microorganisms are an excellent source of new
antimicrobial natural products skeletons because of their capability to synthesize a plethora of
small molecules with fascinating chemical structures and potent biological properties. Indeed,
four antibacterial small molecules approved by the U.S. FDA during 2011 to 2014 were either
microbial derivedꢀnatural products or synthetic derivatives thereof.2 As part of an ongoing
collaboration between Fraunhofer IME and Sanofi, we have screened a part of the Sanofi’s
microbial extract library for antibacterial activity against opportunistic microbial pathogens. In
particular, methanol extracts of a Streptomyces sp. (DSM 14386) strongly inhibited the growth of
Eschericia coli, MRSA, and Mycobacterium smegmatis. Bioassay and UPLCꢀHRMSꢀguided
fractionation led to the isolation of five new chlorinated peptides termed svetamycins AꢀE (1ꢀ5)
together with the known chlorinated pentalenolactone AAꢀ57.3 Additionally, to determine if
strain DSM 14386 was biosynthetically capable of producing other halogenated peptides, DSM
14386 was cultivated with a nutrient medium supplemented with sodium bromide leading to the
production of two brominated congeners named svetamycin F (6) and G (7). Their structures
were elucidated by extensive NMR and HRꢀESIꢀMS analysis along with quantum mechanical
calculations, chemical derivatization, and circular dichroism experiments.
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RESULTS AND DISCUSSION
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Large scale fermentation (20 L) of DSM14386 was carried out, and the culture supernatant
was treated with a mixture of Amberlite® XADꢀ7 and ꢀ16. The bound compounds were
extracted with methanol and the dried extract was fractionated by Sephadex LHꢀ20 column
chromatography, and further subjected to semi preparative HPLC (Cꢀ12 column) to yield pure
compounds 1−5. An additional fermentation was conducted on agar plates in which the NaCl of
the fermentation media was replaced with an equal amount of NaBr. Accordingly, fractionation
of the generated methanol extracts afforded the brominated compounds 6 and 7.
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The most abundant compound among this group was svetamycin A (1). Its molecular formula
was determined as C24H35ClN8O10 based on HRꢀESIꢀMS and NMR data (see Table 1), indicating
11 degrees of unsaturation. The HSQC spectrum of 1 exhibited resonances characteristic of a
peptide bearing oxygenated and imine functionalities including four αꢀamino methines (δH 4.90,
δc 50.9; δH 5.73, δc 49.8; δH 5.33, δc 43.1; δH 4.71, δc 56.9), two oxymethines (δH 3.93, δc 62.6; δH
4.06, δc 63.8), two oxymethylene (δH 3.67, 3.82, δc 63.6;. δH 4.64, 5.30, δc 61.5), and one olefinic
methine (δH 6.97, δc 147.2). Besides, the 13C NMR spectrum showed resonances ascribable to six
carbonyls at δ 166.6 ꢀ 172.7 and one quaternary carbon at δ 60.4. Further analysis of the 2D
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NMR data allowed us to determine the presence of the amino acids alanine, αꢀmethlyserine (αꢀ
MeSer), piperazic acid (Pip), and γꢀchloroꢀpiperazic acid (γꢀClPip) together with a hydroxyacetic
acid residue (Haa). Additionally, TOCSY and COSY spectra showed that the remaining spin
system comprised an imine proton (Hꢀ5β,γꢀOHdPip) two oxymethines (Hꢀ3β,γꢀOHdPip and Hꢀ4β,γꢀ
OHdPip), two hydroxyl protons (OHꢀ3β,γꢀOHdPip and OHꢀ4β,γꢀOHdPip), and a αꢀamino methine (Hꢀ2β,γꢀ
OHdPip) (Figure 1). This evidence in combination with key HMBC correlations from Hꢀ3β,γꢀOHdPip
to Cꢀ1β,γꢀOHdPip (δ 166.6), Cꢀ2β,γꢀOHdPip (δ 56.9), Cꢀ4β,γꢀOHdPip (δ 62.6) and Cꢀ5β,γꢀOHdPip (δ 147.2)
revealed the presence of a rare 4,5ꢀdihydroxyꢀ2,3,4,5ꢀtetrahydropyridazineꢀ3ꢀcarboxylic acid
(β,γꢀOHdPip). While monohydroxylated δ,εꢀdehydropiperazic acids have been reported in
kutznerides,4a luzopeptides,4b and quinoxapeptides,4c to the best of our knowledge, this report
represents the first occurrence of a β,γꢀdihydroxydehydro piperazic acid.
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Longꢀrange correlations between αꢀprotons to carbonyl carbons of adjacent amino acids led us
to establish the following partial sequence for 1: γꢀClPipꢀAlaꢀβ,γꢀOHdPipꢀHaa. Moreover, interꢀ
residue ROE correlations of NHꢀ2αꢀMeSer (δ 7.68) to Hꢀ2Pip (δ 4.90) and of NHꢀ5Pip (δ 4.67) to Hꢀ2
(δ 5.73), together with HMBC correlations from NHꢀ2αꢀMeSer to Cꢀ1Pip (δ 168.9) and NHꢀ
γꢀClPip
5Pip to Cꢀ1 γꢀClPip (δ 171.8), linked the partial sequence αꢀMeSerꢀPip to the Cꢀterminus of γꢀClPip.
Finally, the downfield chemical shifts of the oxygenated methylene protons at δ 5.30 and 4.64
along with a HMBC correlation from the later protons to δ 171.9 indicated an ester linkage
between Cꢀ2Haa and Cꢀ1αꢀMeSer. Upon these spectroscopy data, the structure of 1 was
characterized as an 18ꢀmembered cyclic depsipeptide. With the amino acid sequence of 1 in
hand, it was evident that svetamycin A was structurally related to the piperazimycins, which
were isolated from a marine Streptomyces,5 and to the gerumycins that were isolated from an antꢀ
associated Pseudonocardia.6
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Table 1. NMR spectroscopic data (500 MHz, DMSOꢀd6) for Svetamycin A (1)
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6
7
a
b
position δC
δH (J in Hz)
HMBCc
ROESYd
8
9
αꢀMeSer
171.9
60.4
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1
2
3a
63.6
3.67 dd (11.0, 7.5)
3.82 dd ( 11.0, 5.7)
1.42 s
1, 2, 4
3b, 4, NHꢀ2, OHꢀ3
3a, 4, NHꢀ2, OHꢀ3
3ab, NHꢀ2
3b
4
1, 2, 4
19.7
1, 2, 3
NHꢀ2
7.68 s
1, 2, 3, 4, 1Pip
3ab, 4,OHꢀ3, 2Pip,
3bPip
OHꢀ3
4.57 t (6.5)
4.90 m
2, 3
3ab, 4, NHꢀ2
Pip
1
2
168.9
50.9
1, 3, 4
3a,3b, 4b, NHꢀ5, NHꢀ
2αꢀMeSer
3a
24.4
20.2
46.3
1.72 m
1, 2, 4, 5
2, 3b, 4a, 5a
3b
2.19 m
2, 3a, 4a, NHꢀ2αꢀMeSer
3b, 4b, 5ab
4a
1.35 m
4b
1.68 m
3, 5
2, 3b, 4a, 5b, NHꢀ5
3a, 4a, 5b, 2γꢀClPip
4ab, 3a, 5a, NHꢀ5
3a, 5b, 2γꢀClPip
5a
2.61 m
5b
2.94 br d (12.7)
4.67e
3, 4
NHꢀ5
1γꢀClPip, 5γꢀClPip
γꢀClPip
171.8
49.8
1
2
5.73 dd (5.5, 1.2)
1.90 td (11.8, 5.5)
1, 3, 4, 1Ala
1, 2, 4, 5
3ab, NHꢀ5Pip
2, 3b, 5a
3a
34.5
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3b
2.41 ddd (11.8, 4.6, 1.2) 2, 4, 5
2, 3a, 4
5
6
7
8
4
52.5
54.0
4.36 tt (11.4, 4.6)
3, 5
3, 4
3b, 5b, NHꢀ5
3a, 5b, 3Ala
4, 5a, NHꢀ5
4, 5b
5a
2.66 td (12.8, 11.6)
9
5b
3.38 ddd (11.6, 4.7, 1.8) 3, 4
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NHꢀ5
5.23 dd (12.8, 1.4)
5, 1Ala
Ala
1
172.7
43.1
17.9
2
5.33e dq (8.3, 6.9)
1.18 d (6.9)
1, 3, 1βγꢀOHdPip
1, 2
3, NHꢀ2, 2βγꢀOHdPip
2, NHꢀ2
3
NHꢀ2
8.15 d (8.3)
1, 2, 1βγꢀOHdPip
2, 3, 2βγꢀOHdPip, 3βγꢀ
OHdPip
β,γꢀOHdPip
166.6
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2
3
56.9
4.71 br d (5.4)
4.06 q (4.3)
1, 3, 4
3, 4, OHꢀ3, NHꢀ2Ala
63.8
1, 2, 4, 5
2, 4, 5, NHꢀ2Ala, OHꢀ
3, OHꢀ4
4
62.6
3.93 m
2, 3, 5
3, 4
2, 3, 5, OHꢀ3, OHꢀ4
3, 4, OHꢀ4
2, 3, 4
5
147.2
6.97 d (2.3)
5.27 d (4.2)
5.80 d (8.6)
OHꢀ3
OHꢀ4
1, 2, 3, 4
3, 4, 5
3, 4, 5
Haa
1
166.7
61.5
2a
2b
4.64 d (15.6)
5.30 d (15.6)
1, 1αꢀMeSer
1, 1αꢀMeSer
2b, 5bPip, 2γꢀClPip
2a
aRecorded at 175 MHz; referenced to residual DMSOꢀd6 at δ 39.51 ppm. Recorded at 500
b
c
MHz; referenced to residual DMSOꢀd6 at δ 2.50 ppm. Proton showing HMBC correlation to
indicated carbon. dProton showing ROESY correlation to indicated proton. eSignal overlapped
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Figure 1. Key 2D NMR data to establish the structure of β,γꢀOHdPip and the connectivity of the
residues in 1.
The configurations of the stereogenic centers of 1 were solved using a combination of
methods. The absolute configurations of αꢀMeSer and Ala were deduced as
L by LCꢀMS
comparison of
Lꢀ and DꢀFDLA (1ꢀfluoroꢀ2,4ꢀdinitrophenylꢀ5ꢀL/Dꢀleucinamide) derivatives of the
acid hydrolysate of 1 with authentic standards (Advanced Marfey’s method).7 The
Lꢀ and Dꢀ
FDLA derivatives of Pip were detected at retention times of 31.5 and 29.0 min on the
reconstructed ion chromatogram for m/z 425 [M + H]+, respectively (see Advanced Marfey’s
Analysis, Experimental Section). As reported by Cai et al,8 the
LꢀFDLA derivative of the DꢀPip
should elute before the ꢀPip derivative. Thus, the absolute configuration of Pip was determined
L
to be
L. To establish the absolute stereochemistry of γꢀClPip, we first established its relative
3
configuration by analyzing ROESY spectrum and JH,H coupling constants. The equatorial
3
position of Hꢀ2γꢀClPip was assigned on the basis of its intermediate and small JH,H coupling
constants of (5.5 and 1.2 Hz). In addition, key ROE correlations between Hꢀ2γꢀClPip/Hꢀ3aγꢀClPip, Hꢀ
2γꢀClPip/Hꢀ3bγꢀClPip, Hꢀ5aγꢀClPip/Hꢀ3aγꢀClPip, and Hꢀ4γꢀClPip/NHꢀ5γꢀClPip indicating Hꢀ4γꢀClPip was axial
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(see Figure 2a). We then synthesized an authentic sample of (3R,5S)ꢀ5ꢀchloropiperazic acid by
using a variation of two previously described routes (see Scheme 1),9 and applied the advanced
Marfey’s method which pointed out the presence of (3R,5S)ꢀ5ꢀchloropiperazic acid.
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Scheme 1. Synthesis of (3R,5S)ꢀ5ꢀchloropiperazic acid
O
O
O
O
TBSCl
DCM
NaH, BnBr
KOH, MeOH
reflux, 6h
O
O
HO
OBn
TBSO
OBn
THF/DMF, rt, 4d
OH
OTBS
OH
OBn
11
8
9
10
O
O
O
O
O
PivCl, NEt3
aq. K2CO3
Pd/C, EtOAc
H2
THF, 0 °C
O
O
N
OBn
HO
OBn
N
OH
MeOH/THF, 0 °C
BuLi, THF, -79 °C
OTBS
OTBS
OTBS
Bn
Bn
O
HN
13
14
12
O
O
O
O
O
O
O
N
DBAD, LDA
DMPU, THF
Ph3P, CBr4
THF
OTBS
NaOMe
O
OTBS
Br
MeO
N
DCM, -33°C
OTBS
N
N
N
N
Bn
Bn
Boc
Boc
Boc
Boc
15
16
17
O
O
O
O
Cl
Cl
Cl
TBAF
THF
OH
LiOH, H2O
THF
Ph3P, CCl4
MeCN
TFA
HO
MeO
HO
MeO
DCM
HN
N
N
N
N
N
N
N
Boc
Boc
H
Boc
Boc
Boc
Boc
18
19
20
21
3
Once again ROE correlations and JH,H coupling constants were used to determine the relative
3
configuration of β,γꢀOHdPip. The intermediate JH,H coupling constant between Hꢀ2β,γꢀOHdPip and
Hꢀ3β,γꢀOHdPip (5.5 Hz) along with strong ROE correlations between Hꢀ2β,γꢀOHdPip/Hꢀ4β,γꢀOHdPip and
Hꢀ3β,γꢀOHdPip/NHAla indicated Hꢀ2β,γꢀOHdPip and the hydroxyl group at Cꢀ3β,γꢀOHdPip were
pseudoaxial, whereas the hydroxyl group at Cꢀ4β,γꢀOHdPip was pseudoequatorial (see Figure 2b).10
Together, this NMR data indicated the relative configuration of the stereogenic centers in β,γꢀ
OHdPip as 2R*, 3R*, 4S*. Interestingly, this result highlighted a structural key difference of
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svetamycins with respect to natural product cyclodepsipeptides, in which the αꢀproton of Nαꢀ
3
4
5
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8
acylꢀpiperazic acid or Nαꢀacylꢀδ,εꢀdehydropiperazic acid is located in an equatorial or
pseudoequatorial orientation due to a rotameric A1,3ꢀstrain effect.9b,
In particular, the
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piperazimycins and gerumycins, which have similar amino acid sequences and stereochemistry
at comparable centers to svetamycin A, bear piperazate residues displaying axial orientations for
the αꢀcarboxamide groups.
3
Figure 2. Key ROESY correlations and JHꢀH coupling constants to establish the relative
stereochemistries of a) γꢀClPip, b) β,γꢀOHdPip, and c) δꢀMePip.
Furthermore, we employed a combined quantumꢀmechanical (QM)/NMR approach to compare
1
experimental and calculated 13C and H NMR chemical shifts to corroborate the proposed
configuration for the β,γꢀOHdPip residue. Since the absolute configuration of αꢀMeSer, Pip, and
γꢀClPip were already determined, the QM calculations were performed on eight possible
diastereoisomers of 1 arising from the three stereocenters of β,γꢀOHdPip (compounds 1aꢀ1h, see
Figure S1). An extensive conformational search at the empirical level was carried out for 1aꢀ1h
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by using Monte Carlo Molecular Mechanics (MCMM), LowꢀMode Conformational Sampling
(LMCS), and Molecular Dynamics (MD) simulations (see Computational Details, Experimental
Section). All the obtained conformers were then submitted to a geometry and energy
optimization step at the DFT (density functional theory) level. To obtain information on the
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1
stereochemistry of β,γꢀOHdPip, 13C and H NMR chemical shifts were predicted at the
M062X/6ꢀ31g(d,p) level for 1aꢀ1h, taking account the Boltzmann distribution of the conformers
for each stereoisomer obtained at the same level of theory. Subsequently, the corrected mean
absolute error (CMAE) values were used to compare calculated and experimental values (see
Computational Details, Table 2 and Figure 3). As shown in Figure 3, isomer 1g showed the
13
1
lowest C and H CMAE values (1.60 ppm and 0.21 ppm, respectively), confirming the 2R*,
3R*, 4S* as the most probable relative configuration at β,γꢀOHdPip and suggesting 2S, 3S, 4R
absolute configuration. To further corroborate our findings, we also employed the recently
introduced DP4+ method that has proven to be a powerful tool for the correct stereochemical
assignment of organic compounds.14 Herein, we observed that isomer 1g had the highest sDP4+
probabilities for both 13C and H (99.99% and 99.84%, respectively). These probabilities
1
confirmed the absolute configuration of the residue β,γꢀOHdPip as 2S, 3S, 4R with a high level
of confidence.
In addition, a careful analysis of the energyꢀ and geometryꢀoptimized conformers 1aꢀ1h
pointed out two main different arrangements of the β,γꢀOHdPip residue for each of the
conformers. In the first arrangement, named Conf A, the αꢀproton of β,γꢀOHdPip is
pseudoequatorial oriented, while in the second arrangement, termed Conf B, the proton Hꢀ2β,γꢀ
OHdPip is pseudoaxial oriented (see Figure 4). Analysis of the conformers of 1g on the basis of the
experimental ROE data clearly indicated that only Conf B was in agreement with the ROE
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correlation between Hꢀ2β,γꢀOHdPip/ Hꢀ4β,γꢀOHdPip. Besides, analysis of the energies of the
conformers of 1g at the M062X/6ꢀ31g(d,p) level of theory highlighted specific relative
contributions of the Conf A/Conf B species on the final Boltzmann distribution (see Experimental
section).12,13 Interestingly, Conf B represented the main conformer in 1g (relative weight on the
final Boltzmann distribution: 69%), while Conf A which was not in agreement with the NMR
data, represented the minor conformer (relative weight on the final Boltzmann distribution:
31%). Taken together, the reported data supports 2S, 3S, 4R as the most probable absolute
configuration for β,γꢀOHdPip residue.
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1
Figure 3. 13C (opaque and transparent red bars) and H (opaque and transparent blue bars)
corrected mean absolute errors (CMAE) histograms related to compounds 1aꢀh, as indicated in
Table 2. CMAEs related to compound 1g are highlighted with opaque colored bars.
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Figure 4. 3D representation of the most energetically favored conformers in 1g.
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Table 2. 13C/1H MAE (ppm) values, and DP4+ data reported for all the possible considered
stereoisomers of compound 1 (1aꢀ1h)
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8
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Stereoisomer Configuration at 13C CMAE 1H CMAE
sDP4+ probabilityc
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β,γꢀOHdPip
(ppm)a
(ppm)b
13C data
1H data
all data
2R, 3R, 4R
2R, 3R, 4S
2R, 3S, 4R
2R, 3S, 4S
2S, 3R, 4R
2S, 3R, 4S
2S, 3S, 4R
2S, 3S, 4S
2.46
2.65
2.11
1.98
2.12
2.32
1.51
2.54
0.33
0.29
0.32
0.26
0.24
0.30
0.21
0.32
0.00%
0.00%
0.00%
0.01%
0.00%
0.00%
99.99%
0.00%
0.00%
0.00%
0.00%
0.00%
0.16%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
0.00%
1a
1b
1c
1d
1e
1f
99.84% 100.00%
1g
0.00% 0.00%
1h
a 13C CMAE = (Σ[|(δexp – δscaled)|])/n, summation of the absolute error values (difference of the
13C
absolute values between corresponding experimental, δexp, and scaled calculated, δscaled
,
chemical shifts), normalized to the number of considered chemical shifts (n); the related data are
reported in Table S8. The δscaled values were obtained by the corresponding calculated chemical
shift data (δcalcd, see Computational Details); the latters were produced using the “multi standard”
approach, using TMS as reference compound for sp3 13C atoms, benzene for sp2 13C atoms
(excluding carbonyl carbons), and NꢀMethylformamide for carbonyl carbons. b 1H CMAE =
Σ[|(δexp – δscaled)|]/n, summation of the absolute error values (difference of the absolute values
1
between corresponding experimental, δexp, and scaled calculated, δscaled, H chemical shifts),
normalized to the number of considered chemical shifts (n); the related data are reported in Table
S9 . The δscaled values were obtained by the corresponding calculated chemical shift data (δcalcd
,
see Computational Details); the latters were produced using the “multi standard” approach, using
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TMS as reference compound for sp3 1H atoms, and benzene for 1H atoms bound to sp2 13C atoms.
csDP4+ probabilities related to the set of data reported in Table S8 (13C chemical shift set of
data) and Table S9 (1H chemical shift set of data).
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HRꢀESIꢀMS and NMR data supported the molecular formula of C25H37ClN8O10 and
C26H39ClN8O10 for compounds 2 and 3, respectively. The NMR data clearly indicated that the
amino acid sequences of 2 and 3 are the same as that of 1, except for small changes in the
1
13
resonances ascribable to the piperazic acid residue (see H and C NMR data at Table 3 and
Table 5). Indeed, compound 2 contained an additional methyl group whereas 3 displayed two
additional methyl groups, which explained the 14 and 28 amu mass differences with 1.
Furthermore, COSY and TOCSY correlations of 2 revealed a spin system starting from the
aminomethine proton Hꢀ2 δꢀMePip (δ 4.88) to the secondary amine NHꢀ5δꢀMePip (δ 4.88). Taken this
data together with key HMBC correlations from the methyl protons at δ 1.00 (Meꢀ5δꢀMePip) to the
methylene at 28.2 (Cꢀ4δꢀMePip) and to the methine at 52.3 (Cꢀ5δꢀMePip) established the presence of
an unusual 6ꢀmethylhexahydropyridazineꢀ3ꢀcarboxylic acid (δꢀMePip). Therefore, svetamycin B
(2) was assigned as a Cꢀ5Pip methyl analogue of 1. In similar fashion, interpretation of the 2D
NMR data of svetamycin C (3) allowed us to determine this compound as the Cꢀ5Pip dimethyl
congener of 1. To the best of our knowledge, this is the first report of either a δꢀsubstituted
piperazic acid or a methyl piperazic congener. Up to date, only γꢀchloro and γꢀhydroxy
congeners of piperazic acid have been reported from peptide natural products.15 The absolute
configurations for LꢀαꢀMeSer, 3R,5SꢀγꢀClPip, and LꢀAla in 2 and 3 were determined by using the
advanced Marfey’s method. The relative configuration of δꢀMePip in 2 was determined by
3
analysis of homonuclear coupling constants JH,H and ROE correlations. In particular, the
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intermediate JH,H coupling constant of 5.2 Hz between Hꢀ2δꢀMePip and Hꢀ3aδꢀMePip together with
ROE correlations between Hꢀ3aδꢀMePip/Hꢀ5δꢀMePip, Hꢀ4aδꢀMePip/Meꢀ5δꢀMePip, and Hꢀ4bδꢀMePip/Meꢀ5δꢀ
MePip suggested an equatorial orientation for both Hꢀ2δꢀMePip and Meꢀ5δꢀMePip (see Figure 2c). Also,
the relative configurations of β,γꢀOHdPip in 2 and 3 were deduced as 2R*, 3R*, 4S* on the basis
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of ROE, correlations, JH,H coupling constants, and chemical shifts. Unfortunately, we were not
able to establish the absolute configurations of neither δꢀMePip in 2 nor δ,δꢀdMePip in 3 by
using Marfey’s analysis.
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Table 3. 1H NMR Data (500 MHz, DMSOꢀd6) for Svetamycins BꢀD (2ꢀ4)
5
6
7
8
9
2a
3a
4a
position
δH (J in Hz)
αꢀMeSer
δH (J in Hz)
αꢀMeSer
δH (J in Hz)
αꢀMeSer
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1
2
3a
3.71 dd (10.7, 8.0)
3.81 dd (10.8, 5.6)
1.43 s
3.61 dd (10.8, 6.4)
3.83 dd (10.8, 5.9)
1.37 s
3.61 m
3.88b m
3b
4
1.46 s
NHꢀ2
OHꢀ3
7.55 s
7.78 s
7.43 s
4.44 br. dd (8.0, 5.5)
δꢀMePip
4.76b br. dd (6.2, 5.7)
4.06b m
δ,δꢀdMePip
γ,εꢀdPip
1
2
4.88 br. dd (5.5, 1.4)
1.76 m
4.88 br. dd (5.4, 1.6)
1.96 m
4.99 br. dd (4.3, 1.1)
1.82 m
3a
2.17 br. d (13.5)
1.36 m
2.07 dq (13.5, 2.2)
1.34 dt (13.3, 3.2)
1.42 br. dd (13.3, 4.0)
2.09b m
3b
4a
2.21 m
4b
1.51 br. dd (12.6,4.0)
2.69b m
2.32 br. dd (11.6, 6.6)
7.12 br. d (2.8)
5
1.00b br. d (6.5)
1.00 br, s
1.01 br. s
4.57b s
Meꢀ5a
Meꢀ5b
NHꢀ5
4.14 d (12.2)
γꢀClPip
γꢀClPip
γꢀClPip
1
2
5.8 d (8.8)
5.83 br. s
5.77 br. s
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3a
1.93 td (13.0, 6.0)
2.38 m
1.89 ddd (12.7, 11.2, 6.0)
2.40 br. ddd (12.8, 4.5, 1.7)
4.46 tt (10.9, 4.6)
2.69 td (12.7, 11.0)
3.41 br. ddd (12.8, 4.4, 2.0)
5.21b
2.10b m
2.50b
3b
4
4.36 tt (11.2, 5.4)
2.67b m
4.12 m
2.72b m
5a
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5b
3.39 m
3.35b
5.27b
5.48 br. dd (12.8, 1.4)
Ala
NHꢀ5
Ala
Ala
1
2
5.31 q (7.0)
5.38 q (7.0)
1.18 d (7.0)
7.91 br. d (7.0)
β,γꢀOHdPip
5.23 q (6.9)
3
1.19 br. d (6.9)
7.94 br. d (6.9)
β,γꢀOHdPip
1.19 br. d (6.9)
8.06 br. d (6.9)
β,γꢀOHdPip
NHꢀ2
1
2
4.67 d (4.8)
4.05 q (4.3)
3.94 m
4.56b br. d (5.0)
4.06 q (4.0)
3.95 m
4.88b br. s
4.02 m
3
3.91b m
4
5
6.97 br. d (2.0)
5.27b
6.93 br. d (1.1)
5.22b
7.05 br. d (2.8)
5.41 br. s
6.06 br. d (8.3)
Haa
OHꢀ3
OHꢀ4
5.85 d (8.7)
Haa
5.73 d (8.3)
Haa
1
2a
2b
4.71 d (15.9)
5.25b d (15.9)
4.73b d (16.2)
5.18 d (16.2)
4.87b d (15.9)
5.11 d (15.9)
aRecorded at 500 MHz; referenced to residual DMSOꢀd6 at δ 2.50 ppm. bSignal overlapped
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Table 4. 1H NMR Data for Svetamycins EꢀG (5ꢀ7)
5
6
7
8
5a
6a
7b
position
δH (J in Hz)
αꢀMeSer
δH (J in Hz)
αꢀMeSer
δH (J in Hz)
αꢀMeSer
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10
11
12
13
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23
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33
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1
2
3.53c d (10.8)
3.69 dd (11.0, 7.7)
3.78 dd (11.0, 5.8)
1.43 s
4.06c s
4.06c
3a
3b
3.64 d (10.8)
1.35 s
4
1.54 s
7.72 s
NHꢀ2
OHꢀ1
OHꢀ3
7.96 s
7.59 s
4.55 br. dd (7.7, 5.8)
Pip
δ,δꢀdMePip
δ,δꢀdMePip
1
2
4.88 br. s
2.00c m
2.00c m
1.38c m
1.38c m
4.92 m
1.72c m
2.20 m
1.35 m
1.74c m
2.61 m
2.95 m
5.04 br. dd (5.8, 1.6)
1.99 m
3a
3b
2.07 m
4a
1.46 m
4b
1.76 td (13.3, 3.5)
5a
5b
Meꢀ5a
Meꢀ5b
NHꢀ5
1.03 s
1.08 s
4.64 s
1.14 s
1.16 s
4.60c br. dd (12.7, 1.6)
γꢀBrPip
γꢀClPip
γꢀBrPip
1
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2
5.80 br. dd (5.5, 2.3)
1.97c m
5.70 br. dd (6.2, 1.3)
2.05 td (12.8, 6.2)
2.50c
5.86 br. dd (6.2, 1.5)
3a
3b
2.20 ddd (13.5, 12.4, 6.2)
2.77 ddd (13.5, 4.0, 1.7)
2.65c m
4.32 dddd (12.2, 10.5, 4.5,
4.1)
4
10
11
12
13
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4.24 tt (10.9, 4.5)
4.51 m
2.68c m
2.82 q (12.3)
3.45 m
2.94 dd (13.4, 11.0)
5a
3.36c
3.45 br. dd (13.6, 4.4)
5b
5.28c br. dd (12.3, 1.7)
NHꢀ5
5.49 br. dd (12.3, 1.8)
Ala
Ala
Ala
1
5.35c dq (8.2, 6.9)
1.18 d (6.9)
5.27 q (6.9)
1.33 d (6.9)
2
5.04 dq (8.5, 6.8)
1.16 d (6.8)
3
NHꢀ2
7.59 d (8.5)
8.13 d (8.2)
β,γꢀOHdPip
β,γꢀOHdPip
β,γꢀOHdPip
1
2
4.75 br. d (5.2)
4.00 br. dd (5.2, 3.5)
3.96 t (3.3)
4.71 d (5.4)
4.05 q (4.6)
3.94 ddd (8.6, 4.5, 2.5)
6.97 d (2.5)
5.26c d (4.3)
5.81 d (8.6)
Haa
4.99 br. d (5.7)
3
4.17 br. dd (5.7, 5.3)
4.02 br. dd (5.3, 3.0)
7.07 d (3.0)
4
5
6.86 d (3.0)
OHꢀ3
OHꢀ4
Haa
Haa
1
4.61c d (15.8)
5.37c d (15.8)
4.85c d (16.3)
2a
2b
4.30 d (17.3)
4.35 d (17.3)
5.37 d (16.3)
b
aRecorded at 500 MHz; referenced to residual DMSOꢀd6 at δ 2.50 ppm. Recorded at 500
MHz; referenced to residual CD3OD at δ 3.31 ppm. cSignal overlapped
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Table 5. 13C NMR Data for Svetamycins BꢀG (2ꢀ7).
5
6
7
8
9
2a
3a
δC
4a
δC
5a
δC
6a
δC
7b
δC
position
δC
10
11
12
13
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16
17
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αꢀMeSer
αꢀMeSer
αꢀMeSer
αꢀMeSer
αꢀMeSer
αꢀMeSer
1
2
3
4
172.0
60.5
171.9
59.8
172.2
60.6
173.5
60.1
171.8
60.5
64.3
19.3
Pip
172.4
61.5
63.9
63.4
63.8
64.6
63.0
19.1
19.9
19.0
19.6
17.4
δꢀMePip
δ,δꢀdMePip
γ,εꢀdPip
δ,δꢀdMePip
δ,δꢀdMePip
1
169.0
49.7
24.3
28.2
52.3
169.1
49.7
168.9
50.9
170.2
49.5
169.0
51.2
24.2
20.2
46.4
169.9
49.2
2
3
4
21.4
17.2
22.9
19.5
31.8
19.7
31.9
31.4
5
50.9
145.5
50.9
50.5
Meꢀ5a
Meꢀ5b
28.3
28.4
27.1
19.3
22.7
22.1
21.4
γꢀClPip
γꢀClPip
γꢀClPip
γꢀClPip
γꢀBrPip
γꢀBrPip
1
2
3
4
5
171.4
49.7
34.7
52.6
171.7
48.8
34.2
52.3
170.6
50.2
34.4
52.5
172.0
49.1
34.8
52.5
171.3
51.0
35.6
45.2
173.8
50.3
35.1
41.9
54.1
Ala
54.0
Ala
53.4
Ala
53.6
Ala
54.7
Ala
53.9
Ala
1
2
172.4
43.4
172.3
43.1
172.4
44.0
172.8
45.4
172.0
43.0
173.2
45.1
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3
18.1
17.9
17.9
18.0
18.1
16.7
5
6
7
β,γꢀOHdPip β,γꢀOHdPip β,γꢀOHdPip β,γꢀOHdPip β,γꢀOHdPip β,γꢀOHdPip
8
9
1
2
3
4
5
166.3
56.9
63.7
62.4
146.9
Haa
166.4
56.9
63.7
63.0
147.1
Haa
166.9
55.9
63.0
61.3
146.4
Haa
165.3
55.1
65.2
62.3
144.2
Haa
166.3
56.7
63.8
62.6
146.8
Haa
166.6
55.8
62.9
60.9
145.3
Haa
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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59
60
1
2
166.4
61.9
166.1
61.3
166.8
62.5
172.5
59.9
166.6
61.5
166.6
62.2
aRecorded at 175 MHz; referenced to residual DMSOꢀd6 at δ 39.51 ppm. Recorded at 175
b
MHz; referenced to residual CD3OD at δ 49.15 ppm
Evident from the total ion chromatogram (TIC) obtained from the LC/MS, the methanol
extract also contained two low abundance chlorinated compounds (4ꢀ5) eluting at considerably
shorter retention times compared to 1ꢀ3. The HRꢀESIꢀMS spectrum of svetamycin D (4)
suggested a molecular formula of C24H33ClN8O10 that differed from that of 1 by subtraction of
two hydrogen atoms and required 12 degrees of unsaturation, one more than compound 1.
1
13
Although the 1D and 2D NMR spectra of 4 (see H and C NMR data at Table 3 and Table 5)
contained small impurities, it clearly indicated that the amino acid sequence of 4 is the same as
that of 1 except for the substitution in 4 of δ,εꢀdehydropiperazic acid (δ,εꢀdPip) for a piperazic
acid residue in 1. The HRꢀESIꢀMS of compound 5 displayed a molecular ion at 677.2675
[M+H]+ suggesting a molecular formula of C26H41ClN8O11. Examination of the NMR data of 5
1
13
(see H and C NMR data at Table 4 and Table 5) revealed that its amino acid sequence was
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3
identical to that of 3, although comparison of the H and C NMR chemical shifts of 3 and 5
showed differences in some resonances. In particular, the proton and carbon resonances
corresponding to Hꢀ2Haac in 5 (δH 4.33; δC 59.9) were shifted upfield in comparison to their
respective ones in 3 (δH 4.73, 5.18; δC 61.3). Besides, the molecular formula of 5 required the
presence of 10 degrees of unsaturation, one more than compound 3. Taken together these data,
svetamycin E (5) was determined as the acyclic analogue of svetamycin C (3). It is worth to
mention that the respective acyclic congeners of 1 and 2 were detected by LCꢀHRꢀMS in a
methanol extract of DSM14386. However, their low yields prevented their isolation and
confirmation of their structure by NMR. An insufficient quantity of 5 prevented us from
performing Marfey’s analysis or to acquire ROESY data. Nevertheless, we assumed identical
configurations for 3 and 5 at comparable stereogenic centers since their structures, and optical
rotation values, and NMR data are quite similar.
4
5
6
7
8
9
10
11
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13
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LCꢀMS examination of the methanol extract from the bromideꢀenriched fermentation allowed
us to identify two new brominated peptides. Svetamycin F (6) and svetamycin G (7) displayed in
the ESIꢀMS spectrum an isotopic distribution of the ions [M+H]+, 1:1, at m/z 675/677 and m/z
703/705, respectively, implying the presence of bromine. Their major ion peaks in the HRꢀESIꢀ
MS spectrum were consistent with a molecular formula of C24H35BrN8O10 for 6 and
C26H39BrN8O10 for 7, which in turn differed from the molecular formula of 1 and 3, respectively,
by the replacement of chlorine with a bromine atom. Successively, comparison of the 2D NMR
data (acquired in CD3OD) of 6 to that of 1 determined that the bromine atom was located at the
Cꢀ4γꢀBrPip position (see 1H and 13C NMR data at Table 4 and Table 5). Indeed, the chemical shift
for Cꢀ4γꢀBrPip in 6 was ca 10 ppm upfield when compared to that of Cꢀ4γꢀClPip in 1.16 In similar
fashion, 2D NMR data for 7 clearly determined svetamycin G to be the Cꢀ4γꢀBrPipꢀbromoꢀ
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5
6
7
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9
derivative of 3. We were unable to carry out Marfey’s analysis on 6 and 7 due to the scarce
amount of isolated material. However, we propose similar configurations for 6, 7, and 1 at
comparable stereogenic centers on the basis of their NMR data (chemical shifts, ROE
correlations, and 3JH,H) and optical rotation values.
10
11
12
13
14
15
16
17
18
19
20
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22
23
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60
The biosynthetic origin of the piperazic acid units in the svetamycins was studied by feeding
experiments with labeled precursors. LCꢀHRꢀESIꢀMS/MS analysis of an extract prepared from a
culture of Streptomyces sp. DSM14386 fed with
L
ꢀ[3,3,4,4,5,5ꢀ2H6]ꢀornithine revealed mass
shifts for 1ꢀ3 indicating incorporation of ornithine. Exact determination of the mass shifts was
complicated by the presence of three piperazic acid moieties and the chlorine isotopic pattern
(see Figure S2). However, these results are consistent with the recently report by Walsh et al.
that determined ornithine, and not glutamine, as the starting point in the biosynthesis of piperazic
acid in Kutzneria spp. Indeed, it was demonstrated that formation of N5ꢀOHꢀOrn by the ornithine
Nꢀhydroxylase Ktzl, is the initial step for conversion of ornithine to piperazic acid.17
Additionally, LCꢀHRꢀESIꢀMS/MS analysis of compounds 1ꢀ3 obtained from an L
ꢀ[methylꢀ2H3]ꢀ
methionine feeding experiment showed mass shifts of 3 Da for 2 and of 6 Da for 3, whereas for
compound 1 no mass shift was observed (see Figure S3). These results prove that the
introduction of the methyl groups in the piperazic acid units in the svetamycins is SꢀadenosylꢀLꢀ
methionine (SAM)ꢀdependent.
Microbroth dilution and microbial cell viability assays were used to evaluate the antimicrobial
activity of compounds 1, 3, and 5ꢀ7 towards M. smegmatis, E. coli, C. albicans, and MRSA. The
results are summarized in Table 6 and furnished insights in the structureꢀactivity relationships of
this structural class of piperazic acidꢀcontaining peptides. Svetamycin G (7) showed the most
potent activity against M. smegmatis (IC80 = 2 ꢀg/mL) whereas svetamycin C (3) and 7 were the
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most potent antibiotics towards MRSA (MICs = 16 ꢀg/mL). Svetamycin E (5) inhibited the
5
6
7
8
9
growth of neither M. smegmatis nor MRSA at concentrations as high as 64 ꢀg/mL, indicating
that macrocyclization is needed for the antibiotic activity. Among the cyclic peptides, compound
7 was 4ꢀ16 times more potent than svetamycin F (6) while compound 3 was 4ꢀfold more potent
than svetamycin A (1). All together, these results revealed that methyl substitution of the
piperazic acid unit at δꢀposition increases the activity. Finally, the weaker potency of 3 compared
to 7 showed that replacement of chlorine by bromine has beneficial effects on the antimicrobial
activity. Svetamycins A and C did not inhibit the growth of either E. coli or C. albicans at
concentrations up to 64 µg/mL. Instead, the activity of the extract towards this Gramꢀnegative
pathogen was traced to the known pentalenolactone AAꢀ57.3
10
11
12
13
14
15
16
17
18
19
20
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23
24
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32
33
34
35
36
37
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49
50
51
52
53
54
55
56
57
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59
60
Table 6. Antimicrobial activities for compounds 1, 3, and 5ꢀ7 (µg/mL)
M. smegmatis
MRSA
(MIC)
64
E. coli
(MIC)
> 64
> 64
nd
C. albicans
(MIC)
> 64
> 64
nd
M. tuberculosis
(IC80)
32
(IC80)
65.6
54.0
nd
1
3
5
6
7
8
16
> 64
32
> 64
64
nd
nd
nd
2
16
nd
nd
nd
nd = not determined
Although piperazic acidꢀcontaining cyclic peptides such as hytramycins and lydiamycins have
been reported to exhibit antiꢀMycobacterium tuberculosis activity,8, 18 svetamycins A (1) and C
(3) did show a very weak activity against the standard M. tuberculosis strain H37Rv with IC80
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4
values of 65 and 54 µg/mL, respectively (see Table 6). Also, compounds 1 and 3 were cytotoxic
5
6
to a hepatoma cell line (HepG2) with IC50 values of 11.04 ± 4.02 and 3.59 ± 1.19 µg/mL.
7
8
9
10
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CONCLUSION
In summary, we have discovered seven new halogenated piperazic acidꢀcontaining peptides,
svetamycins AꢀG, that add to a small class represented by the piperazimycins and gerumycins.
Svetamycins contain one hydroxyacetic acid residue, one alanine, and three tailored piperazate
moieties, among which are the previously undescribed 6ꢀmethylꢀ and 6,6ꢀ
dimethylhexahydropyridazineꢀ3ꢀcarboxylic acids along with the unusual 4,5ꢀdihydroxyꢀ2,3,4,5ꢀ
tetrahydropyridazineꢀ3ꢀcarboxylic acid. The latter, is the first example of a piperazyl scaffold
that in a peptide displays a pseudoequatorial conformation for the αꢀcarboxamide group. In
closing, this study also showed the utility of using a combined QM/NMR approach together with
the DP4+ probability method for solving the configuration of stereogenic centers in natural
products.
EXPERIMENTAL SECTION
General Experimental Procedures.
Optical rotations were measured with a Kruess LCꢀSerie 1 polarimeter, and IR spectra were
obtained using a Fourier Transform (FT) Infrared Spectrometer (Japan Bunkou Ltd., JASCO
FT/IRꢀ420, vmax 600ꢀ4.000 cmꢀ1, 20 °C) equipped with a Diamond ATR accessory. NMR spectra
were recorded in DMSOꢀd6 and CD3OD on a Bruker Avance 500 MHz spectrometer equipped
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5
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with a 5 mm TXI cryoprobe. CQFꢀ COSY, 2DꢀHOHAHA, HSQC, HMBC, and ROESY
experiments were recorded using standard pulse programs. HSQC experiments were optimized
7
8
9
1
2,3
for JCꢀH = 145 Hz, and HMBC spectra were optimized for
J
CꢀH
= 8 and 6 Hz. UPLCꢀHRꢀMS
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11
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data were obtained on a quadrupole time of flight spectrometer (LCꢀQTOF maXis II, Bruker
Daltronics, Bremen, Germany) or a micrOTOFꢀLC spectrometer (Bruker Daltronics, Bremen,
Germany) using a BEH C18 (150 x 2.1 mm, 1.7 µm column, Waters, Germany) with a linear
gradient of 5ꢀ95% ACN + 0.1% FA at 450 ꢁL/min in 18 min with UV detection in 205ꢀ640 nm
range. Mass spectra were acquired using the ESI source in the range from 50–2000 m/z. HPLC
for separation and purification was performed on a semiꢀpreparative Agilent 1200 HPLC system
equipped with a Jupiter Proteo C12 column (250 x 25 mm, 4 µm, DAD at 220 and 254 nm). All
solvents used for separation and purification were analytical grade. The Marfey’s analysis was
performed in positive mode on an Agilent Series LC/MSD VL mass spectrometer (Agilent
Technologies, Santa Clara, California).
Bacterial Strains and Cell Line. Streptomyces sp. DSM14386 was bought from the Leibniz
Institute DSMZꢀGerman Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany. Staphylococcus aureus ATCC 33582, Escherichia coli ATCC 53218, Mycobacterium
smegmatis ATCC 607 and Mycobacterium tuberculosis H37Rv ATCC 27294 and the human
liver cancer cell line HepG2 ATCC HB8065 were bought from the American Type Culture
Collection, Manassas, Virginia. Candida albicans FH 2173 is from the strain collection of
SanofiꢀAventis Deutschland GmbH, Industriepark Höchst, Germany.
Cultivation and Extraction. Preꢀcultures of Streptomyces sp. DSM14386 were conducted in
0.3 L Erlenmeyer flasks with 0.1 L of preꢀculture medium (20 g/L glucose, 5 g/L meat extract, 5
g/L yeast extract, 5 g/L peptone, 3 g/L caseine, 1.5 g/L NaCl, at pH 7.5) followed by incubation
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2
3
4
5
6
7
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9
for 4ꢀ7 days on a rotary shaker at 180 rpm and 28°C. The inoculation of the main fermentation
with the preꢀcultures was conducted in forty 2 L Erlenmeyer flasks, containing 0.5 L of the mainꢀ
culture medium (30 g/L glycerol, 5 g/L CaCO3, 15 g/L soybean meal, 2 g/L NaCl, at pH 7.5)
followed by incubation on the rotary shaker at 180 rpm and 28°C for 7 days. After 7 days’
fermentation, the cells and the supernatant were separated by centrifugation, followed by
addition of a mixture of 2 % Amberlite® XAD 16 and XAD 7 (1:1) to the supernatant and
stirring overnight. The resin was harvested afterwards, washed with H2O, lyophilized, extracted
with MeOH and dried under reduced pressure to yield 4 g.
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Cultivation on Agar plates and Extraction. Mainꢀculture medium supplemented with 1.5%
of agar was used to conduct static fermentations in twenty agar plates (24 x 24 cm, Nunc). After
the incubation at 28°C for 7 days, the agar cultures were harvested and extracted by adding 4 L
H2O and homogenizing by vigorous stirring. Once homogenized, extraction was performed by
the addition of 4 L EtOAc and stirring overnight, followed by centrifugation and evaporation of
the EtOAc to yield 18 g of dry extract.
Cultivation on NaBr Agar plates and Extraction. An additional static fermentations was
conducted on ten agar plates (24 x 24 cm, Nunc) replacing 1.5 g/L NaCl with 1.5 g/L NaBr.
After the incubation at 28°C for 7 days, the agar cultures were harvested and extracted by adding
2.0 L H2O and homogenizing by vigorous stirring. Once homogenized, extraction was performed
by the addition of 2.0 L EtOAc and stirring overnight, followed by centrifugation and
evaporation of the EtOAc to yield 6.1 g of dry extract.
Isolation. The extracts prepared from liquid and solid fermentations were fractionated over a
Sephadex LHꢀ20 column (150 x 10 cm), respectively, using MeOH as eluting solvent. Fractions
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containing the peptides were purified by semiꢀpreparative reversedꢀphase HPLC (Jupiter Proteo
C12, 250 x 25 mm, 4 µm, DAD at 220 and 254 nm) eluting with a linear gradient of 50ꢀ85%
MeOH/H2O with 0.05% TFA in 35 min to yield compounds 1 (3.69 mg, tR = 17.3 min), 2 (0.42
mg, tR = 22.4 min), 3 (0.93 mg, tR = 24.9 min), 4 (0.25 mg, tR = 13.2 min), 5 (0.40 mg, tR = 15.3
min), 6 (0.81 mg, tR = 19.5 min) and 7 (0.26 mg, tR = 26.2 min).
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Svetamycin A (1). colorless, amorphous powder; [α]23 ꢀ3.3 (c 0.009, MeOH); LCꢀUV
D
[(MeOH in H2O+0.05% TFA)] λmax 213, 240 nm; IR (film) vmax 3265, 2949, 2359, 2341, 1734,
1
13
1672, 1630, 1558, 1419, 1247, 1202, 1126, 1018, 913 cmꢀ1; H and C NMR data, see Table 1;
HRMS (ESIꢀTOF) m/z: [M+Na]+ calcd for C24H35ClN8O10Na 653.2057; found 653.2059.
Svetamycin B (2). colorless, amorphous powder; [α]23 ꢀ5.2 (c 0.002, MeOH); LCꢀUV
D
[(MeOH in H2O+0.05% TFA)] λmax 213, 240 nm; IR (film) vmax 3264, 2948, 2360, 2341, 1739,
1
1689, 1641, 1540, 1436, 1205, 1135, 842 cmꢀ1; H NMR data, see Table S2; HRMS (ESIꢀTOF)
m/z: [M+Na]+ calcd for C25H37ClN8O10Na 667.2213; found 667.2225.
Svetamycin C (3). colorless, amorphous powder; [α]23 ꢀ2.4 (c 0.004, MeOH); LCꢀUV
D
[(MeOH in H2O+0.05% TFA)] λmax 213, 240 nm; IR (film) vmax 3269, 2967, 2359, 2341, 1745,
1673, 1633, 1541, 1417, 1244, 1200, 1128, 1055, 939 cmꢀ1; 1H NMR data, see Table S3; HRMS
(ESIꢀTOF) m/z: [M+Na]+ calcd for C26H39ClN8O10Na 681.2369; found 681.2374).
Svetamycin D (4). colorless, amorphous powder; [α]23 ꢀ8.8 (c 0.001, MeOH); LCꢀUV
D
[(MeOH in H2O+0.05% TFA)] λmax 213, 240 nm; IR (film) vmax 3360, 2937, 2360, 2341, 1678,
1
13
1635, 1558, 1421, 1244, 1200, 1126, 1058, 963 cmꢀ1; H and C NMR data, see Table 3 and
Table 5; HRMS (ESIꢀTOF) m/z: [M+Na]+ calcd for C24H33ClN8O10Na 651.1900; found
651.1864.
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