A new member of the fusaricidin family - Structure elucidation and synthesis of fusaricidin E

Article abstract of DOI:10.3762/bjoc.13.140

Two hitherto unknown fusaricidins were obtained from fermentation broths of three Paenibacillus strains. After structure elucidation based on tandem mass spectrometry and NMR spectroscopy, fusaricidin E was synthesized to confirm the structure and the suggested stereochemistry. The synthesis was based on a new strategy which includes an efficient access to the 15-guanidino-3-hydroxypentadecanoyl (GHPD) side chain from erucamide.

Full text of DOI:10.3762/bjoc.13.140

A new member of the fusaricidin family – structure
elucidation and synthesis of fusaricidin E
Marcel Reimann1, Louis P. Sandjo1,2, Luis Antelo3, Eckhard Thines3,4, Isabella Siepe5
and Till Opatz*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 1430–1438.
1Institute of Organic Chemistry, Johannes Gutenberg-University,
Duesbergweg 10–14, 55128 Mainz, Germany, 2Departamento de
Ciências Farmacêuticas, Centro de Ciências da Saúde, Bloco J/K,
Universidade Federal de Santa Catarina, Florianópolis 88040-900,
SC, Brazil, 3Institute of Biotechnology and Drug Research, Erwin
Schrödinger-Str. 56, 66776 Kaiserslautern, Germany, 4Institute of
Molecular Physiology, Microbiology and Wine Research, Johannes
Gutenberg University Mainz, Johann-Joachim-Becher-Weg 15, 55128
Mainz, Germany and 5BASF SE, 67056 Ludwigshafen, Germany
doi:10.3762/bjoc.13.140
Received: 03 May 2017
Accepted: 07 July 2017
Published: 20 July 2017
This article is part of the Thematic Series "Lipids: fatty acids and
derivatives, polyketides and isoprenoids".
Guest Editor: J. S. Dickschat
Email:
Till Opatz* - opatz@uni-mainz.de
© 2017 Reimann et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
cyclodepsipeptides; fusaricidins; lipopeptides; structure elucidation;
total synthesis
Abstract
Two hitherto unknown fusaricidins were obtained from fermentation broths of three Paenibacillus strains. After structure elucida-
tion based on tandem mass spectrometry and NMR spectroscopy, fusaricidin E was synthesized to confirm the structure and the
suggested stereochemistry. The synthesis was based on a new strategy which includes an efficient access to the 15-guanidino-3-
hydroxypentadecanoyl (GHPD) side chain from erucamide.
Introduction
Fusaricidins are lipid-modified non-ribosomal cyclic hexadepsi- essential part of the fusaricidins most likely shows similarity to
peptides containing four D-amino acids and two L-amino acids. the fatty acid synthesis pathway [2].
All of them carry an L-threonine linked to a unique 15-guani-
dino-3-hydroxypentadecanoic acid side chain through the All fusaricidins have three amino acids (L-Thr, D-allo-Thr, and
N-terminus. This particular ω-functionalized lipid side chain is D-Ala) in common and are mostly isolated in pairs which differ
of key importance for the antibiotic activity of the fusaricidins in a single amino acid (asparagine vs glutamine). There are
and their selective inhibition of bacterial cells due to the interac- several known members of the fusaricidin family which were
tion with phospholipid cell membranes [1]. Genetic analysis of isolated from several strains of Paenibacillus polymyxa [3,4].
the producing organisms suggests that the biosynthesis of this Fusaricidins exhibit antimicrobial activity against Gram-posi-
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tive bacteria and a wide range of fungi including Leptosphaeria were inoculated with 4–5 pieces of a well grown agar culture
maculans, a plant pathogenic fungus responsible for the and cultivated on an orbital shaker at 120 rpm at room tempera-
blackleg disease on Brassica crops [5-7].
ture. Large scale fermentations were carried out in 20 L
fermenters with 15 L GYM medium inoculated with 250 mL
Two new compounds were obtained from fermentation of well grown liquid culture at room temperature with agitation
Paenibacillus sp. strain Lu16774 as an inseparable mixture of (120 rpm) and aeration (3 L/min) for 5 to 8 days. To prevent
two homologous cyclic depsipeptides containing either gluta- excessive foaming, a few mL of silicone antifoam were added
mine (in 1) or asparagine (in 2). Initially, the absolute and rela- prior to sterilization of the medium.
tive configuration of the isoleucine residue was unknown
(Figure 1). Thus, an assumed stereoisomer (containing D-allo- An equal volume of isopropanol was added to the culture. After
Ile) of the new fusaricidin member was synthesized based on agitation for 2 h, 200 g/L sodium chloride was added to the
analogy to known members of the series and compared to the mixture until phase separation of the organic and aqueous phase
natural product [8].
was visible. The isopropanol layer was concentrated in vacuo.
The resulting extract was dissolved in methanol, centrifuged for
better precipitation of salt residues, and the organic phase was
concentrated in vacuo again. This step was repeated until no
further salt precipitates were visible.
A portion of the extract (30 g) was dissolved in methanol and
adsorbed to silica gel (Merck, K60, 70–230 mesh, 50 g), dried
at 40 °C and applied onto silica gel (1 kg, column 10 cm diame-
ter, 30 cm height). Elution was carried out in four steps as
follows: ethyl acetate, ethyl acetate/methanol (3:1, v/v), ethyl
acetate/methanol (1:1, v/v) and methanol. The third fraction
containing the active compounds, was dried in vacuo and dis-
solved in 40% methanol (MeOH) in 0.1% formic acid (FA, con-
centration: 100 mg/mL). The other fractions were discarded. A
fraction (20 mL) of the sample was loaded onto a previously
equilibrated (with 40% MeOH in 0.1% FA) Chromabond HR-X
cartridge (Macherey-Nagel, 1000 mg). The cartridge was
washed with 100 mL 40% MeOH in 0.1% FA and eluted with
60 mL 70% MeOH in 0.1% FA. The sample was dissolved in
DMSO (concentration: 200 mg/mL) and 300 μL thereof were
applied to a Sunfire C18 column (19 × 250 mm, 5 μm, Waters)
and eluted as follows: 16 min at 10 mL/min, isocratic 70%
0.2% FA; 30% acetonitrile (MeCN), 1 min at 14 mL/min,
gradient to 65% 0.2% FA; 35% MeCN, 5 min at 14 mL/min,
isocratic 65% 0.2% FA; 35% MeCN. The five resulting frac-
tions were dried in vacuo and re-dissolved in DMSO (concen-
tration: 125 mg/mL). Further purification was performed using
the same column and isocratic conditions (flow: 10.5 mL/min)
adjusted for every fraction (12.5 mg per run). In the second
fraction 68% 0.2% FA; 32% MeCN was used and a single peak
was detected, consisting of an inseparable mixture of two novel
Figure 1: Structure of fusaricidins E (1) and F (2).
Results and Discussion
Isolation and structure elucidation
The Paenibacillus strain was cultivated on agar plates contain- fusaricidins which were named fusaricidin E (1) and fusaricidin
ing GYM medium (10 g/L glucose, 4 g/L yeast extract, 10 g/L F (2). The mixture (6.0 mg) was composed of 3 parts of 2 and
malt extract; pH 5.5, adjusted before autoclaving) and 20 g/L 7 parts of its higher homologue 1 and had an optical rotation of
agar. The submerged cultivation was carried out for 10 to [α]D25 +20.9 (c 0.6, DMSO-d6). The mass difference between
20 days at room temperature. For maintenance, agar slants con- both metabolites is 14 amu. This observation was supported by
taining the same medium were used and stored at 4 °C. Small two ion peaks observed in the ESIMS spectrum at m/z 961.6
scale liquid cultures (250 mL GYM medium in 500 mL flasks) and m/z 975.6, respectively.
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Through extensive analysis of the 1 and 2D NMR data of the δ 3.87 and the carbonyl of Thr2 at δ 170.6. All data suggested a
major component 1, six amino acids including tyrosine (Tyr), cyclodepsipeptidic structure for 1. The N-atom of Thr1 was
glutamine (Gln), alanine (Ala), two threonines (Thr1 and Thr2) bound to a guanidine β-hydroxy fatty acid as a key correlation
and isoleucine (Ile) were identified. NMR experiments like was observed between the signal of its α-methine proton at
COSY, NOESY and HMBC (Figure 2) clarified the sequence of δ 4.46 and the resonance of a carbonyl at δ 171.9. This carbon-
the amino acids through two or three bonds correlation.
yl showed HMBC correlation with α-methylene protons at
δ 2.35 and the β-methine proton at δ 3.77. Additionally correla-
tions between methylene protons at δ 3.03 and the guanidine
carbon at δ 157.2 were found. The length of the side chain be-
tween the β-hydroxy and the guanidine group was affirmed by
the fragment ion observed in the APCI–MS–MS spectrum of
the parent [M + H]+ ion at m/z 256.2. Likewise, this spectrum
provided information (Figure 3) which confirmed the connec-
tion sequence of amino acids and led to elucidate the structure
of 1 and 2 as shown below.
Signals of geminal hydrogen atoms at δ 2.80, 2.52 revealed a
direct correlation with the carbon atom δ 36.3 in the HSQC
spectrum. Based on long-range H–H and H–C interactions,
these resonances were assigned to the β-CH2 group of
asparagine (Asn) in the lower homologue 2. Thus, the conclu-
sion was supported by reported data for Asn in other fusari-
cidins [9] in conjunction to fragments obtained from the tandem
mass of the parent peak at m/z 961.6 (Figure 3).
Retrosynthetic plan
For our synthesis, a novel and efficient access to the GHPD side
chain starting from erucamide (6) as an inexpensive natural
source for the required C13-fragment was developed. After
ozonolysis of the Fmoc-protected amine obtained by reduction
and alkoxycarbonylation from 6, the stereocenter should be
generated by nucleophilic addition of an allyl anion equivalent
to the resulting aldehyde 5.
Guanidine formation and ozonolysis with subsequent oxidation
to the carboxylic acid would then furnish the protected GHPD
side chain building block 3 which can then be coupled to the
cyclodepsipeptide fragment to give the desired product 1
Figure 2: NOESY /COSY and HMBC correlations of compound 1.
The spectra revealed correlations from the Thr2 NH at δ 8.50 to (Scheme 1). For the synthesis of the cyclodepsipeptide portion,
the α-CH of Thr2 at δ 3.94 and the carbonyl of Tyr at δ 166.7. a convergent route was envisaged in which the complete GHPD
The same correlations were found for the Tyr NH at δ 8.52, the side chain should be attached in solution after assembly of the
Tyr α-CH at δ 2.60 and the carbonyl of Ile at δ 170.4. The spec- cyclodepsipeptide on solid support. The hitherto only published
tra also showed a strong correlation of the Ile α-CH at δ 4.16 synthesis of a natural fusaricidin by the Jolliffe group em-
with the carbonyl signal of Ile at δ 170.4 and a weak correla- ployed a ring closure via a lactonization in solution and subse-
tion with the carbonyl of Thr1 at δ 168.6. Furthermore, the quent attachment of the side chain to the cyclized depsipeptide
β-methine proton at δ 5.30 of Thr1 correlated with the carbonyl [10]. Since the macrolactonization approach suffered from dia-
signal at δ 170.4 of Ala and additionally the NH of Ala at δ 7.27 stereoselectivity issues and low yield, it was decided to perform
had the expected contact with Ala’s α-CH at δ 4.20. The latter an on-resin head-to-tail macrolactamization instead [11]. A sim-
proton showed correlations with the carbonyl of the same amino ilar strategy had been used by Cudic and co-workers to synthe-
acid and the one of Gln. The NH proton of Gln at δ 8.20 size analogs of fusaricidin A with an on resin coupling of a
displayed correlations with the methine hydrogen of Gln at 12-aminododecanoic acid combined with a late stage guani-
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Figure 3: Fragmentation pattern of compounds 1 and 2.
Scheme 1: Retrosynthetic plan for the depsipeptide and GHPD side chain.
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dinylation to give the unnatural 12-guanidinyldodecanoic acid Maruoka–Keck allylation in another total synthesis, the conver-
side chain [12]. In order to reduce the number of linear steps, sion was not satisfying in this case [15]. After protection and
the protected guanidino group was included in the side chain guanidinylation with triflylguanidine 14, the homoallylic
building block in our case. This strategy would allow to alcohol 12 was subjected to ozonolysis and Pinnick oxidation to
assemble the complete peptide core in a solid-phase synthesis furnish the protected GHPD acid 3 in an overall yield of 14.3%.
and to perform the solution-phase coupling without a large
excess of the GHPD side chain building block. Thus, Cudic’s The peptide core was synthesized manually according to a stan-
SPPS approach should be combined with the advantages of the dard SPPS Fmoc protocol using HATU and NMM in NMP
late stage coupling employed by Jolliffe.
[16]. For protection of the threonine unit, it was converted into
a 2,2-dimethylated pseudoproline (ΨMe,Me'Pro). This method
has been reported to improve yields of macrolactamizations
Synthesis
The C13-fragment was prepared starting from erucamide (6) in through stabilizing the cis-configuration of the amide bond and
three simple operations. Reduction of the amide with lithium al- working as a turn inducer in peptides [17-20]. The ΨMe,Me'Pro
uminium hydride, followed by Fmoc-protection and ozonolysis unit turned out not to be completely stable during the esterifica-
furnished aldehyde 5 in 57% yield over three steps (Scheme 2). tion with DIC/DMAP [12] as the double acylation product 18
could be detected (Scheme 3).
The homoallylic alcohol was prepared by an enantioselective
Duthaler–Hafner allylation [13] with the titanium-complex 15 Further investigations suggested DMAP to be responsible for
in high yield and 94% ee. Attempts to perform a catalytic Keck the cleavage of the ΨMe,Me'Pro and after reduction of the
allylation with BINOL-titanium catalysts failed due to low amount of DMAP to 5 mol %, nearly no doubly acylated prod-
conversion [14]; however, in spite of good experience with the uct was found.
Scheme 2: a) LiAlH4, THF, reflux, 12 h, quant.; b) Fmoc-OSu, NaHCO3, 1,4-dioxane, H2O, 0 °C to rt, 87%; c) 1: O3, DCM, –78 °C, 2: Zn, HOAc,
–78 °C to 10 °C, 66%; d) allylmagnesium chloride, 15, Et2O, –78 °C, 84%, 94% ee; e) MOMCl, DIPEA, DCM 0 °C to rt; f) piperidine, DCM, rt; g) 14,
NEt3, DCM, 49% (over 3 steps); h) 1: O3, DCM, –78 °C, 2: PPh3, –78 °C → rt, 77%; i) NaClO2, NaH2PO4, amylene, t-BuOH, H2O, rt, 80%.
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Scheme 3: Ester bond formation with 2,2-dimethylated pseudoproline including peptide 16.
Besides the doubly acylated product, several other uncharacter-
ized byproducts were formed. Nevertheless, O-deallylation with
Pd(PPh3)4 and BH3·NHMe2 gave a high conversion to the
deprotected peptide [1]. The cyclization with PyBOP, HOBt
and DIPEA in DMF led again to the formation of numerous by-
products. In contrast to literature reports [12], it was found that
cleavage from the resin with “reagent K” [12,21] was inferior to
a TFA/TIS/H2O mixture (95:2.5:2.5). Due to the instability of
ΨMe,Me'Pro during esterification and cyclization, the isolated
yield of 19 was only 6% (Scheme 4).
To improve this, O-tert-butyl-protected D-allo-Thr was em-
ployed instead. For the O-tert-butyl-protected D-allo-Thr, clean
and complete conversion was observed during both esterifica-
tion and deprotection. This time, cyclization was performed
with PyAOP and NMM in NMP to provide 19 in an isolated
yield of 37% (Scheme 5).
Unfortunately, we observed two signal sets in NMR spectra (in-
tensity 3:1) for the product which could not be attributed to
conformers or rotamers as evidenced by variable temperature
and NOESY NMR experiments.
These results, and the fact that Cochrane also reported prob-
lems due to epimerization during macrolactonization suggested
Scheme 4: Cyclization with 2,2-dimethylated pseudoproline including
peptide 16.
that a partial loss of stereochemical integrity had taken place
during either cyclization or esterification [11]. The site of epi-
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Scheme 5: Depsipeptide cyclization and coupling with GHPD side chain.
merization could not be determined with certainty, but most product 25 resulting from a ring opening reaction of the solvent
likely, the D-Ala residue was affected.
(Figure 4). This side reaction has been reported for unstabilized
THF [22] while stabilized THF was used in our case.
As the assumed diastereomers could not be observed or separat-
ed by HPLC, the next steps were performed with the mixture. Due to solubility problems in 1,4-dioxane and other solvents
Performing the removal of the Cbz group with H2/Pd-C in THF, suitable for hydrogenolysis, the deprotection was thus per-
we encountered the formation of the N-(4-hydroxybutylated) formed in DMF [23,24]. Unfortunately, an O→N acyl shift to
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Beilstein J. Org. Chem. 2017, 13, 1430–1438.
formed with extensive NMR spectroscopy and tandem mass
spectrometry. The full stereostructure of the major component,
fusaricidin E, could be confirmed by total synthesis. It included
a macrolactamization approach combined with a late stage
attachment of the GHPD side chain which was synthesized by a
newly developed and efficient sequence starting from
erucamide. Compared to the Jolliffe strategy, yields were im-
proved and the number of solution phase transformations was
reduced. On the other hand, the yields of the Cudic synthesis
could not be reached and more steps required purification of the
respective products.
Supporting Information
Supporting Information File 1
Procedures for the synthesis and characterisation data of the
compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-13-140-S1.pdf]
Acknowledgements
This work was supported by the Rhineland Palatinate Center for
Natural Products Research as well as by BASF SE. We thank
Dr. J. C. Liermann (Mainz) for NMR spectroscopy and Dr. N.
Hanold (Mainz) for mass spectrometry.
Figure 4: Byproducts from removal of Cbz group in THF and DMF.
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