Efficient microwave-assisted one shot synthesis of peptaibols using inexpensive coupling reagents

Article abstract of DOI:10.1021/ol5003253

A diisopropylcarbodiimide/Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) coupling cocktail was successfully incorporated into the automated microwave-assisted synthesis of two peptaibols and one analog, whose previously reported syntheses were complicated by steric hindrance. This method utilizes commercially available reagents and affords alamethicin F50/5 and bergofungin D in high yields and purities along with an appreciable reduction of synthesis time and cost when compared to previously reported methods.

Full text of DOI:10.1021/ol5003253

Letter
pubs.acs.org/OrgLett
Efficient Microwave-Assisted One Shot Synthesis of Peptaibols Using
Inexpensive Coupling Reagents
Khoubaib Ben Haj Salah and Nicolas Inguimbert*
́
Universite de Perpignan Via Domitia, Centre de Recherche Insulaire et Observatoire de l’Environnement (CRIOBE) USR CNRS
3278, Centre de Phytopharmacie, batiment T, 58 avenue P. Alduy, 66860 Perpignan, France
S
* Supporting Information
ABSTRACT: A diisopropylcarbodiimide/Oxyma (ethyl 2-
cyano-2-(hydroxyimino)acetate) coupling cocktail was suc-
cessfully incorporated into the automated microwave-assisted
synthesis of two peptaibols and one analog, whose previously
reported syntheses were complicated by steric hindrance. This
method utilizes commercially available reagents and affords
alamethicin F50/5 and bergofungin D in high yields and
purities along with an appreciable reduction of synthesis time
and cost when compared to previously reported methods.
α-Helices account for 30% of a protein’s secondary structure
while β-strands account for 20%; therefore, considerable
interest in the design and application of small helical de novo
peptides can be observed in the literature.1,2 For example,
peptide helices have been exploited as protein/protein
interaction disruptors,3,4 and studying their self-association
provides clues toward understanding their pathogenicity.5
These exquisite properties allow helices to find numerous
applications in medicine as well as in the design of
nanomaterials.6,7
recently COMU.13 Most of the reported syntheses of
peptaibols rely on in-solution strategies based on segment
condensation that lead to tedious flash chromatography
separations to purify each fragment and the final com-
pound.14,15 It is noteworthy that this arduous solution-based
approach is still a popular method for synthesizing
peptaibols.16,17 Consequently, solid-phase peptide synthesis
(SPPS) is a convenient alternative that avoids lengthy
intermediate purification steps.19 However, few examples of
peptaibols synthesized on solid-phase resin have been reported
thus far.20,21 The present paper focuses on the synthesis of two
peptaibols: alamethicin F50/5, which is considered the
archetype of peptaibols, and bergofungin D, which has been
synthesized previously with only an 11% yield.21−25 Using the
method herein both compounds were obtained cost effectively
by SPPS in high yield and purity.
To compare de novo peptaibols on the basis of architectural
design it is necessary to optimize the synthetic strategy for
preparing peptaibols of varying lengths. A cost-effective and
automated synthetic strategy would avoid the following
characteristics: (1) The use of expensive coupling reagents
such as HATU or TFFH (TFFH was shown to be efficient for
the synthesis of ampullosporin A, but was not sufficiently
automated21,22); (2) the preparation of ready-to-couple amino
acids such as α-amino acid fluoride;20,21 (3) reprograming the
peptide synthesizer or changing coupling reagents during the
synthesis;21,22 (4) the use of double-coupling steps for Aib
residues or subsequent residues also sterically hindered by
Aib.21
Studies concerning the development of peptides designed to
self-associate or to adopt a stable helical confirmation led us to
consider peptaibols as a template. Peptaibols are a class of
nonribosomally synthesized peptides from a diverse set of fungi
that include Trichoderma, Gliocladium, and Stibella.8 Usually
linear, these peptides contain 4 to 20 residues and are
characterized by a high content of α,α-dialkyl α-amino acids
such as α-aminoisobutyric acid (Aib, U) and D- or L-isovaline
(Iva). They possess an acetylated N-terminus along with a β-
amino alcohol, generally phenylalaninol (Fol) or leucinol (Lol),
at their C-terminus. These peptides show antibiotic activity
through formation of voltage-dependent pores in biological
membranes.9 Pore formation of this type requires that peptides
have a high propensity to form 310- or α-helical structures, a
conformation sterically induced by the constrained Cα carbon
of the Aib residue.10,11 Because of their intriguing channel
forming properties and small size, peptaibols have attracted
considerable interest as model peptides and numerous
syntheses have been reported.12 However, the synthesis of
peptaibols is significantly hampered by steric hindrance in
residues such as Aib, where reactivity during the coupling
reaction is diminished in comparison to other amino acid
residues. Overcoming the poor reactivity of such residues
requires the use of very efficient but expensive coupling
reagents that sufficiently activate the Aib carboxylic functional
group. These include HATU, HCTU, TFFH, and more
An Fmoc-based strategy in conjunction with a DIPC/ethyl 2-
cyano-2-(hydroxyimino) acetate (Oxyma) coupling cocktail
proved ideal for accomplishing these goals. Encouraging results
Received: February 14, 2014
Published: March 12, 2014
© 2014 American Chemical Society
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dx.doi.org/10.1021/ol5003253 | Org. Lett. 2014, 16, 1783−1785

Organic Letters
Letter
have been observed previously by Albericio and co-workers for
the coupling of Aib residues in model peptides such as the
enkephalin analog YUUFLNH2 and by our group for the
cyclization of marine peptide; however, this approach has yet to
be applied to peptaibols.28,29 Among the numerous advantages
of incorporating these reagents are reduced racemization and a
lower cost, making them 15 and 30 times cheaper than HATU
and TFFH, respectively. As reported for the enkephalin analog,
it was necessary to use a 4 h double-coupling step at the Aib
residue to drive the synthesis.28 More recently, microwave
irradiation has been effective in shortening the reaction time
and increasing product purity, while being compatible with
both oxyma-derived COMU and DIC/Oxyma activation
methods.30,31
Table 1. Analysis and Purity of Synthesized Peptides
crude
purity
(%)
reported
crude
reported
purified
a
compd t (h)
purity (%) purified yields (% - mg) yields (%)
b
b
1
2
3
15
10
10
91
97
95
53
24
−
35 - 68
50 - 72
24
11
b
b
49 - 68
b
a
Total duration of the synthesis (h: hour). Reference 21.
To test the DIC/Oxyma and microwave combination
approach, two challenging peptaibols were selected. Alamethi-
cin F50/5 (1) (Figure 1) was chosen for its 20-residue length
Figure 1. Structures of alamethicin F50/5 (1), bergofungin D (2), and
its analog (3). U: Aib, O: Hyp.
which contains eight Aib (U) residues, two of which are located
at adjacent positions and two of which are coupled to sterically
hindered proline. Bergofungin D (2) (Figure 1) is challenging
because it contains two UU motifs, one of which is followed by
hydroxyproline (O) as well as another UO motif. Previous
methods offered only satisfactory crude purity (<24%) and low
purified yield (11%) despite synthesis optimization.21 Com-
pound 3 is an analog of compound 2 in which the (2S,4R)-4-
hydroxyproline residue is replaced with a proline residue for
initial synthesis optimization at lower cost.
Solid-phase synthesis was carried out using a CEM Liberty
One peptide synthesizer. Compounds 1, 2, and 3 were
assembled on 0.28 g, 0.1 mmol of a preloaded Fmoc-
phenylalaninol o-chlorotrityl resin (0.36 mmol/g). The
optimized protocol for the synthesis of the target peptides
was accomplished on a 0.1 mmol scale using Fmoc-protected
amino acids, DIC, and Oxyma each in 5-fold excess with regard
to the resin capacity. The most efficient reaction conditions
affording complete coupling of Aib were found to be microwave
irradiation at 70 °C for 20 min. A 20% solution of piperidine in
DMF was used for Fmoc deprotection after each coupling step,
and the fulvene−piperidine adduct was quantified by UV
absorption (301 nm) to estimate coupling efficacy (Supporting
Information (SI)). Acetylation was performed on a solid
support by treatment with a solution of acetic anhydride in
DMF for 10 min. After completion peptides were cleaved from
the resin using dilute TFA in dichloromethane to ensure
complete peptide cleavage without degradation of the acid
labile Aib-Pro bond.
Compounds 1, 2, and 3 were obtained in high crude purity
(Table 1), as determined by HPLC for which an evaporative
light scattering detector was used to detect even small amounts
of impurities (Figure 2).33,34
The three compounds were purified by semipreparative
HPLC, and single products were obtained. LC/MS electrospray
analysis provided the singly, doubly, and triply charged
pseudomolecular ions [M + H]+, [M + 2H]2+, and [M +
3H]3+ for each of the compounds (Table 2 and SI). During
Figure 2. HPLC profiles of the crude alamethicin F50/5 (1),
bergofungin D (2), and its analog (3) with ELSD detection. The
HPLC analysis was conducted on a Waters 2695 HPLC system with a
Grace Vydac 218MS 5 μM C-18 column (250 mm × 4.6 mm) using a
gradient mixture of water with 0.1% FA (Buffer A) and ACN with
0.1% FA (Buffer B). All compounds were eluted with a 0.9 mL/min
flow rate from 10% B to 100% B over 50 min.
Table 2. Observed Ions in Full ESI Mass Spectra of
Compounds 1, 2, and 3
compd [M + H]+ [M + 2H]2+ [M + 3H]3+
b ions
y ions
1
1963.52
981.95
654.92
1425.97
714.26
1393.92
697.61
b13: 1189.09
y7: 774.22
2
3
b8: 751.05
b11: 1076.85
b8: 751.06
b11: 1060.83
y6: 676.15
y3: 350.07
y6: 644.11
y3: 334.07
analysis the labile Aib-Pro peptide bond breaks to yield the
expected fragments b13 and y7 of 1 even in full scan mode.35
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Organic Letters
Letter
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These results demonstrate a rapid and efficient method for
the synthesis of peptaibols offering products of higher purities
and yields than the previously reported methods (Table 1).
This method allows the use of inexpensive resins and of a low
cost DIC/Oxyma-based activation scheme with standard
reagent excesses. These results highlight the effectiveness of
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speed and efficiency, particularly for the synthesis of difficult
sterically hindered peptides. Future synthesis of these peptides
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* Supporting Information
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UV quantification of the fulvene−piperidine adduct obtained
after Fmoc deprotection with piperidine. HPLC trace of
purified peptides. LCMS analysis of pure peptides. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Albericio, F. Chem.Eur. J. 2009, 15, 9394.
(29) Boyaud, F.; Mahiout, Z.; Lenoir, C.; Tang, S.; Wdzieczak-Bakala,
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: nicolas.inguimbert@univ-perp.fr.
Notes
The authors declare no competing financial interest.
(30) Pedersen, S. L.; Tofteng, A. P.; Malik, L.; Jensen, K. J. Chem. Soc.
Rev. 2012, 41, 1826.
ACKNOWLEDGMENTS
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(31) Coantic, S.; Subra, G.; Martinez, J. Int. J. Pept. Res. Ther. 2008,
14, 143.
(32) Subiros-Funosas, R.; Acosta, G. A.; El-Faham, A.; Albericio, F.
Tetrahedron Lett. 2009, 50, 6200.
(33) Megoulas, N. C.; Koupparis, M. A. Crit. Rev. Anal. Chem. 2005,
35, 301.
This work was supported by grants of la Ligue Nationale
Contre le Cancer and the Bonus Qualite
́
Recherche of
Perpignan University. The spectroscopic experiments have
been performed using the Biodiversite et Biotechnologies
Marines (Bio2Mar, http://bio2mar.obs-banyuls.fr/fr/index.
html) facilities at the University of Perpignan.
́
(34) Adnani, N.; Michel, C. R.; Bugni, T. S. J. Nat. Prod. 2012, 75,
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