A macrolactonization approach to the total synthesis of the antimicrobial cyclic depsipeptide LI-F04a and diastereoisomeric analogues

Article abstract of DOI:10.3762/bjoc.8.154

The cyclic peptide core of the antifungal and antibiotic cyclic depsipeptide LI-F04a was synthesised by using a modified Yamaguchi macrolactonization approach. Alternative methods of macrolactonization (e.g., Corey-Nicolaou) resulted in significant epimerization of the C-terminal amino acid during the cyclization reaction. The D-stereochemistry of the alanine residue in the naturally occurring cyclic peptide may be required for the antifungal activity of this natural product.

Full text of DOI:10.3762/bjoc.8.154

A macrolactonization approach to the total synthesis
of the antimicrobial cyclic depsipeptide LI-F04a
and diastereoisomeric analogues
James R. Cochrane, Dong Hee Yoon, Christopher S. P. McErlean
and Katrina A. Jolliffe*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 1344–1351.
School of Chemistry, The University of Sydney, 2006, NSW, Australia;
Tel: +61-2-93512297; Fax: +61-2-93513329
doi:10.3762/bjoc.8.154
Received: 02 May 2012
Accepted: 16 July 2012
Published: 21 August 2012
Email:
Katrina A. Jolliffe* - kate.jolliffe@sydney.edu.au
* Corresponding author
This article is part of the Thematic Series "Antibiotic and cytotoxic
peptides".
Keywords:
antifungal; cyclic depsipeptide; epimerization; lipopeptide;
macrolactonization; peptides
Guest Editor: N. Sewald
© 2012 Cochrane et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
The cyclic peptide core of the antifungal and antibiotic cyclic depsipeptide LI-F04a was synthesised by using a modified
Yamaguchi macrolactonization approach. Alternative methods of macrolactonization (e.g., Corey–Nicolaou) resulted in significant
epimerization of the C-terminal amino acid during the cyclization reaction. The D-stereochemistry of the alanine residue in the
naturally occurring cyclic peptide may be required for the antifungal activity of this natural product.
Introduction
The LI-F or fusaricidin class of cyclic depsipeptides are are D-Asn, L-Val and D-Val. A unique 15-guanidino-3-
produced by a number of strains of Bacillus (Paenebacillus) hydroxypentadecanoyl (GHPD) side chain is appended to the
and exhibit antifungal and antibacterial activity against a range cyclic peptide core through the nitrogen atom of the L-Thr
of clinically relevant species, including Candida albicans, residue. There has been recent interest in the synthesis of the
Cryptococcus neoformans, Staphylococcus aureus and Micro- LI-F family of cyclic depsipeptides due to their antifungal
coccus luteus [1-7]. These compounds have a cyclic hexadep- activity. Biosynthetic processes have been employed to this end,
sipeptide core, in which three amino acids, L-Thr, D-allo-Thr although these provide mixtures of depsipeptides, which makes
and D-Ala are conserved throughout the series, while there are it difficult to determine structure–activity relationships [7,8].
slight variations in the other three amino acids. In LI-F04a these More recently, the solid-phase synthesis of a number of
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
analogues of the fusaricidins has been reported. However, in all
cases, the side chain 3-hydroxy group was not incorporated into
the structure [9]. By total synthesis of both side-chain epimers
of this structure we have recently established that the absolute
configuration of this side-chain hydroxy group is (R) in the
naturally occurring LI-F04a 1 [10]. We employed a late-stage
coupling of the cyclic peptide core 2 with the GHDP side chain
3 to enable ready access to both side-chain epimers (Scheme 1).
While macrocyclization to give the core 2 could be performed
at any of the amide or ester bonds [10], we chose to use a
macrolactonization approach to enable ready access to
analogues of the LI-F04a core through straightforward Fmoc
solid-phase peptide synthesis of the linear precursors. We report
here our optimization of these macrolactonization conditions,
together with the synthesis of several analogues of LI-F04a
using this approach, and an investigation of the antifungal
activity of these synthetic lipodepsipeptides.
Results and Discussion
The required linear precursor for the synthesis of 2 by a macro-
lactonization approach is 4 (Scheme 1), in which the N-terminal
amino group of the L-Thr residue is protected while the side-
chain hydroxy group is free. The Cbz group was chosen as a
suitable protecting group for the N-terminus. The D-Asn and
D-allo-Thr residues were the only amino acids requiring side-
chain protection. Given previous reports that 2,2-dimethylated
pseudoprolines (ΨMe,Me'Pros) [11-13] are useful turn-inducers
for improving yields of macrolactamization reactions [14-17],
we chose to prepare two linear precursors, 5 and 6 (Scheme 2),
in which the D-allo-Thr protecting group was either tert-butyl
or ΨMe,Me'Pro, respectively, to investigate whether a turn-
inducer might assist the macrolactonization reaction. Linear
peptides 5 and 6 were prepared by standard Fmoc solid-phase
peptide synthesis protocols using PyBOP/Hünigs base as the ac-
tivation reagent and 2-chlorotritylchloride resin to allow
cleavage of the peptide from the solid support with side-chain
protecting groups intact. In the case of 6, the ΨMe,Me'Pro was
introduced by coupling the known dipeptide Fmoc-Val-D-a-
Thr(ΨMe,Me'Pro)-OH [18] into the growing peptide chain.
Scheme 1: Retrosynthetic strategy.
While there are a large number of methods available for macro-
lactonization reactions, those most commonly employed include
the Corey–Nicolaou [19], Boden–Keck [20] and Yamaguchi significantly under the three sets of conditions, with the major
[21] lactonization procedures. We initially chose to screen these diastereomer formed under the Yamaguchi conditions differing
three procedures for the macrolactonization of 5. In all three from the major product obtained in the other reactions.
cases (Table 1, entries 1–3), analysis of the crude product
mixtures showed that mixtures of cyclic diastereoisomers were In order to establish which set of conditions gave the highest
obtained, indicating that the C-terminal amino acid underwent ratio of the desired product 7, the mixture of cyclic depsipep-
epimerization during the macrolactonization reactions (see tides 7 and 8 obtained from macrolactonization under the
Supporting Information File 1 for full experimental details) highest yielding conditions (Corey–Nicolaou) was separated,
[22]. However, the ratio of the two diastereoisomers differed and the major diastereomer was subjected to hydrolysis upon
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
Scheme 2: Macrolactonization reactions of seco acids 5 and 6 (for reagents and yields see Table 1 and Table 2).
treatment with MeOH/H2O/aq NH4OH (4:1:1 v/v/v). Compari- focussed on this and related procedures (Table 1). The best
son of the crude peptide, so obtained, to authentic samples of yields of the desired cyclic depsipeptide 7 (58% isolated yield,
both 5 and the C-terminal epimer 9 (which was independently with 5–12% of epimer 8 also observed) were obtained using a
prepared by solid-phase peptide synthesis), indicated that the modification of Yonemitsu’s conditions [23] in which linear
major product obtained upon macrolactonization of 5 under the peptide 5 was added slowly to a solution of DMAP, 2,4,6-
Corey–Nicolaou conditions was the undesired cyclic trichlorobenzoyl chloride and triethylamine in toluene at room
depsipeptide 8, in which the C-terminal Ala residue had epimer- temperature. Similar yields and low epimerization (52% isol-
ized (Figure 1). Since the Yamaguchi conditions gave impro- ated yield, 6% epimer observed) were obtained using 2-methyl-
ved ratios of the desired/epimerized cyclic depsipeptides, 6-nitrobenzoic anhydride (MNBA) [24] as the activating agent
subsequent optimization of the macrolactonization conditions in place of 2,4,6-trichlorobenzoyl chloride.
Table 1: Reaction conditions for macrocyclization of 5.
entry reaction conditions
yield of major isomera ratio of 7:8b
1
2
3
4
5
6
7
8
9
dithiopyridine, triphenylphosphine, MeCN, 80 °C
56%
14%
20%
33%
33%
16%
58%
52%
30%
13:87
45:55
61:39
69:31
89:11
82:18
92:8c
94:6
DCC, DMAP, camphorsulfonic acid, CH2Cl2
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 110 °C
2,4,6-trichlorobenzoyl chloride, DMAP, iPr2NEt, toluene, 80 °C
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, THF, 25 °C
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C, slow addition of 5
2-methyl-6-nitrobenzoic anhydride, DMAP, NEt3, toluene, 25 °C, slow addition of 5
cyanuric chloride, MeCN, 25 °C
80:20
aIsolated yield; bas determined by integration of analytical HPLC traces of the crude reaction mixtures; caverage value (5–12% epimerization was
observed upon repeat reactions).
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
Figure 1: Analytical HPLC traces of linear peptides. (a) Compound 9 (retention time = 31.5 min); (b) Compound 5 (retention time = 30.7 min); (c)
co-injection of mixture of 9 and 5 (2:1); (d) Crude reaction mixture after hydrolysis of major cyclic peptide from Corey–Nicolaou cyclization, indicating
that the major product of hydrolysis is 9; ▲ indicates the unreacted cyclic peptide (retention time = 34.0 min) and ● is a peak attributed to hydrolysis of
both the cyclic peptide ester bond and Cbz protecting group (LCMS m/z = 902 [M + H]+).
To investigate whether the ΨMe,Me'Pro would assist macrolac- Thr(O-t-Bu) protected cyclic peptides 7 and 8. With cyclic
tonization, linear precursor 6 was subject to cyclization under a peptides 7 and 8 in hand, we chose to attach the GHPD side
range of conditions (Table 2). However, in all cases, the cycliz- chain to both compounds to enable the effect of peptide stereo-
ation yields were found to be lower for the ΨMe,Me'Pro- chemistry on the biological activity of the LI-F cyclic peptides
containing peptide than for the tert-butyl protected precursor 5, to be assessed. Additionally, to investigate the effect of the
with higher amounts of C-terminal epimerization also observed, 3-hydroxy group of the GHPD side chain on the biological
except in the case of cyclization using cyanuric chloride [25]. activity of this class of cyclic peptide, we prepared the dehy-
Unfortunately, the yield could not be improved above 17% in droxy side-chain analogue 12 for attachment to the cyclic
this case, so all further reactions were performed using the peptide core (Scheme 3). Notably, a previously synthesised
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
Table 2: Reaction conditions for macrocyclization of 6.
entry reaction conditions
yield of major isomera
ratio of 10:11b
1
2
3
4
5
dithiopyridine, triphenylphosphine, MeCN, 80 °C
24%
19%
7%
<5 to >95
50:50
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 110 °C
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C
80:20
2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C, slow addition of 5 24%
cyanuric chloride, MeCN, 25 °C 17%
79:21
>95 to <5
aIsolated yield; bas determined by integration of analytical HPLC traces of the crude reaction mixtures.
LI-F04a analogue with a twelve-carbon side chain lacking the 18, 19 [10] and 12, by using HATU as the coupling agent. The
hydroxy group has been observed to have antimicrobial activity resulting compounds were not isolated but immediately
[9]. Thus, hydrolysis of pentadecanolide 13 [26] was followed subjected to global deprotection upon treatment with trifluoro-
by esterification to give the methyl ester 14 in excellent yield. acetic acid/CH2Cl2/H2O (90:5:5 v/v/v) to give LI-F04a (1),
Reaction of 14 with di(tert-butoxycarbonyl)guanidine under side-chain epimer 20, dehydroxy analogue 21 and the two side-
Mitsunobu conditions [27] proceeded smoothly to give 15 in chain epimers of the L-Ala derivative, 22 and 23.
86% yield. Hydrolysis of the methyl ester followed by acidic
work up to enable extraction of the resulting carboxylic acid The antifungal activity of 1 and 20–23 was evaluated by using a
gave 12, in which one of the guanidino Boc protecting groups standardised serial dilution sensitivity assay [28] against refer-
was also removed.
ence strains of Candida albicans, Cryptococcus neoformans and
Aspergillus fumigatus (Table 3). Synthetic LI-F04a was found
to exhibit good activity against both C. albicans and C.
neoformans, but only modest activity against A. fumigatus,
consistent with the previously reported activity of the natural
product [1,3,5]. The side-chain epimer 20 exhibited signifi-
cantly lower activity than 1 against C. albicans and C.
neoformans, indicating that the stereochemistry of the side-
chain hydroxy group is important for the antifungal activity of
the compounds. Removal of the hydroxy group, as in 21,
resulted in a further small decrease in activity against these
species. Notably, compounds 22 and 23, prepared from the
C-terminal-epimerised cyclic peptide did not exhibit antifungal
activity against any of the species tested. This suggests that the
conformation of the cyclic peptide core is important in deter-
mining the antifungal activity of these compounds, since inver-
sion of the stereocentre of one of the amino acids in the macro-
cycle is expected to result in a significantly different peptide
conformation [29]. Modelled structures of the side-chain-acyl-
ated cyclic peptides 24 and 25 obtained by using Monte Carlo
conformational searches in Macromodel [30] suggest that these
cyclic peptides adopt significantly different conformations with
different arrangements of hydrogen bonds (Figure 2). The
temperature dependence of the chemical shifts of the signals
attributable to the amide NHs of 1 in d6-DMSO was deter-
mined experimentally and confirmed the involvement of the
D-Ala and D-Asn amide protons in hydrogen bonds [31] as
suggested by the modelling studies of the acetamide analogue
Scheme 3: Synthesis of the dehydroxy side chain 12.
Hydrogenolysis of the Cbz protecting groups of 7 and 8 gave (see Supporting Information File 1 for details). This indicates
the corresponding amines 16 and 17, respectively (Scheme 4). that these hydrogen bonds may be important in locking the
These were coupled with the previously synthesised side chains cyclic depsipeptide into a biologically active conformation.
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
Scheme 4: Synthesis of LI-F04a (1) and analogues 20–23.
Table 3: Antifungal activity.
C. albicans
ATCC 10231
(MIC µM)a
C. neoformans
ATCC 90112
(MIC µM)a
A. fumigatus
ATCC 204305
(MIC µM)a
compound
1
5.5
22
2.8
11
44
20
21
22
23
22
44
22
22
>88
>88
>88
>88
>88
>88
aMICs for amphotericin B: C. albicans 0.35 µM; C. neoformans 0.35 µM; A. fumigatus 0.70 µM.
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
Figure 2: Structures and lowest-energy conformers of 24 (left) and 25 (right) obtained using Macromodel. Hydrogen bonding is highlighted in yellow.
Conclusion
Supporting Information
In summary, macrolactonization to form the cyclic depsipeptide
core of LI-F04a was achieved in good yields by using either
Supporting Information File 1
modified Yonemitsu conditions or similar conditions, in which
the 2,4,6-trichlorobenzoyl chloride activating agent was
replaced with MNBA. Slow addition of the linear seco-acid to
the activating agents was found to be the key factor to minim-
izing epimerization of the C-terminal amino acid during the
macrolactonization reaction. Synthetic LI-F04a was found to
exhibit similar antifungal activity to that reported for the natur-
ally occurring material. The antifungal activity of 1 was reduced
upon either inversion of the stereochemistry, or deletion of the
side-chain hydroxy group. Inversion of the D-Ala residue in the
Experimental details for all new compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-154-S1.pdf]
Supporting Information File 2
1H, 13C and 2D NMR data for all new compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-154-S2.pdf]
cyclic depsipeptide core resulted in complete loss of antifungal Acknowledgements
activity, indicating that the cyclic peptide conformation may be We thank the University of Sydney for financial support and the
important in the biological activity of this class of cyclic award of a postgraduate scholarship to JRC; and Prof. Tania
lipodepsipeptide.
Sorrell and Dr. Julianne Djordjevic (Centre for Infectious
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Beilstein J. Org. Chem. 2012, 8, 1344–1351.
27.Dodd, D. S.; Kozikowski, A. P. Tetrahedron Lett. 1994, 35, 977–980.
doi:10.1016/S0040-4039(00)79943-6
Diseases and Microbiology, Westmead Hospital, University of
Sydney) for assistance with the antifungal assays.
28.Reference Method for Broth Dilution Susceptibility Testing of Yeasts:
Approved Standard. NCCLS document M27-A (ISBN 1-56238-186-5).
National Committee for Clinical Laboratory Standards, Pennsylvania,
USA, 1997; Reference Method for Broth Dilution Susceptibility Testing
of Filamentous Fungi: Approved Standard. NCCLS document M38-A
(ISBN 1-56238-186-5). National Committee for Clinical Laboratory
Standards, Pennsylvania, USA, 2002.
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