Total synthesis of polydiscamides B, C, and D via a convergent native chemical ligation-oxidation strategy

Article abstract of DOI:10.1021/ol502045u

The first total syntheses of the marine sponge-derived cyclic depsipeptide natural products Polydiscamides B, C, and D are described. The molecules were constructed through the convergent fusion of cyclic and linear fragments via an unprecedented native chemical ligation-oxidation protocol.

Full text of DOI:10.1021/ol502045u

Letter
pubs.acs.org/OrgLett
Total Synthesis of Polydiscamides B, C, and D via a Convergent
Native Chemical Ligation−Oxidation Strategy
Gajan Santhakumar and Richard J. Payne*
School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
S
* Supporting Information
ABSTRACT: The first total syntheses of the marine sponge-
derived cyclic depsipeptide natural products Polydiscamides B,
C, and D are described. The molecules were constructed
through the convergent fusion of cyclic and linear fragments
via an unprecedented native chemical ligation−oxidation
protocol.
olydiscamides B, C, and D (1−3) were isolated by Quinn
and co-workers from a marine sponge Ircinia sp., collected
interesting structural features described above, a number of
P
members of this family have been shown to exhibit promising
antimicrobial3,4 and antifungal activity.7,8 Discodermin A has
also been shown to exhibit a permeabilizing effect on the
plasma membrane,11 while polydiscamide A has been reported
to possess inhibitory activity against a human lung cancer cell
line.2
from the Great Barrier Reef in Australia (Figure 1).1 These
In this study, we set out to develop a robust and convergent
synthetic strategy to access all three cyclic depsipeptides 1−3
and thus facilitate profiling of the antimicrobial activity of the
natural products against a range of clinically relevant Gram
positive and Gram negative bacterial strains.
From the outset, we envisaged that all three natural products
could be accessed via a novel native chemical ligation−
oxidation strategy (Scheme 1). Specifically, it was proposed that
a common cyclic depsipeptide fragment bearing a pendant D-
cysteine moiety (7) could be fused to linear peptide thioesters
4−6 (unique to each natural product) under native chemical
ligation conditions.12 Following the ligation event, oxidation of
the cysteine residue would then afford polydiscamides B−D
(1−3). For this synthetic strategy we deemed that protection of
the side chain of tryptophan was crucial to prevent over-
oxidation of the indole ring,13,14 and therefore peptide
thioesters 4−6 were targeted with side chain protecting groups
intact.
Synthesis of the cyclic depsipeptide fragment 7 commenced
with loading of 2-chlorotrityl chloride resin with Fmoc-Pro-OH
(Scheme 2). Elongation to the linear resin-bound peptide 8 was
achieved via Fmoc-solid phase peptide synthesis (SPPS),
whereby Fmoc-deprotection was facilitated through treatment
with 10 vol % piperidine in DMF, and amino acids were
coupled using (benzotriazol-1-yloxy)tripyrrolidinophospho-
Figure 1. Polydiscamides B−D (1−3).
natural products were shown to exhibit potent agonist activity
against a human neuron-specific G-protein coupled receptor
involved in the modulation of pain. Key structural features of
1−3 include a hydrophilic cyclic depsipeptide fragment that
contains two D-amino acids (valine and aspartic acid) with a
lactone functionality occurring between the side chain of L-
threonine and the α-carboxylate of the D-aspartic acid residue.
The cyclic depsipeptide of 1−3 is linked through the α-amine
of L-threonine to a linear hydrophobic peptide chain possessing
p-bromo-L-phenylalanine and D-cysteic acid residues. In
addition, polydiscamides B and C have a β-methyl-L-isoleucine
in this linear portion, while polydiscamides B and D contain
tert-D-leucine and tert-L-leucine residues respectively, notably at
different sites in the linear chain. The polydiscamides belong to
a family of sponge-derived natural products that includes
polydiscamide A,2 discodermins A−H,3−6 halicylindramides A−
D,7,8 and microspinosamide,9 and to date, only halicylindra-
mide A has succumbed to total synthesis.10 In addition to the
Received: July 13, 2014
© XXXX American Chemical Society
A
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Scheme 1. Retrosynthetic Analysis of Polydiscamides B−D
(1−3)
Scheme 2. Synthesis of Cyclic Depsipeptide Fragment 7
nium hexafluorophosphate (PyBOP) as the coupling agent and
N-methylmorpholine as the base (see Supporting Information
for full details). Notably, coupling of Fmoc-L-Thr-OH to the N-
methylated L-glutamine residue required modified conditions of
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyri-
dinium 3-oxid hexafluorophosphate (HATU) as the coupling
reagent and iPr2NEt as the base. Following the assembly of
resin-bound peptide 8 the free side-chain hydroxyl moiety of
the L-threonine residue was esterified with Fmoc-D-Asp(OtBu)-
OH en bloc using Steglich esterification conditions [N,N′-
diisopropylcarbodiimide (DIC), 4-dimethylaminopyridine
(DMAP), DMF]. Fmoc-deprotection followed by cleavage of
the side-chain protected peptide from the resin by treatment
with 30 vol % hexafluoroisopropanol (HFIP) in CH2Cl2 yielded
the crude, suitably protected peptide 9, ready for cyclization.
Macrolactamization between the free carboxylic acid of the C-
terminal L-proline residue and the free N-terminal amine of the
D-aspartic acid residue was facilitated by HATU and iPr2NEt in
CH2Cl2 at a final concentration of 0.001 M.15 The cyclization
reaction was monitored by HPLC-MS and upon completion
(16 h) was subjected to an acidic cocktail comprising TFA/
iPr3SiH/H2O (90:5:5 v/v/v) to remove the side-chain
protecting groups. Following reversed-phase HPLC purification
the cyclic pentapeptide 7 was isolated in 12% yield over 14
steps (based on the initial resin loading).
of the linear peptide fragments, on-resin formylation of the N-
terminal D-alanine residue was effected by treatment with p-
nitrophenyl formate in DMF. Mild acidolytic cleavage of the
resin-bound peptide with 30 vol % HFIP in CH2Cl2 yielded the
crude side-chain protected peptides (14−16). These were not
purified but rather subjected to low temperature thioester-
ification with ethyl-3-mercaptopropionate using PyBOP as the
coupling reagent and iPr2NEt as the base at −30 °C.17,18
Following reversed-phase HPLC purification the target side-
chain protected peptide thioester fragments 4−6 were isolated
in good yields (21−26% over 15 steps, based on the original
resin loading).
With the cyclic depsipeptide 7 and linear peptide thioester
fragments 4−6 in hand, efforts now turned to the assembly of
the natural products via the proposed ligation−oxidation
strategy. A mixed solvent system [1:1 v/v 6 M Gn·HCl, 1.0
M 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
(HEPES)/N-methylpyrrolidone (NMP), pH 7.2]19 was used
to ensure complete solubility of both fragments. To a solution
of cyclic peptide 7 and peptide thioesters 4, 5, or 6 was added
thiophenol as an aryl thiol catalyst. After 16 h at 37 °C the
reactions were judged by HPLC-MS analysis to be complete
and purification by flash column chromatography on normal
phase silica yielded 17−19 in good yields (51−72%). These
compounds were then subjected to oxidation conditions to
convert the thiol functionality of the cysteine side chains (used
Synthesis of the linear peptide thioester fragments 4−6
began with loading of Fmoc-L-Arg(Pbf)-OH onto 2-chlorotrityl
chloride resin (Scheme 3). Fmoc-SPPS of the linear sequence
included incorporation of several nonproteinogenic amino
acids, namely, Fmoc-L-tert-Leu-OH 10, Fmoc-D-tert-Leu-OH
11, Fmoc-L-β-MeIle 1216 (see Supporting Information for the
synthetic details), and Fmoc-L-p-Br-Phe-OH 13. After assembly
B
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Scheme 3. Synthesis of Linear Peptide Thioester Fragments
4−6
Scheme 4. Total Synthesis of Polydiscamides B−D (1−3)
as an auxiliary in the ligation reactions) to the corresponding
cysteic acid residues found in the natural products. Optimal
conditions involved treatment with a 1 vol % aqueous
performic acid solution20 for 1 h at 0 °C which effected clean
oxidation as judged by HPLC-MS (Scheme 4; see Supporting
Information). Subsequent treatment with an acidic cocktail of
TFA/iPr3SiH/H2O (90:5:5 v/v/v) to remove the side-chain
protecting groups, followed by reversed-phase HPLC purifica-
tion, afforded polydiscamide B (1), polydiscamide C (2), and
polydiscamide D (3) in good yields over the three steps (54−
70%). The spectroscopic data obtained for synthetic poly-
discamides B−D (1−3) were in complete accordance with
those reported for the isolated natural products1 (see
Supporting Information for NMR spectral comparsions).
Furthermore, the measured specific rotations were also in
agreement in terms of both magnitude and sign. These data
validate the full stereochemical assignment of polydiscamides
B−D reported by Quinn and co-workers.1,21 Having prepared
polydiscamides B−D (1−3) we were next interested in
investigating the antimicrobial activity. To this end, the
compounds were screened against a panel of 15 clinically
relevant Gram positive and Gram negative bacterial strains
using a high-throughput screening methodology recently
reported by Linington and co-workers (see Supporting
Information).22 Interestingly, despite the structural similarities
with the discodermin family of natural products that possesses
activity against strains of Bacillus subtilis and Pseudomonas
aeruginosa, 1−3 showed no antibacterial activity against any of
the 15 strains up to a concentration of 100 μM. As such, future
work in our laboratory will focus on the application of our
native chemical ligation−oxidation strategy to access these
natural products, and others in the family, to identify the subtle
structural features that lead to antimicrobial activity.
In summary, we have successfully completed the first total
syntheses of the cyclic depsipeptide natural products
polydiscamides B, C, and D (1−3). A key feature of our
synthetic approach involved the late stage, high yielding
assembly of the natural products by native chemical ligation
of a common cyclic depsipeptide fragment to a variety of linear
peptide thioesters, followed by chemoselective oxidation of a D-
cysteine residue to a D-cysteic acid moiety. Using this strategy
the natural products were isolated in 30−50% yield following a
three-step ligation−oxidation−deprotection sequence. Given
the efficient and convergent nature of the synthetic strategy
described herein, it is anticipated that it will find future utility in
the total synthesis of structurally related natural products
C
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including the discodermins,3,4 halicylindramides,7 and micro-
spinosamide.9
(20) Cao, R.; Liu, Y.; Chen, P.; Lv, R.; Song, Q.; Sheng, T.; He, Q.;
Wang, Y.; Wang, X.; Liang, S. Anal. Biochem. 2010, 407, 196.
(21) The spectroscopic data of synthetic polydiscamide B, C, and D
were consistent with those reported for the isolated natural products.
However, no isolated material was available for a direct spectroscopic
comparison.
ASSOCIATED CONTENT
* Supporting Information
■
S
(22) Wong, W. R.; Oliver, A. G.; Linington, R. G. Chem. Biol. 2012,
19, 1483.
Full characterization of polydiscamides B−D and all novel
intermediates. 1H and 13C NMR spectra, analytical HPLC
chromatograms, and high resolution mass spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: richard.payne@sydney.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Ian Luck and Dr. Nick Proschogo from the
University of Sydney for technical support (NMR spectroscopy
and mass spectrometry, respectively). We would also like to
thank Prof. Roger Linington and Dr. Weng Wong from the
University of California, Santa Cruz, USA for carrying out the
antimicrobial screening assays. Finally, we would like to
acknowledge the Henry Bertie and Florence Mabel Gritton
Research Scholarship for PhD support (G.S.) and an ARC
Future Fellowship to R.J.P. (FT130100150).
REFERENCES
■
(1) Feng, Y.; Carroll, A. R.; Pass, D. M.; Archbold, J. K.; Avery, V. M.;
Quinn, R. J. J. Nat. Prod. 2007, 71, 8.
(2) Gulavita, N. K.; Gunasekera, S. P.; Pomponi, S. A.; Robinson, E.
V. J. Org. Chem. 1992, 57, 1767.
(3) Ryu, G.; Matsunaga, S.; Fusetani, N. Tetrahedron 1994, 50,
13409.
(4) Ryu, G.; Matsunaga, S.; Fusetani, N. Tetrahedron Lett. 1994, 35,
8251.
(5) Matsunaga, S.; Fusetani, N.; Konosu, S. Tetrahedron Lett. 1984,
25, 5165.
(6) Matsunaga, S.; Fusetani, N.; Konosu, S. Tetrahedron Lett. 1985,
26, 855.
(7) Li, H.-Y.; Matsunaga, S.; Fusetani, N. J. Med. Chem. 1995, 38,
338.
(8) Li, H.-Y.; Matsunaga, S.; Fusetani, N. J. Nat. Prod. 1996, 59, 163.
(9) Rashid, M. A.; Gustafson, K. R.; Cartner, L. K.; Shigematsu, N.;
Pannell, L. K.; Boyd, M. R. J. Nat. Prod. 2001, 64, 117.
(10) Seo, H.; Lim, D. J. Org. Chem. 2009, 74, 906.
(11) Sato, K.; Horibe, K.; Amano, K.; Mitusi-Saito, M.; Hori, M.;
Matsunaga, S.; Fusetani, N.; Ozaki, H.; Karaki, H. Toxicon 2001, 39,
259.
(12) Dawson, P.; Muir, T.; Clark-Lewis, I.; Kent, S. Science 1994, 266,
776.
(13) Dai, J.; Wang, J.; Zhang, Y.; Lu, Z.; Yang, B.; Li, X.; Cai, Y.;
Qian, X. Anal. Chem. 2005, 77, 7594.
(14) Dai, J.; Zhang, Y.; Wang, J.; Li, X.; Lu, Z.; Cai, Y.; Qian, X. Rapid
Commun. Mass Spectrom. 2005, 19, 1130.
(15) Wen, S.; Packham, G.; Ganesan, A. J. Org. Chem. 2008, 73, 9353.
(16) Pigza, J. A.; Molinski, T. F. Org. Lett. 2010, 12, 1256.
(17) Kajihara, Y.; Yoshihara, A.; Hirano, K.; Yamamoto, N.
Carbohydr. Res. 2006, 341, 1333.
(18) Wilkinson, B. L.; Stone, R. S.; Capicciotti, C. J.; Thaysen-
Andersen, M.; Matthews, J. M.; Packer, N. H.; Ben, R. N.; Payne, R. J.
Angew. Chem., Int. Ed. 2012, 51, 3606.
(19) Payne, R. J.; Ficht, S.; Greenberg, W. A.; Wong, C.-H. Angew.
Chem., Int. Ed. 2008, 47, 4411.
D
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