A Novel Cyclodepsipeptide, HA23, from a Fusarium sp

Article abstract of DOI:10.1021/ol0260167

(Matrix Presented) HA 23, a novel cyclodepsipeptide (1) of mixed peptide-polyketide origins, was isolated from a fungal isolate of a Fusarium sp. The structure was determined from 1D and 2D NMR and mass spectral data.

Full text of DOI:10.1021/ol0260167

ORGANIC
LETTERS
2002
Vol. 4, No. 12
2095-2096
A Novel Cyclodepsipeptide, HA23, from
a Fusarium sp.
,†
Yunjiang Feng,† John W. Blunt,† Anthony L. J. Cole,‡ and Murray H. G. Munro*
Departments of Chemistry and Plant and Microbial Sciences, UniVersity of
Canterbury, PriVate Bag 4800, Christchurch, New Zealand
m.munro@chem.canterbury.ac.nz
Received April 15, 2002
ABSTRACT
HA 23, a novel cyclodepsipeptide (1) of mixed peptide−polyketide origins, was isolated from a fungal isolate of a Fusarium sp. The structure
was determined from 1D and 2D NMR and mass spectral data.
During an investigation of the biologically active metabolites
produced by fungi, the extract from a Fusarium sp. showed
strong antifungal activity. Bioassay-guided fractionation and
purification of the culture extract led to the isolation of a
novel cyclodepsipeptide HA 23 (1) and the known antifungal
secondary metabolite W 493-B (2).1 We report here the
isolation and structural elucidation of (1).2
† Department of Chemistry.
‡ Department of Plant and Microbial Sciences.
(1) Nihei, K.; Itoh, H.; Hashimoto, K.; Miyairi, K.; Okuno, T. Biosci.
Biotechnol. Biochem. 1998, 62(5), 858-863.
(2) Compound 1 was obtained as a colorless oil: [R]20D -46° (c 0.001,
MeOH); UV (MeOH) λmax (log ꢀ) 232 (3.23), 278 (2.80); IR (chloroform)
γmax 3692, 3638, 3414, 2943, 2839, 2367, 2336, 1730, 1676, 1634, 1603,
Fusarium sp. (CANU-HA 23) was fermented in half
1510, 1464, 1335, 1267, 1182, 1126 cm-1; HRESIMS 601.3858 (M + H+)
(calcd for C34H53N2O7, 601.3853); 1H NMR (CDCl3, 500 MHz) δ 7.12
(1H, d, 9.0, H16), 6.83 (1H, d, 9.0, H17), 6.56 (1H, brs, NH), 5.47 (1H, t,
5.0, H20), 5.40 (1H, d, 5.0, H7), 5.32 (1H, m, H13), 4.77 (1H, dt, 10.0,
3.0, H5), 4.47 (2H, d, 6.5, H19), 4.14 (1H, m, H3), 3.98 (1H, brd, 14.0,
H11a), 3.77 (1H, m, H30), 3.16 (1H, dd, 13.0, 9.5, H14a), 2.88 (1H, dd,
13.0, 5.5, H14b), 2.69 (1H, t, 12.0, H11b), 2.56 (1H, m, H2), 2.10 (1H,
brd, 14.0, H8a), 1.96 (1H, m, H25), 1.78 (3H, brs, H22), 1.73 (3H, brs,
H23), 1.69 (2H, m, H4), 1.63 (1H, m, H10b), 1.57 (1H, m, H26a), 1.55
(1H, m, H9a), 1.54 (1H, m, H10a), 1.47 (1H, t, 7.5, H29a), 1.36 (2H, m,
H28), 1.33 (1H, m, H8b), 1.32 (1H, m, H29b), 1.30 (2H, m, H27), 1.18
(3H, d, 6.0, H31), 1.08 (1H, m, H26b), 1.07 (3H, d, 7.0, H24), 0.86 (1H,
m, H9b), 0.80 (3H, d, 6.0, H32); 13C APT NMR (CDCl3, 125 MHz) δ
173.7 (C1), 171.5 (C12), 169.7 (C6), 157.7 (C18), 138.1 (C21), 129.9 (C16),
128.7 (C15), 119.6 (C20), 114.8 (C17), 77.0 (C5), 68.9 (C3), 68.1 (C30),
strength Sabouraud dextrose yeast broth under static condi-
tions at 26 °C for 28 days. The ethyl acetate extract (273
mg) from both the mycelium and culture filtrate (1 L) was
chromatographed on a flash reverse phase (rp) column using
64.8 (C19), 51.1 (C7), 48.8 (C13), 47.8 (C2), 45.1 (C11), 38.8 (C29), 36.0
(C14), 34.4 (C25), 33.1 (C4), 31.8 (C26), 26.3 (C27), 25.8 (C22), 25.7
(C10), 25.4 (C28), 24.6 (C8), 23.6 (C31), 19.9 (C9), 18.2 (C23), 15.8 (C32),
7.3 (C24). NOE correlations: H2/NH, H2/H3, H2/H24, H3/NH, H3/H4,
H3/H25, H4/H5, H4/H24, H5/H26b, H5/H32, H7/H8a, H7/H8b, H8a/H8b,
H9a/H9b, H11a/H11b, H11a/H13, H13/H14a, H13/H14b, H13/H16, H14a/
H14b, H14a/H16, H14b/H16, H17/H19, H19/H20, H19/H23, H20/H22,
H25/H32, H28/H30, H30/H31.
10.1021/ol0260167 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/21/2002

a sharp, stepped gradient from water through methanol to
dichloromethane. The fraction that eluted off the rp column
with methanol/water (9:1) was partitioned between petroleum
ether and methanol. The methanol fraction was repeatedly
chromatographed on diol eluting with a petroleum ether/ethyl
acetate gradient (4:1 to 3:2 to 1:1) to yield 1 (1.8 mg) and
2 (2.2 mg).
The cyclodepsipeptide (1) was obtained as a colorless oil
with [R]D ) -46°. The UV spectrum of 1 displayed
absorption maxima at 232 and 278 nm, suggesting the
presence of an oxygen-substituted aromatic system. IR peaks
at 3692, 3414, 1730, 1676, and 1634 cm-1 indicated the
presence of alcohol, amine, and amide (or ester) function-
alities. The ES positive ion mass spectrum of 1 showed
strong M + H+ and M + Na+ peaks at m/z 601 and 623.
High-resolution mass measurement on the M + H+ (m/z
601.3858) in the ESI mass spectrum, in combination with
1H and 13C NMR data2 supported the molecular formula
C34H52N2O7 (10 double bond equivalents).
The 1H NMR spectrum of 12 contained signals assignable
to a 1,4-disubstituted phenyl ring system, an exchangeable
proton at 6.56 ppm, six methines (five were heteroatom-
substituted), two heteroatom-substituted methylenes, and five
methyl groups. The APT NMR experiment2 exhibited 32
carbon signals, comprising 5 CH3, 11 CH2, 10 CH, and 6
quaternary carbon signals. In addition to the four aromatic
carbon signals for a 1,4-disubstituted phenyl group, two other
sp2 carbon signals accounted for a further double bond. Three
quaternary carbon signals at 169.7, 171.5, and 173.7 ppm
were assigned as amide (or ester) carbonyl groups.
f through two methylene groups was also established by 1D
and 2D TOCSY experiments.
A detailed analysis of the CIGAR experimental data
allowed the assembly of the subunits into an O-prenyl-
substituted tyrosine residue (I), a pipecolinic acid residue
(II), and the polyketide residue (III) (Figure 1).
The residues I-III accounted for all but one degree of
unsaturation required by the molecular formula, suggesting
that 1 is a cyclic depsipeptide. The assembly of I-III was
determined by a second CIGAR experiment using a different
solvent, pyridine-d5. In this experiment an additional cor-
relation from the amide NH in I to the carbonyl group in
III was observed (Figure 1), allowing the assignment of HA
23 as 1.
The absolute stereochemistry of the amino acid residues
in the molecule was determined. Acid hydrolysis of 1 with
6 N HCl at 110° C provided tyrosine and pipecolinic acid.
HPLC analysis of the FDAA-derivatized amino acids
confirmed that each amino acid had an S-configuration.
NOESY experiments on 1 were illuminating but not con-
clusive. Although a range of NOE correlations were ob-
served,2 uncertainty on the conformations of the 12-
membered depsipeptide reduced the reliability of the
possibilities of any stereochemical conclusions to speculation
only. Because of the limited amount of sample available,
no other chemical reactions were attempted to determine the
configurations of the remaining stereogenic centers in the
polyketide portion.
The second metabolite, W 493-B (2), had been previously
reported by a Japanese group in 1998. The NMR spectral
data were identical to those reported in the literature.1
Compound 1 is a unique depsipeptide containing a 14-
carbon polyketide unit, a substituted tyrosine, and pipecolinic
acid. This metabolite of mixed peptide-polyketide origins
possesses a structurally novel polyketide moiety and is
composed of just three residues, whereas depsipeptides (and
cyclic peptides) typically incorporate either four or more than
five residues.4
A series of COSY, HSQC, and CIGAR3 experiments
established several sequences of partial connectivities and
defined the subunits b-f (Figure 1 in bold). The assignments
Biological studies on natural products with similar struc-
tural features indicated that they possess a variety of
biological activities.5 In this present study, W 493-B showed
antifungal activity against Cladosporium resinae, whereas
HA 23 was not active in either antimicrobial or cytotoxicity
bioassays.
Figure 1. CIGAR correlations in subunits I, II, and III: (solid
line) in chloroform-d; (dash line) in pyridine-d5.
Acknowledgment. Financial support from the University
of Canterbury for a Postdoctoral fellowship (Y.F.) is
acknowledged and from Instituto BioMar SA for technical
support. We thank Mr. Bruce Clark for mass spectrometric
analyses, Ms. Gill Ellis for bioactivity assays, and Mr. Nick
Cummings for fungal isolation and fermentation.
of two hydroxyls at C3 and C30 and one ester (or lactone)
1
substituent at C5 in subunits e and f were based on the H
and 13C chemical shifts at the three positions2 and supported
by the molecular formula. Subunit a was established by the
COSY correlations from two broad methyl singlets to the
methine group, and the CIGAR correlations from the protons
at the two methyl groups to the two sp2 carbons (Figure 1).
A series of 1D TOCSY experiments confirmed the spin
systems in subunits a-d. The connectivity of subunits e and
Supporting Information Available: Detailed description
1
of experimental procedures and H and 13C APT NMR
spectra for 1. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL0260167
(4) Kleinkauf, H.; von Dohren, H. Eur. J. Biochem. 1990, 192, 1-15.
(5) Hegde, V. R.; Puar, M. S.; Chan, T. M.; Dai, P.; Das, P. R.; Patel,
M. J. Org. Chem. 1998, 63, 9584-9586.
(3) Hadden, C. E.; Martin, G. E.; Krishnamurthy, V. V. Magn. Reson.
Chem. 2000, 38, 143-147.
2096
Org. Lett., Vol. 4, No. 12, 2002