Chemical mediators: The remarkable structure and host-selectivity of depsilairdin, a sesquiterpenic depsipeptide containing a new amino acid

Article abstract of DOI:10.1021/ol0478786

(Chemical equation presented) The chemical structure determination of depsilairdin, a highly selective phytotoxin produced by the plant pathogenic fungus Leptosphaeria maculans/Phoma lingam, is described. The elucidation of the unusual chemical structure

Full text of DOI:10.1021/ol0478786

ORGANIC
LETTERS
2004
Vol. 6, No. 24
4615-4617
Chemical Mediators: The Remarkable
Structure and Host-Selectivity of
Depsilairdin, a Sesquiterpenic
Depsipeptide Containing a New Amino
Acid
M. Soledade C. Pedras,* Paulos B. Chumala, and J. Wilson Quail
Department of Chemistry, UniVersity of Saskatchewan, 110 Science Place,
Saskatoon SK, S7N 5C9, Canada
s.pedras@usask.ca
Received October 13, 2004
ABSTRACT
The chemical structure determination of depsilairdin, a highly selective phytotoxin produced by the plant pathogenic fungus Leptosphaeria
maculans/Phoma lingam, is described. The elucidation of the unusual chemical structure used a combination of NMR spectral data and X-ray
crystallography. The absolute configuration was established using chemical degradation and synthesis of (3S,6R)-3,6-diisopropyl-2,5-
morpholinedione and its (3R,6S) and (3R,6R) stereoisomers. Similar to the fungal pathogen, depsilairdin caused strong lesions only on brown
mustard leaves but not on related species.
The chemical mediators of molecular interactions between
pathogenic fungi and their host plants are of great signifi-
cance to the understanding and control of plant fungal
diseases.1 Yet, the chemical structures and biological effects
of potential mediators of many fungal diseases remain to be
discovered. The task is indeed daunting, as fungi appear to
possess a complex chemical arsenal that enables them to
elude the plants’ counterattack weaponry.
range of the fungus includes some of the most economically
important cruciferous oilseed crops (e.g., canola, Brassica
napus). Our studies of blackleg fungal isolates4 revealed that
this plant pathogen produces various phytotoxic metabolites,
including phomalirazine (1),5 sirodesmins (2),6 phomalide
(3),7 and phomalairdenone (4).8 Importantly, 3 was toxic only
to plants that host fungal isolates producing it, e.g., canola
(3) Howlett, B. J.; Idnurm, A.; Pedras, M. S. C. Fungal Genet. Biol.
2001, 33, 1-14.
In this context, the “blackleg” fungus [Leptosphaeria
maculans (Desm.) Ces. et de Not., asexual stage Phoma
lingam (Tode ex Fr.) Desm] has been a major player in
defeating plants designed to resist its invasion.2,3 The host-
(4) For a recent review, see: Pedras, M. S. C. Rec. Res. DeV. Phytochem.
2001, 5, 109-117.
(5) Pedras, M. S. C.; Abrams, S. R.; Se´guin-Swartz, G.; Quail, J. W.;
Jia, Z. J. Am. Chem. Soc. 1989, 111, 1904-1905.
(6) Pedras, M. S. C.; Se´guin-Swartz, G.; Abrams, S. R. Phytochemistry
1990, 29, 777-782.
(7) Pedras, M. S. C.; Taylor, J. L.; Nakashima, T. T. J. Org. Chem. 1993,
58, 4778-4780.
(8) Pedras, M. S. C.; Erosa-Lo´pez, C. C.; Quail, J. W.; Taylor, J. L.
Bioorg. Med. Chem. Lett. 1999, 9, 3291-3294.
* Telephone: 1-306-966-4772. Fax: 1-306-966-4730.
(1) For reviews on plant-pathogen interactions, see for example: (a)
Jackson, A. O.; Taylor, C. B. Plant Cell 1996, 8, 1651-1668. (b) Knogge,
W. Plant Cell 1996, 8, 1711-1722.
(2) Gugel, R. K.; Petrie, G. A. Can. J. Plant Pathol. 1992, 14, 36-45.
10.1021/ol0478786 CCC: $27.50
© 2004 American Chemical Society
Published on Web 10/29/2004

type.7,9 By contrast, a new group of blackleg fungal isolates
able to produce 4, caused disease only on brown mustard
but not on canola.10 This finding was rather disturbing
because brown mustard was known to be resistant to blackleg
disease. The leaf lesions observed on blackleg-infected
mustard suggested that colonization by these “new” blackleg
isolates (Laird 2 and Mayfair 2) would be mediated by a
rather potent metabolite. However, none of the metabolites
isolated from these new isolates, 4-6,8 appeared to be
involved in causing the disease symptoms observed on brown
mustard.
Figure 1. Substructures A and B, and selected HMBC correlations
observed for depsilairdin (7). A: H-8 (δH 5.29) and H3-12 (δH 3.36)
to C-13 (δC 171.1), and H-14 (δH 5.34) and H3-18 (δH 2.74) to
C-19 (δC 175.4). B: H-2 (δH 4.47) to C-3 (δC 76.6) and C-5 (δC
52.8).
five of the eight degrees of unsaturation of the molecule;
the remaining three unsaturations unaccounted for by the
NMR data suggested the presence of three rings.
The 1H-1H COSY spectrum displayed several spin
systems, as shown in Figures 1 and 2. Three isopropyl spin
A comprehensive metabolite search for new phytotoxic
compounds produced by isolates Mayfair 2 and Laird 2 led
to the discovery of a highly selective phytotoxic metabolite
that we named depsilairdin (7). Here we describe the
structure elucidation and the chemistry involved in the
determination of the absolute configuration of this chemical
mediator. In addition to the unique structural combination
tripeptide-sesquiterpene, 7 contains a (2S,3S,4S)-3,4-dihy-
droxy-3-methylprolyl residue, which represents an amino
acid hitherto undescribed. The remarkable selectivity of 7
to brown mustard was observed over a broad concentration
range from µM to mM.
Figure 2. Substructures C, D, and E, and selected HMBC
correlations observed for depsilairdin (7). E: H3-14′ (δH 1.00) to
C-3′ (δC 40.5), C-4′ (δC 71.3), and C-5′ (δC 53.1); H3-15′ (δH 1.09)
to C-1′ (δC 83.3), C-5′ (δC 53.1), C-9′ (δC 40.3), and C-10′ (δC
38.9).
systems coupled to three methine protons at δH 5.34 (H-14,
d, J ) 11 Hz), 5.29 (H-8, d, J ) 11 Hz), and 4.22 (H-20, br
d, J ) 7 Hz) could be connected using HMBC correlations
summarized for structural subunit A, Figure 1. Another spin
system containing a proton at δH 3.68 (H-4), coupled to two
methylene protons at δH 4.48 and 3.89 (m, H2-5), showed
HMBC correlations summarized for subunit B, Figure 1. Two
additional spin systems C and D, Figure 2, were observed
through H-H coupling. Selected HMBC correlations are
summarized in Figure 2. The connectivity of subunits B
(Figure 1) and E (Figure 2) was established on the basis of
an HMBC correlation between proton H-1′ (δ 4.89) of E
and carbonyl carbon C-1 (δC 171.1) of B. Because subunit
A did not show a correlation with any other unit, there was
only one possibility to connect units A and B and assign the
structure of depsilairdin as 7. Depsilairdin was crystallized
to yield crystals, monoclinic system, P21 space group.11 The
data obtained from X-ray crystallographic analysis of a
single-crystal allowed the assignment of the relative but not
the absolute stereochemistry of 7. To establish the absolute
stereochemistry of depsilairdin (7), a mild degradation
The molecular formula of 7, C38H65N3O9 based on MS
(HREI-MS) and NMR (1H and 13C NMR and HMQC) data,
indicated eight degrees of unsaturation. The 1H NMR
spectrum displayed signals for six methyl singlets and six
methyl doublets; two of the methyl singlets (δH 3.36, H3-
12, and 2.74, H3-18) could be attributed to N-methyl groups.
The 13C NMR spectrum displayed four carbon signals at δC
171.7, 175.4, 170.1, and 171.1, indicating the presence of
amide or ester carbonyl groups, and two signals at δC 108.4
and 150.1 attributable to olefinic carbons. These four
carbonyl groups and the C-C double bond accounted for
(9) Plants’ defenses were found to inhibit the biosynthesis of sirodesmins.
Pedras, M. S. C.; Taylor, J. L. J. Nat. Prod. 1993, 56, 731-738.
(10) Isolates producing sirodesmins (2) are classified as group A, but
the new isolates Laird 2 and Mayfair 2 remain unclassified, cf. ref 3.
(11) Compound 7 was crystallized from hexane-acetone (7:3). An X-ray
crystallographic data file (CIF) can be found in Supporting Information.
4616
Org. Lett., Vol. 6, No. 24, 2004

configuration of all stereogenic centers of 7 could be
established as 2S, 3S, 4R, 8S, 14S, 20R, 1′S, 4′S, 5′S, 7′S,
and 10′S.
Scheme 1. Syntheses of
3,6-Diisopropyl-4-methyl-2,5-morpholinediones 8, 14, and 17
Depsilairdin (7) caused strong necrotic and chlorotic
lesions18 only on brown mustard leaves,19 whereas no damage
was observed on canola or white mustard leaves. The high
selectivity of 7 appears to mimic the pathogenicity range of
the producing isolates, and the lesions resemble the disease
symptoms, which is consistent with a chemically mediated
interplay between host and pathogen.20 It is remarkable that
7 was selective over a wide range of concentrations (µM-
mM), whereas most phytotoxins are selective only at much
narrower ranges (µM).19 It is expected that a highly selective
mediator such as 7 can be an excellent probe to detect, e.g.,
genes and/or receptors targeted by the pathogen to defeat
the plant.
Acknowledgment. We thank Prof. D. E. Ward, Chem-
istry, University of Saskatchewan, for a generous gift of 9.
Financial support from the Natural Sciences and Engineering
Research Council of Canada (Discovery Grant to M.S.C.P)
and the University of Saskatchewan (graduate teaching
assistantship to P.B.C.) is gratefully acknowledged.
procedure12 yielded product X in an amount sufficient for
spectroscopic analysis. The molecular formula (C11H19O3N,
HREI-MS) and the NMR data of X suggested trans-3,6-
diisopropyl-4-methyl-2,5-morpholinedione.
Supporting Information Available: General experimen-
tal, experimental procedures including the synthesis of 8,
X-ray crystallographic data (CIF) for depsilairdin (7),
characterization data for compounds 7 (copies of 1H and 13
C
Next, to determine the absolute configuration of trans-
morpholinedione X (3S,6R or 3R,6S), three stereoisomers
were synthesized as summarized in Scheme 1. Analysis of
the 1H NMR spectra of 8, 14, and 17 confirmed that isomers
8 and 17 displayed proton signals identical to the product
NMR, COSY, HMQC, HMBC spectra) 8, 14, and 17. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0478786
1
X, whereas 14 was clearly different. Subsequently, the H
NMR spectra of 8, 17, and X were obtained separately in
the presence of an NMR chiral solvating agent ((R)-(-)-
TFAE).13 The 1H NMR spectrum of X was identical to that
(14) Koch, C.; Simonyiova´, S.; Pabel, J.; Ka¨rtner, A.; Polborn, K.;
Wanner, K. T. Eur. J. Org. Chem. 2003, 1244-1263.
(15) Li., W.; Ewing, W. R.; Harris, B. D.; Joullie´, M. M. J. Am. Chem.
Soc. 1990, 112, 7659-7672.
1
of 8. Furthermore, the H NMR spectrum of X spiked with
(16) Campbell, A. D.; Raynham, T. M.; Taylor, J. K. Synthesis 1998,
1707-1709.
(17) Cheung, S. T.; Benoiton, N. L. Can. J. Chem. 1977, 55, 906-910.
(18) Bioassays described in Supporting Information, as previously
reported for other toxins: Pedras, M. S. C.; Biesenthal, C. J.; Zaharia, I. L.
Plant Sci. 2000, 156, 185-192.
(19) Depsilairdin (7) caused lesions on brown mustard (blackleg
susceptible) ranging from 12 mm (10-3 M) to 6 mm (5 × 10-6 M) in
diameter, whereas canola or white mustard (blackleg resistant) were not
affected, even at 10-3 M. For a multiauthor review on phytotoxins, see:
Graniti, A. Experientia 1991, 47, 751-755.
(20) We suspect that blackleg isolates similar to Mayfair 2 and Laird 2
are not common because their favorite host is not widely cultivated.
Nonetheless, because new oilseed varieties of brown mustard are com-
mercially available, the new blackleg type may soon become widespread.
17 was different, showing two signals for H-6, at δH 4.55
and 4.52. These results confirmed that X was identical to 8.
Since X was a structural fragment of depsilairdin (7), and
the relative configurations of 7 were established by X-ray
crystallography, the overall assignment of the absolute
(12) Depsilairdin (7) was allowed to stand in a solution of DCl in CD3-
OD. After completion of the reaction (15 days), the reaction mixture was
concentrated to dryness and the residue separated by chromatography.
(13) 1H NMR (0.12 M of (R)-(-)-TFAE in CDCl3) for H-6 of (3S,6R)-
enantiomer was at δH 4.55, whereas that of the (3R,6S)-enantiomer was at
δH 4.52.
Org. Lett., Vol. 6, No. 24, 2004
4617