Synthesis and biological activity of a stereoisomeric mixture of the mating hormone of Phytophthora

Article abstract of DOI:10.1016/j.tetlet.2007.04.109

The first synthesis of a stereoisomeric mixture of hormone α1, the mating hormone of Phytophthora, was achieved, and the synthetic mixture was confirmed to be hormonally active.

Full text of DOI:10.1016/j.tetlet.2007.04.109

Tetrahedron Letters 48 (2007) 4601–4603
Synthesis and biological activity of a stereoisomeric mixture
of the mating hormone of Phytophthora
Arata Yajima,a,* Naoki Kawanishi,a Jianhua Qi,b Tomoyo Asano,b Youji Sakagami,b
Tomoo Nukadaa and Goro Yabutaa
aDepartment of Fermentation Science, Faculty of Applied Biological Science, Tokyo University of Agriculture (NODAI),
Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502, Japan
bGraduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Received 23 March 2007; revised 20 April 2007; accepted 24 April 2007
Available online 27 April 2007
Abstract—The first synthesis of a stereoisomeric mixture of hormone a1, the mating hormone of Phytophthora, was achieved, and
the synthetic mixture was confirmed to be hormonally active.
Ó 2007 Elsevier Ltd. All rights reserved.
Phytophthora, whose name translates as ‘plant-destroyer’,
is one of the most destructive pathogens in the world. In
the mid-1840s, late blight, the plant disease caused by a
member of this fungus-like genus, destroyed potato
crops in Europe and the United States and caused the
Irish potato famine.1 This pathogen, which devastates
potato and tomato crops, is known as Phytophthora
infestans. Another species, Phytophthora ramorum, is
an aggressive pathogen which is the cause of extensive
mortality among oak trees in California;2 the disease is
known as sudden oak death. In Europe, this disease
has not been detected in oaks, but has been found to
cause twig blight in other plants.3 In the 20th century,
these diseases were managed by effective use of fungi-
cides. However, certain virulent and fungicide-resistant
strains have recently undergone extensive migration,
causing a worldwide resurgence of late blight in
potatoes.1
tion of both. After sexual reproduction, the oogonia
develop into sexual spores called oospores, which can
survive harsh conditions such as drying or freezing for
months or years in the absence of a living host plant.
However, during its asexual phase, Phytophthora is an
obligate parasite and requires the presence of a living
host tissue. In 1929, Ashby proposed that sexual repro-
duction in Phytophthora was regulated by a hormone-
like compound,4 and data were later reported by Gallo-
way and Kouyears supporting a mechanism of chemical
stimulation for oospore formation.5 A factor secreted by
the A1 mating type induces the formation of oospores in
the A2 mating type, while a factor secreted by A2
induces the formation of oospores in A1. These factors
are known as hormones a1 and a2, respectively.
Although extensive studies have been conducted with
the aim of isolating these hormones, their structures
are still obscure due to their scarcity.6 Recently, Ojika
and co-workers finally succeeded in isolating hormone
a1 from 1830 L of culture broth of the A1 mating type
of another species, Phytophthora nicotianae.7 The struc-
ture of hormone a1 was determined as 1 (Fig. 1), a novel
diterpene with four stereogenic centers, by extensive MS
The life cycle of Phytophthora species, which are some-
times called water molds as their development is favored
by wet conditions, includes characteristic biological
events, including sexual reproduction.1 Each individual
is bisexual, capable of producing both female (oogonia)
and male (antheridia). There are two mating types, A1
and A2, with sexual reproduction requiring the interac-
Keywords: Phytophthora, Mating hormone; Hormone a1; Synthesis.
*
Figure 1. Structures of hormone a1 (1) and its bis-p-bromobenzoate
derivative 2.
Corresponding author. Tel.: +81 3 5477 2264; fax: +81 3 5477
2622; e-mail: ayaji@nodai.ac.jp
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.04.109

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A. Yajima et al. / Tetrahedron Letters 48 (2007) 4601–4603
and NMR studies of the natural product and its bis-p-
bromobenzoate derivative. No information about the
relative or absolute configuration of natural 1 was
reported. Surprisingly, hormone a1 was found to
induce oospore formation not only in P. nicotianae
but also in P. capsici, P. cambivora, and P. infestans.
These results indicate that hormone a1 (1) is a universal
mating hormone in the heterothallic species of Phytoph-
thora. We became interested in synthesizing hormone a1
(1) in order to confirm its structure and to investigate
its biological activity. This Letter describes the synthe-
sis and biological activity of a stereoisomeric mixture
of 1.
of 3 yielded a tosylate which gave the corresponding
iodide on treatment with NaI (73% in two steps),9 and
alkylation of ethyl acetoacetate with the resulting iodide
furnished a b-ketoester derivative which was saponified
to give ketone 4 (67% in two steps). After protection
of the carbonyl group (95%), the benzyl ether was re-
moved by hydrogenolysis (98%), and the resulting alco-
hol was converted to iodide 5 by tosylation followed by
treatment with NaI (77% in two steps). Halogen–metal
exchange of 5 with t-BuLi followed by coupling with
known aldehyde 610 (=E) afforded 7 in 90% yield. Acid
hydrolysis of the ketal of 7 gave ketone 8 (=B). The pre-
cursor (10) of fragment A was derived from known alco-
hol 911 (=C) in four steps: protection of the hydroxyl
group as a benzyl ether (77%), hydroboration–oxidation
(54%), tosylation (84%) and treatment with NaI (99%).
The iodide 10 was lithiated with t-BuLi, and the result-
ing organolithium was coupled with ketone 8 to afford
11 in 78% yield. Deprotection of the two benzyl groups
by hydrogenolysis gave tetraol 12 (91%). As the p-bro-
mobenzoyl derivative (2) of the natural hormone a1
(1) has already been prepared by Ojika et al. and its
physical properties reported,7 a p-bromobenzoyl group
was selected for protection of the two primary hydroxyl
groups (60%). Oxidation of the remaining secondary
hydroxyl group with Dess–Martin periodinane gave 212
(83%), and hydrolysis of the p-bromobenzoyl groups
gave a stereoisomeric mixture of 1 in 83% yield.13 The
overall yield of 1, based on 3, was 9.2% after 15 steps.
The 1H and 13C NMR spectra of synthetic 1 and 2 were
in good agreement with the previously reported data.7
The structure of the natural hormone a1 (1) was con-
firmed by these results. Unfortunately, both of the syn-
thetic samples gave simple spectra on 1H and 13C NMR
analyses, as though they contained only a single diaste-
reomer. Therefore, no information about the relative
stereochemistry of 1 was obtained; in other words, it
was impossible to discriminate between the diastereo-
mers of 1 and 2 by NMR analysis.
We selected a stereoisomeric mixture of 1 as a target for
synthesis. Since diastereomers often give different NMR
spectra, it was thought likely that information about the
relative configuration of 1 could be obtained by NMR
study of the stereoisomeric mixture. Scheme 1 shows a
retrosynthetic analysis of 1. Hormone a1 (1) would be
synthesized from fragments A and B via nucleophilic
addition. Fragment B would in turn be derived from
nucleophilic addition of fragment D to the known alde-
hyde E. The known and easily prepared compounds C,
E, and F were selected as starting materials. Scheme 2
summarizes our synthesis of 1. First, fragment D was
prepared from the known alcohol 38 (=F). Tosylation
The oospore-inducing activity of a sample of synthetic 1
was evaluated according to the previously reported pro-
cedure.7 Figure 2 shows the results of oospore induction,
Scheme 1. Retrosynthetic analysis of hormone a1 (1).
Scheme 2. Synthesis of hormone a1 (1). Reagents, conditions and yields: (a) (i) p-TsCl, py, (ii) NaI, acetone (73%); (b) ethyl acetoacetate, K2CO3,
acetone, reflux (74%); (c) KOH aq, EtOH reflux, then HCl aq (90%); (d) HO(CH2)2OH, PTSA, benzene, reflux (95%); (e) H2, Pd/C, MeOH (98%); (f)
p-TsCl, py (77%); (g) NaI, NaHCO3, acetone (quant.); (h) t-BuLi, ether, À78°C, then 6 (90% based on 6, 65% based on 5); (i) PTSA, EtOH, H2O
(quant.); (j) NaH, BnBr, THF (77%); (k) NaBH4, BF3OEt2, THF, then H2O2 aq (54%); (l) p-TsCl, py (84%); (m) NaI, acetone (99%); (n) t-BuLi,
ether, À78°C, then 8 (78% based on 8); (o) H2, Pd/C, MeOH (91%); (p) p-Br-C6H4COCl, py (60%); (q) Dess–Martin periodinane, CH2Cl2 (83%); (r)
K2CO3, MeOH (83%).

A. Yajima et al. / Tetrahedron Letters 48 (2007) 4601–4603
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5. (a) Galloway, L. D. Sci. Rep. Agr. Res. Inst., Pusa 1936,
1934–1935, 120–130; (b) Kouyeas, V. Ann. Inst. Phyto-
pathol. Benaki 1953, 7, 40–53.
6. (a) Stamps, D. J. Trans. Br. Mycol. Soc. 1953, 36, 255–259;
(b) Apple, J. L. Phytopathology 1959, 49, 37–43; (c)
Haasis, F. A.; Nelson, R. R. Plant Dis. Rep. 1963, 48, 705–
709; (d) Marx, D. H.; Haasis, F. A.; Nelson, R. R. J.
Elisha Mitchell Sci. Soc. 1965, 81, 75–76; (e) Brasier, C. M.
Trans. Br. Mycol. Soc. 1972, 58, 237–251; (f) Chang, S. T.;
Shepherd, C. J.; Pratt, B. H. Aust. J. Bot. 1974, 22, 669–
679; (g) Ko, W. H. J. Gen. Microbiol. 1978, 107, 15–18; (h)
Ko, W. H. J. Gen. Microbiol. 1983, 129, 1397–1401; (i)
Chern, L. L.; Tang, C. S.; Ko, W. H. Bot. Bull. Acad. Sin.
1999, 40, 79–85.
Figure 2. The dose-dependent increase of oospore formation in the A2
mating type of P. nicotianae induced by synthetic sample (ng/disc) in
comparison with natural hormone a1 (a1) at doses of 10 and 30 ng/
disc. Values are means of four replicates. C: control.
7. Qi, J.; Asano, T.; Jinno, M.; Matsui, K.; Atsumi, K.;
Sakagami, Y.; Ojika, M. Science 2005, 309, 1828.
8. Jung, S. H.; Yeo, M. S.; Kim, S. O.; Cheong, C. S. Bull.
Korean Chem. Soc. 1997, 18, 344–347.
9. All new compounds gave satisfactory spectral and ele-
mental analysis data (combustion and/or HRMS).
10. Van Middlesworth, F.; Wang, Y. F.; Zhou, B.; DiTullio,
D.; Sih, C. J. Tetrahedron Lett. 1985, 26, 961–964.
with doses of 50–450 ng/disk, for the A2 mating type of
P. nicotianae. The values for the natural product were
recorded at 10 and 30 ng/disk. The synthetic stereoiso-
meric mixture of 1 did exhibit oospore-inducing activity,
but its activity was estimated to be about five times
weaker than that of the natural product. This result indi-
cates that some stereoisomers of 1 show hormonal activ-
ity, but the other isomers do not inhibit activity.
´
11. Cherest, M.; Felkin, H.; Frajerman, C.; Lion, C.; Roussi,
G.; Swierczewski, G. Tetrahedron Lett. 1966, 875–879.
12. Properties of synthetic 2: colorless oil; IR (KBr) mmax
(cmÀ1) = 3545 (br s, –OH), 2940 (br s), 2355 (w), 1715 (s,
C@O), 1590 (s), 1485 (m), 1465 (m), 1395 (s), 1270 (s),
1175 (m), 1115 (s), 1070 (m), 1010 (m), 965 (w), 850 (m),
755 (s), 710 (w), 685 (m), 630 (w), 475 (w); 1H NMR
(400 MHz, CDCl3): d = 0.84 (m, 3H, 19-CH3), 1.03 (d,
J = 6.8 Hz, 3H, 17-CH3), 1.10–1.60 (m, 15H), 1.16 (s, 3H,
18-CH3), 1.17 (d, J = 6.8 Hz, 3H, 20-CH3), 1.77 (m, 1H, 2-
CHH), 1.94 (m, 1H, 15-CH), 2.20 (m, 1H, 2-CHH), 2.36–
2.57 (m, 2H, 5-CH2), 2.73 (sxt, J = 6.8 Hz, 1H, 3-H), 4.11
(dd, J = 6.0, 10.7 Hz, 1H, 16-CHH), 4.21 (dd, J = 6.8,
10.7 Hz, 1H, 16-CHH), 4.31 (br t, J = 6.3 Hz, 2H, 1-CH2),
7.57–7.92 (m, 8H, Ar-H); 13C NMR (100 MHz, CDCl3):
d = 16.9, 19.18, 19.21, 21.1, 26.7, 30.4, 31.3, 32.2, 32.3,
32.6, 33.9, 37.2, 37.3, 39.0, 41.9, 42.0, 43.01, 43.04, 63.2,
69.9, 72.5, 127.8, 128.0, 128.9, 129.2, 131.0, 131.56, 131.61,
165.6, 165.8, 213.7; HRMS (FAB): calcd. for
C34H4679Br2O6Na 731.1559, found 731.1556 [M+Na]+.
1H and 13C NMR spectra are in good accordance with
those of the reported natural product.
In summary, we achieved the first synthesis of a stereo-
isomeric mixture of hormone a1 (1). The synthetic mix-
ture was found to induce the formation of oospores.
Since the activity of synthetic 1 was about five times
weaker than that of the natural product, it was con-
cluded that some stereoisomers of 1 show hormonal
activity. The development of a stereoselective synthesis
of 1 and investigation of the relationship between stereo-
chemistry and biological activity are currently under
way.
Acknowledgment
13. Properties of synthetic 1: colorless oil; IR (KBr) mmax
(cmÀ1) = 3395 (br s, –OH), 2935 (br s), 2350 (w), 1705 (s,
C@O), 1465 (w), 1370 (w), 1055 (m), 565 (br m); 1H NMR
(400 MHz, CD3OD): d = 0.80 (d, J = 6.3 Hz, 3H, 19-
CH3), 0.82 (d, J = 6.8 Hz, 3H, 17-CH3), 0.98 (d,
J = 6.8 Hz, 3H, 20-CH3), 0.99 (m, 1H, 14-CHH) 1.04 (s,
3H, 18-CH3), 1.05 (m, 1H 8-CHH), 1.15–1.56 (m, 15H),
1.80 (m, 1H, 2-CHH), 2.45 (m, 2H, 5-CH2), 2.68 (sxt,
J = 6.8 Hz, 1H, 3-CH), 3.22 (dd, J = 6.8, 10.7 Hz, 1H, 16-
CHH), 3.32 (dd, J = 5.9, 10.7 Hz, 1H, 16-CHH), 3.43 (br
t, J = 6.8 Hz, 2H, 1-CH2); 13C NMR (100 MHz, CD3OD):
d = 16.9 (C-20), 17.1 (C-17), 19.9 (C-19), 22.3 (C-9,13),
26.9 (C-18), 31.7 (C-6), 33.6 (C-7), 35.0 (C-14), 36.7 (C-2),
36.9 (C-15), 38.6 (C-8), 40.0 (C-5), 43.0 (C-10,12), 44.0
(C-3), 60.6 (C-1), 68.4 (C-16), 73.4 (C-11), 217.5
(C-4); HRMS (FAB): calcd. for C20H40O4Na 367.2825,
We thank Dr. T. Tashiro (RIKEN) for MS
measurements.
References and notes
1. Fry, W. E.; Goodwin, S. B. Bioscience 1997, 47, 363–371.
2. Lane, C. R.; Beales, P. A.; Hughes, K. J. D.; Griffin, R. L.;
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2003, 52, 414.
3. (a) Werrens, S.; Marwitz, R.; Man in’t Veld, W. A.; De
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1
found 367.2823 [M+Na]+. H and 13C NMR spectra are
in good accordance with those of the reported natural
product.
4. Ashby, S. F. Trans. Br. Mycol. Soc. 1929, 14, 18–38.