Stereochemical analysis of α1, a mating hormone of the phytopathogen Phytophthora

Article abstract of DOI:10.1016/j.tetasy.2007.07.024

The stereochemistry of α1, the first identified mating hormone of the plant pathogen Phytophthora, has been unknown due to its acyclic flexible nature. Here, two stereogenic centers of α1 are determined to be (3RS,15R)-configuration by NMR analysis of the

Full text of DOI:10.1016/j.tetasy.2007.07.024

Tetrahedron: Asymmetry 18 (2007) 1763–1765
Stereochemical analysis of a1, a mating hormone of the
phytopathogen Phytophthora
Makoto Ojika,* Jianhua Qi, Yuko Kito and Youji Sakagami
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Received 25 June 2007; accepted 18 July 2007
Abstract—The stereochemistry of a1, the first identified mating hormone of the plant pathogen Phytophthora, has been unknown due to
its acyclic flexible nature. Here, two stereogenic centers of a1 are determined to be (3RS,15R)-configuration by NMR analysis of the
Mosher’s esters of a1 and a synthetic model compound. The information obtained here will be helpful for reducing the burden of the
researchers who are trying to synthesize all the possible stereoisomers of a1 to elucidate its full stereochemistry.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
the two terminal stereogenic centers, C-3 and C-15, by
the NMR analysis of the Mosher’s esters of 1.
The fungus-like microorganisms of the genus Phytophthora
include notorious plant pathogens that can devastate
important agricultural products, such as potatoes and
tomatoes. The heterothallic members of Phytophthora uti-
lize mating hormones for their sexual reproduction, result-
ing in the formation of sexual spores known as oospores,1,2
which greatly contribute to their durability and pathogenic
success.3 In 2005, one of the mating hormones, a1 1, was
identified and characterized from the A1 mating type of
Phytophthora nicotianae.4 This substance induced oospores
in the counter mating type A2 at a nanogram level. The
mating hormone a1 1 is an acyclic oxygenated diterpene,
with four discrete stereogenic centers. Although the deter-
mination of the absolute configuration is essential for
further chemical biological researches, the flexibility of
the molecular skeleton, as well as the unavailability of 1
(1.2 mg from ca. 1800 L culture broth),4 hampers the
stereochemical analysis. Recently, an isomeric mixture that
could contain all possible stereoisomers of 1 has been syn-
thesized and its hormonal activity demonstrated,5 suggest-
ing that the synthetic mixture contains 1. However, the
stereoselective total synthesis of the 16 possible stereoiso-
mers will be needed without any stereochemical informa-
tion on the natural specimen of 1. To aid the future total
synthesis of 1, we examined the absolute configuration of
HO
OR
3
7
11
RO
15
O
1: R = H
(R)-2: R = (R)-MTPA
(S)-2: R = (S)-MTPA
2. Results and discussion
A hundred micrograms of the mating hormone a1 1 were
first converted to bis-[a-methoxy-a-(trifluoromethyl)phen-
ylacetate] (bis-MTPA) esters, (R)-2 and (S)-2.6 The proton
chemical shifts of the oxymethylene groups (H-1 and H-16)
were affected by the neighboring MTPA esters, providing
us with valuable information about their stereochemistry.
1
The H NMR spectra indicated that both (R)-2 and (S)-2
were mixtures of stereoisomers, suggesting that a1 1 was
originally an epimeric mixture. This could be supported
by the facts that the epimers ratio was not particularly
affected by the esterification time (data not shown) and that
the synthetic model ketone 3 was converted to the corres-
ponding MTPA esters without epimerization (vide infra).
The major isomer of (R)-MTPA ester (R)-2 (60:40 ratio)
indicated splitting methylene signals both for H-1 (d 4.23
and 4.37, Dd = 0.14) and for H-16 (d 4.08 and 4.25,
Dd = 0.17) (Fig. 1a). On the other hand, the major isomer
of (S)-MTPA ester (S)-2 (64:36 ratio) indicated overlap-
ping methylene signals both for H-1 (d 4.34, Dd = 0) and
*
Corresponding author. Tel./fax: +81 52 789 4284; e-mail: ojika@agr.
nagoya-u.ac.jp
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.07.024

1764
M. Ojika et al. / Tetrahedron: Asymmetry 18 (2007) 1763–1765
known, the absolute configuration of d-oxo compounds
(R = R0CO–), such as a1 1 has not been examined. There-
fore, a model compound that possesses the same substruc-
ture (C-1 to C-6) as that of 1, (S)-1-hydroxy-3-methyl-4-
octanone 3, was synthesized (Scheme 1). (S)-2-Methyl-
1,4-butandiol was converted to monosilyl ether 411 with
3:1 regioselectivity. After chromatographic separation, 4
was oxidized to aldehyde 5.11 Addition reaction with
buthyllithium followed by oxidation gave ketone 6.12
Deprotection of the silyl group furnished the desired keto
alcohol 3.13 Esterification of 3 with MTPA chlorides gave
(R)- and (S)-MTPA esters, (R)-7 and (S)-7, respectively.
1
The H NMR of (R)-7 and (S)-7 indicated overlapping
(Dd = 0) and splitting (Dd = 0.14) oxymethylene signals,
respectively, (Table 1, Fig. 2). Since the opposite pheno-
menon was observed for the oxymethylene protons (H-1)
of the major isomers of (R)-2 (Dd = 0.14) and (S)-2
(Dd = 0) (Table 1, Fig. 1), the (3R)-configuration of the
major epimer of a1 1 was also determined.
Figure 1. Partial 1H NMR spectra of (R)-2 (a) and (S)-2 (b) in CDCl3. The
signals due to the minor epimers [40% in (R)-2, 36% in (S)-2] are indicated
with asterisks.
Table 1. Chemical shift differences of two oxymethylene protons of
MTPA esters
MTPA esters
Dd (R-MTPA)
Dd (S-MTPA)
(R)-2, (S)-2 (H-16)
(S)-RCH(CH3)CH2OMTPA6
(R)-2, (S)-2 (H-1)
(R)-7, (S)-7
0.17
0.00–0.05
0.14
0.03
0.14–0.20
0.00
0.00
0.14
for H-16 (d 4.15 and 4.18, Dd = 0.03) (Fig. 1b). Kobayashi
et al. studied the absolute configuration of the primary
alcohols with a b-methyl branch [R–CH(CH3)–CH2OH]
and reported that the (R)- and (S)-MTPA esters of
(S)-alcohols indicated overlapping (Dd = 0–0.05) and split-
ting (Dd = 0.14–0.20) methylene signals, respectively,
(Table 1).7 Since the opposite phenomenon was observed
for the oxymethylene protons (H-16) of (R)-2 (Dd = 0.17)
and (S)-2 (Dd = 0.03) (Table 1), the (15R)-configuration
of a1 1 was determined.
Figure 2. 1H NMR signals due to the oxymethylene protons of (R)-7 (a)
and (S)-7 (b) in CDCl3.
3. Conclusion
To determine the absolute configuration of C-3, the NMR
data for the MTPA esters of c-methyl-branched primary
alcohols [R–CH(CH3)–CH2CH2OH] are needed. Although
some examples of simple alcohols (R = alkyl)8,9 and a
d-hydroxy compound (R = R0CH(OH)–)10 are already
The absolute configuration of the Phytophthora mating
hormone a1 1 was partially determined as 3RS,15R (3R/
3S = ca. 3:2) by NMR analysis of the Mosher’s esters of
1) TBDPSCl, imidazole
PCC
DMF, -50oC
O
OH
OH
CH2Cl2, rt
(91%)
TBDPSO
HO
TBDPSO
2) chromatography
(26%)
4
5
1) BuLi, THF, -78oC
(64%)
BuNF, THF, rt
(88%)
RO
TBDPSO
2) PCC, CH2Cl2, rt
(87%)
O
3; R = H
(R)-7: R = (R)-MTPA
(S)-7: R = (S)-MTPA
O
6
MTPACl,
pyridine
Scheme 1. Synthesis of model compound 3 and its MTPA esters (R)-7 and (S)-7.

M. Ojika et al. / Tetrahedron: Asymmetry 18 (2007) 1763–1765
1765
Under the same conditions, but using (R)-MTPA chloride, 1
(50 lg) was converted to (S)-MTPA ester (S)-2 (ca. 0.1 mg,
tR = 38.5 min): H NMR (600 MHz, CDCl3) d 7.51 (m, 4H,
1 and the synthetic model compound 3. This stereochemi-
cal outcome has limited the number of the possible stereo-
isomers of 1 from sixteen to four. The total synthesis of the
four stereoisomers, as well as the evaluation of their
hormonal activity, will be essential for determining the full
stereochemistry of a1 (1).
1
Ph), 7.41 (m, 6H, Ph), 4.30 (t, J = 6.0 Hz, 2H, H-1), 4.18 (dd,
J = 10.6, 6.0 Hz, 1H, H-16b), 4.15 (dd, J = 10.6, 5.4 Hz, 1H,
H-16a), 3.55 (s, 3H, OMe), 3.53 (s, 3H, OMe), 2.59 (m, 1H,
H-3), 2.42 (m, 1H, H-5b), 2.34 (m, 1H, H-5a), 2.08 (m, 1H, H-
2b), 1.87 (m, 1H, H-15), 1.67 (m, 1H, H-2a), 1.58 (m, 1H, 6b),
1.42-1.22 (m, 13H, H-6a, 7, 8b, 9, 10, 12, 13, 14b), 1.15 (m,
1H, H-14a), 1.14 (s, 3H, 11-Me), 1.10 (m, 1H, H-8a), 1.09 (d,
J = 7.2 Hz, 3H, 3-Me), 0.93 (d, J = 6.6 Hz, 3H, 15-Me), 0.86
(d, J = 6.6 Hz, 3H, 7-Me).
Acknowledgment
This work was financially supported by a Grant-in-Aid for
Scientific Research (B) (18380073) from the Ministry of
Education, Culture, Sports, Science, and Technology.
7. Tsuda, M.; Toriyabe, Y.; Endo, T.; Kobayashi, J. Chem.
Pharm. Bull. 2003, 51, 448.
8. Oikawa, H.; Matsuda, I.; Kagawa, T.; Ichihara, A.; Kohm-
oto, K. Tetrahedron 1994, 50, 13347.
References
9. Tsuda, M.; Izui, N.; Shimbo, K.; Sato, M.; Fukushi, E.;
Kawabata, J.; Katsumata, K.; Horiguchi, T.; Kobayashi, J. J.
Org. Chem. 2003, 68, 5339.
1. Ko, W. H. J. Gen. Microbiol. 1978, 107, 15.
10. Eguchi, T.; Kobayashi, K.; Uekusa, H.; Ohashi, Y.; Mizoue,
K.; Matsushima, Y.; Kakinuma, K. Org. Lett. 2002, 4,
3383.
2. Chern, L. L.; Tang, C. S.; Ko, W. H. Bot. Bull. Acad. Sin.
1999, 40, 79.
3. Judelson, H. S.; Blanco, F. A. Nat. Rev. Micobiol. 2005, 3, 47.
4. Qi, J.; Asano, T.; Jinno, M.; Matsui, K.; Atsumi, K.;
Sakagami, Y.; Ojika, M. Science 2005, 309, 1828.
11. Uenishi, J.; Kawahama, R.; Yonemitsu, O. J. Org. Chem.
1997, 62, 1691.
26
12. Compound 6: colorless oil; ½aꢁD ¼ þ8:0 (c 0.13, CHCl3); IR
5. Yajima, A.; Kawanishi, N.; Qi, J.; Asano, T.; Sakagami, Y.;
Nukada, T.; Yabuta, G. Tetrahedron Lett. 2007, 48, 4601.
6. Compound a1 1 (50 lg) was treated with (S)-MTPA chloride
(6 lL) in dry pyridine (0.2 mL) for 19 h. The crude product
was purified by HPLC [Develosil ODS-UG-5 (/ 4.6 ·
250 mm), 75–100% MeOH in H2O in a 60 min linear gradient,
flow rate 1 mL minꢀ1, detection at 220 nm] to give (R)-
(film) 1713, 1112, 702, 504 cmꢀ1
;
1H NMR (CDCl3,
400 MHz) d 7.64 (m, 4H), 7.38 (m, 6H), 3.65 (t, J = 6.0 Hz,
2H), 2.79 (sextet, J = 6.8 Hz, 1H), 2.42 (m, 2H), 1.94 (m, 1H),
1.51 (m, 3H), 1.28 (m, 2H), 1.05 (s, 9H), 1.03 (d, J = 7.2 Hz,
3H), 0.89 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d
214.6, 135.5 (4C), 133.8 (2C), 129.6 (2C), 127.6 (4C), 61.7,
42.5, 41.0, 35.5, 26.9 (3C), 25.8, 22.4, 19.2, 16.3, 13.9. Anal.
Calcd for C25H36O2Si: C, 75.75; H, 9.09; N, 0.00. Found: C,
75.67; H, 9.28; N, 0.00.
1
MTPA ester (R)-2 (ca. 0.1 mg, tR = 39.3 min): H NMR for
the major isomer (600 MHz, CDCl3) d 7.51 (m, 4H, Ph), 7.41
(m, 6H, Ph), 4.37 (dt, J = 11.4, 6.2 Hz, 1H, H-1b), 4.25 (dd,
J = 10.6, 5.8 Hz, 1H, H-16b), 4.24 (m, 1H, H-1a), 4.08 (dd,
J = 10.6, 6.6 Hz, 1H, H-16a), 3.55 (s, 3H, OMe), 3.49 (s, 3H,
OMe), 2.55 (m, 1H, H-3), 2.46 (m, 1H, H-5b), 2.30 (m, 1H,
H-5a), 2.08 (m, 1H, H-2b), 1.87 (m, 1H, H-15), 1.67 (m, 1H,
H-2a), 1.58 (m, 1H, 6b), 1.42–1.22 (m, 13H, H-6a, 7, 8b, 9, 10,
12, 13, 14b), 1.17 (m, 1H, H-14a), 1.14 (s, 3H, 11-Me), 1.11
(m, 1H, H-8a), 1.08 (d, J = 7.2 Hz, 3H, 3-Me), 0.92 (d,
J = 6.6 Hz, 3H, 15-Me), 0.84 (d, J = 6.6 Hz, 3H, 7-Me).
25
13. Compound 3: colorless oil; ½aꢁD ¼ ꢀ7:7 (c 0.25, CHCl3); IR
;
(film) 3421 (br), 1709, 1054 cmꢀ1 1H NMR (CDCl3,
400 MHz) d 3.63 (m, 2H), 2.74 (sextet, J = 7.2 Hz, 1H),
2.48 (m, 2H), 1.94 (m, 1H), 1.65–1.50 (m, 3H), 1.31 (sextet,
J = 7.2 Hz, 2H), 1.12 (d, J = 7.2 Hz, 3H), 0.91 (t, J = 7.2 Hz,
3H); 13C NMR d 215.3, 60.6, 43.2, 40.9, 35.4, 25.8, 22.4, 16.6,
14.0. Anal. Calcd for C9H18O2: C, 68.35; H, 11.39; N, 0.00.
Found: C, 68.35; H, 11.48; N, 0.00.