Structure of an unsaturated fatty acid with unique vicinal dimethyl branches isolated from the Okinawan soft coral of the genus Sinularia

Article abstract of DOI:10.1248/cpb.56.861

A new unsaturated fatty acid with unique vicinal dimethyl branches was isolated from the Okinawan soft coral of the genus Sinularia. The structure of the compound was determined based on the results of spectroscopic analysis and chemical conversion. The absolute configuration was deduced by applying the Ohrui-Akasaka method.

Full text of DOI:10.1248/cpb.56.861

June 2008
Notes
Chem. Pharm. Bull. 56(6) 861—863 (2008)
861
Structure of an Unsaturated Fatty Acid with Unique Vicinal Dimethyl
Branches Isolated from the Okinawan Soft Coral of the Genus Sinularia
,a
a
a
Ryota MAKINO, Haruko TAKAHASHI, Kazuo IGUCHI, ,a Hiroshi OHRUI,b and
*
*
Kinzo WATANABE,
b
Kazuaki AKASAKA
a School of Life Sciences, Tokyo University of Pharmacy and Life Sciences; Horinouchi, Hachioji, Tokyo 192–0392, Japan:
and b Graduate School of Life Sciences, Tohoku University; Aoba-ku, Sendai 981–8555, Japan.
Received January 9, 2008; accepted April 2, 2008; published online April 10, 2008
A new unsaturated fatty acid with unique vicinal dimethyl branches was isolated from the Okinawan soft
coral of the genus Sinularia. The structure of the compound was determined based on the results of spectroscopic
analysis and chemical conversion. The absolute configuration was deduced by applying the Ohrui–Akasaka
method.
Key words soft coral; Sinularia sp.; unsaturated fatty acid; Ohrui–Akasaka method
Soft corals have been noted as a rich source of structurally ence of a carboxylic acid group was suggested by the IR
unique and biologically active natural products1,2) such as ter- absorptions of 3200—2500 (broad) and 1714 cmꢀ1 together
penoids, steroids and prostanoids. During the course of our with the 13C signal of 180.3 (C) ppm. The 1H-NMR spectrum
studies3—9) on the constituents of Okinawan marine inverte- (Table 1) showed the signals due to one primary methyl, one
brates, a new unsaturated fatty acid with unique vicinal di- secondary methyl, one olefinic methyl and one olefinic pro-
methyl branches, (E,R)-6,7-dimethylhexadec-7-enoic acid ton. These spectral data, coupled with two degrees of unsatu-
(1), was isolated. This paper describes the structural determi- ration, suggested that compound 1 was an acyclic unsatu-
nation of the fatty acid based on the results of spectroscopic rated fatty acid with two methyl branches.
analysis and chemical conversion involving the application
The HMQC analysis revealed the assignment of each di-
1
of the Ohrui–Akasaka method.10)
rect C–H bond in 1 as summarized in Table 1. The H–1H
Compound 1 was isolated as a colorless oil from the correlations obtained from H–1H COSY are shown by the
1
methanol extract of the genus Sinularia collected from a bold lines in Fig. 2 to give four partial structures (a—d),
coral reef off Ishigaki Island (see Experimental).
which were connected by the HMBC correlations as shown
The molecular formula of compound 1 was found to be by the broken arrows in Fig. 2. The HMBC correlation from
C18H34O2 using the high-resolution electrospray mass spec- H3-16 to C-14 (CH2) indicated the presence of a propyl
trum (HR-ESI-MS) and 13C-NMR data. The 13C-NMR spec- group. The HMBC correlations from H3-18 to C-7 and C-8
trum (Table 1) disclosed the signals due to three methyls, exhibited the presence of a trisubstituted double bond with a
eleven sp3 methylenes, one sp3 methine, one sp2 methine, one methyl group. The presence of another methyl group at the
sp2 quaternary carbon and one carbonyl carbon. The pres- position (C-6) adjacent to the trisubstituted double bond was
demonstrated by the HMBC correlations from H3-18 to C-6
Table 1. NMR Dataa) for 1
and from H3-17 to C-7. The structural unit from the car-
boxylic acid (C-1) to C-5 was indicated by the HMBC corre-
lations from H2-2 to C-1, and from H2-3 to C-4 and C-5. The
1
Position
remaining five methylenes should automatically be con-
nected between C-9 and C-14, leading to the gross structure
dC
dH
1
2
3
4
5
180.3 (C)
34.1 (CH2)
24.8 (CH2)
27.1 (CH2)
34.5 (CH2)
2.33 (2H, t, 7.5)
1.61 (2H, m)
1.22 (2H, m)
1.21 (1H, m)
1.36 (1H, m)
6
7
42.6 (CH)
138.6 (C)
2.06 (1H, sext, 6.9)
8
9
124.5 (CH)
27.7 (CH2)
29.32 (CH2)
29.35 (CH2)
29.54 (CH2)
29.85 (CH2)
31.9 (CH2)
22.7 (CH2)
14.1 (CH3)
19.8 (CH3)
12.1 (CH3)
5.12 (1H, t, 6.9)
1.96 (2H, m)
1.2—1.4 (8H, m)
Fig. 1. Structure of New Fatty Acid 1
10—13
14
15
16
17
18
1.26 (2H, m)
1.28 (2H, m)
0.88 (3H, t, 7.0)
0.96 (3H, d, 6.9)
1.48 (3H, s)
Fig. 2. Gross Structure, 1H–1H Correlations (Bold Lines), Key HMBC
Correlations (Broken Arrows) and Key NOE Correlations (Real Arrows) of
1
a) 13C-NMR: 125 MHz in CDCl3, 1H-NMR: 500 MHz in CDCl3. J in Hz. Assign-
ments of 13C and 1H signals were made based on HMQC.
∗ To whom correspondence should be addressed. e-mail: kinzo@ls.toyaku.ac.jp; onocerin@ls.toyaku.ac.jp
© 2008 Pharmaceutical Society of Japan

862
Vol. 56, No. 6
methylhexadecanoic acid, was cited in the literature as a
patent11) describing surfactant properties of mid-chain
branched fatty acids, although the compound was not a natu-
ral product.
Experimental
General Procedures Optical rotation was measured using a JASCO
DIP-370 automatic polarimeter. IR spectra were recorded using a Perkin-
Elmer FT-IR PARAGON 1000 spectrophotometer. All NMR spectra were
taken using a Bruker DRX-500 (1H; 500 MHz, 13C; 125 MHz) spectrometer.
1H–1H COSY, NOESY, HMQC and HMBC spectra were measured using
standard Bruker pulse sequences. Chemical shifts are given on a d (ppm)
scale with CHCl3 (1H; 7.26 ppm) and CDCl3 (13C; 77.0 ppm) as the internal
standard. High-resolution ESI mass spectra were taken using a Micromass
LCT spectrometer.
Chart 1. Chemical Conversion of 1
Extraction and Isolation Wet specimens (1.6 kg) of the soft coral of
the genus Sinularia, collected from the coral reef off Ishigaki Island (Oki-
nawa, Japan), were extracted with MeOH (4.0 l, three times). Each MeOH
extract was concentrated under reduced pressure. The combined MeOH ex-
tract (71.2 g) was partitioned between EtOAc and H2O. The EtOAc soluble
portion (9.4 g) was chromatographed on a silica gel column (100 g) eluted
with hexane (600 ml, fraction A), hexane–EtOAc (3 : 1, 600 ml, fraction
B), hexane–EtOAc (1 : 1, 600 ml, fraction C), EtOAc (600 ml, fraction
D), and MeOH (600 ml, fraction E), to give five fractions. A part (2.0 g)
of fraction B (4.5 g) was subjected to flash column chromatography
(hexane–EtOAcꢁ9 : 1, 8.5 : 1.5, 8 : 2, 7.5 : 2.5, 7 : 3, 6.5 : 3.5 and EtOAc) to
give 13 fractions. The third fraction (580 mg) was further purified by nor-
mal-phase (hexane–EtOAcꢁ8 : 2) and reversed-phase HPLC (ODS,
MeOH–H2Oꢁ95 : 5, 9 : 1, tetrahydrofuran–H2Oꢁ6 : 4) to give compound 1
(124 mg).
Fig. 3. Structures of RR- and SS-2Acyclo-OH of 1
for 1.
(E,R)-6,7-Dimethylhexadec-7-enoic Acid (1): Colorless oil; [a]D25 ꢀ1.0°
(cꢁ3.86, CHCl3); IR (film) nmax 3200—2500 (broad), 2920, 1714 cmꢀ1
;
13C-
The position of the trisubstituted double bond in 1 was
confirmed by the following chemical conversion (Chart 1).
Compound 1 was converted to methyl ester 2, which was
treated with osmium oxide (VIII) and periodic acid hydrate
at room temperature to give aldehyde 3 and ketoester 4.
Without purification of the products, the mixture was treated
with 2,4-dinitrophenylhydrazine to give corresponding 2,4-
dinitrophenylhydrazones 5 and 6. The structures of 5
(C15H22N4O4) and 6 (C16H22N4O6) were elucidated by the MS
and NMR data. Thus the presence of a trisubstituted double
bond between C-7 and C-8 in 1 was established.
1
and H-NMR, see Table 1; HR-ESI-MS m/z: 283.2633 [MꢂH]ꢂ (Calcd for
C18H35O2, 283.2637).
Conversion of 1 to Phenylhydrazones 5 and 6 Compound 1 was
methylated to methyl ester 2 by the treatment of 1 with CH2N2 in diethyl
ether. A small amount of H5IO4 (total 34 mg) and 30% AcOH solution (total
50 ml) were added to a mixture of 2 (17 mg) and H2O–t-BuOH (1 : 1,
500 ml). The reaction mixture was stirred at room temperature for 2 h. After
the addition of 0.05 M aqueous NaOH solution to pH 6, 2,4-dinitrophenylhy-
drazine (65 mg) in a 5% aqueous H3PO4 solution was added. The reaction
mixture was extracted with diethyl ether and the organic layer was washed
successively with water twice and brine twice, and was dried over anhydrous
MgSO4. After filtration, the filtrate was concentrated under reduced pres-
sure. The residue was chromatographed on a silica gel column. Elution with
hexane–EtOAc (9 : 1) gave two fractions. Each fraction was further purified
by normal-phase HPLC (hexane–EtOAcꢁ9 : 1) to give compound 5 (3.5 mg)
from the first fraction and compound 6 (5.3 mg) from the second fraction.
The E configuration of the double bond at C-7 was dis-
closed by the NOE correlations between H-8 and H3-17 and
H2-9 and H3-18 (Fig. 2). The absolute stereochemistry of the
chiral center at C-6 bearing a methyl group was determined
1
Compound 5: Yellow amorphous solids; H-NMR (300 MHz, CDCl3), d
based on the Ohrui–Akasaka method.10) The method clarifies ppm: 0.89 (3H, s), 1.24—1.38 (10H, m), 1.60 (2H, m), 2.42 (2H, dt, Jꢁ5.4,
7.3 Hz), 7.53 (1H, t, Jꢁ5.4 Hz), 7.92 (1H, d, Jꢁ9.6 Hz), 8.29 (1H, dd,
both the position and absolute configuration of a branched
methyl group on a long-chain fatty acid based on the reten-
tion times of ester derivatives prepared from a fatty acid
Jꢁ2.6, 9.6 Hz), 9.12 (1H, d, Jꢁ2.6 Hz), 11.02 (1H, s, NH); 13C-NMR
(75 MHz, CDCl3), d ppm: 14.1 (CH3), 22.6 (CH2), 26.3 (CH2), 29.2 (CH2),
29.3 (CH2), 29.7 (CH2), 31.8 (CH2), 32.5 (CH2), 116.5 (CH), 123.5 (CH),
and RR- and SS-trans-2-(2,3-anthracenedicarboximido)cyclo-
hexanol in reverse-phase HPLC. When compound 1 has a 6R
configuration, RR-trans-2-(2,3-anthracenedicarboximido)cy-
clohexanol (RR-2Acyclo-OH) of 1, in which the 6R-CH3 is
oriented over the plane of anthracene (Fig. 3), is predicted to
be eluted from reversed-phase column faster than the corre-
sponding SS derivative (SS-2Acyclo-OH) of 1, in which the
6R-CH3 is oriented off the plane of anthracene, based on the
theory of the Ohrui–Akasaka method. In fact, the reversed-
phase HPLC of the ester derivatives showed that RR-2Acy-
clo-OH derivative 7 was eluted faster than the SS-2Acyclo-
OH derivative 8, indicating the R configuration at C-6 in 1.
From these findings, compound 1 was assigned to be (E,R)-
6,7-dimethylhexadec-7-enoic acid. Compound 1 is the first
natural unsaturated fatty acid with a 2,3-dimethyl-1-propenyl
128.8 (C), 130.0 (CH), 137.7 (C), 145.1 (C), 152.7 (CH); HR-ESI-MS m/z:
323.1721 [MꢂH]ꢂ (Calcd for C15H23N4O4, 323.1719).
1
Compound 6: Yellow amorphous solids; H-NMR (300 MHz, CDCl3), d
ppm: 1.17 (3H, d, Jꢁ6.9 Hz), 1.32 (2H, m), 1.48 (1H, m), 1.64 (1H, m), 2.42
(2H, dt, Jꢁ5.4, 7.3 Hz), 7.53 (1H, t, Jꢁ5.4 Hz), 7.92 (1H, d, Jꢁ9.6 Hz), 8.29
(1H, dd, Jꢁ2.6, 9.6 Hz), 9.12 (1H, d, Jꢁ2.6 Hz), 11.02 (1H, s, NH); 13C-
NMR (75 MHz, CDCl3), d ppm: 13.5 (CH3), 17.9 (CH3), 24.9 (CH2), 26.9
(CH2), 33.6 (CH2), 33.8 (CH2), 42.4 (CH2), 51.5 (OCH3), 116.5 (CH), 123.5
(CH), 129.1 (C), 130.0 (CH), 137.7 (C), 145.3 (C), 161.3 (C), 173.6 (CO);
HR-ESI-MS m/z: 367.1637 [MꢂH]ꢂ (Calcd for C16H23N4O6, 367.1608).
Conversion of 1 to RR-2Acyclo-OH Derivative 7 and SS-2Acyclo-
OH Derivative 8 Compound 1 (1.3 mg) was reacted with RR-trans-2-
(2,3-anthracenedicarboximido)cyclohexanol (RR-2Acyclo-OH, 1.6 mg) in
toluene–CH3CN (1 : 1, 0.5 ml) in the presence of 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDC, 2.1 mg) and 4-dimethyl-
aminopyridine (DMAP, 1.3 mg) at 40 °C for 13 h. The reaction mixture was
concentrated under reduced pressure and the residue was dissolved in
CHCl3–MeOH (1 : 1). The solution was passed through a silica gel short col-
unit. The structure of the dihydro derivative of 1, 6,7-di- umn, and the eluate was concentrated under reduced pressure. The residue

June 2008
863
was separated by normal phase HPLC eluted with CHCl3–MeOH (9 : 1) to
obtain the RR-2Acyclo-OH derivative 7 (2.2 mg). The SS-2Acyclo-OH de-
rivative 8 (3.8 mg) was also prepared by the reaction of compound 1
(2.4 mg) with SS-2Acyclo-OH (3.0 mg) in the presence of EDC (3.8 mg) and
DMAP (2.4 mg) in toluene–CH3CN (1 : 1, 0.5 ml) at 40 °C for 13 h.
6) Watanabe K., Tsuda Y., Hamada M., Omori M., Mori G., Iguchi K.,
Naoki H., Fujita T., Van Soest R. W. M., J. Nat. Prod., 68, 1001—1005
(2005).
7) Iwasaki J., Ito H., Aoyagi M., Sato Y., Iguchi K., J. Nat. Prod., 69, 2—
6 (2006).
8) Ito H., Iwasaki J., Sato Y., Aoyagi M., Iguchi K., Yamori T., Chem.
Pharm. Bull., 55, 1671—1676 (2007).
References and Notes
1) Faulkner D. J., Nat. Prod. Rep., 19, 1—48 (2002).
2) Blunt J. W., Copp B. R., Hu W.-P., Munro M. H. G., Northcote P. T.,
Prinsep M. R., Nat. Prod. Rep., 24, 31—86 (2007).
3) Watanabe K., Sekine M., Iguchi K., J. Nat. Prod., 66, 1434—1440
(2003).
4) Iguchi K., Fukaya T., Takahashi H., Watanabe K., J. Org. Chem., 69,
4351—4355 (2004).
5) Iguchi K., Fukaya T., Yasumoto A., Watanabe K., J. Nat. Prod., 67,
577—583 (2004).
9) Watanabe K., Mori G., Iguchi K., Suzuki M., Van Soest R. W. M., Nat.
Prod. Res., 21, 710—720 (2007).
10) Ohrui H., Akasaka K., Shichijyukari S., Matsuoka S., Murata S., Me-
guro H., Biosci. Biotechnol. Biochem., 64, 1842—1846 (2000).
11) Conner D. S., Scheibel J. J., Back D. J., Trinh T., Vinson P. K., Burck-
ett-St. Laurent J. C. T. R., Sivik M. R., Wahl E. H., Frankenbach G.
M., Demeyere H. J. M., Cripe T. A., Declercq M. J., Severson R. G.,
CODEN: PIXXD2 WO 9920722 A2 (1999).