Identification of 2-methoxyhexadecanoic acid in Amphimedon compressa

Article abstract of DOI:10.1021/np970578v

The phospholipid fatty acid composition of Amphimedon compressa was reinvestigated and the 2-methoxyhexadecanoic acid was identified for the first time in nature. Structure characterization was accomplished by means of gas chromatography-mass spectrometry and total synthesis from commercially available 2-hydroxyhexadecanoic acid.

Full text of DOI:10.1021/np970578v

J . Nat. Prod. 1998, 61, 675-676
675
Id en tifica tion of 2-Meth oxyh exa d eca n oic Acid in Am ph im ed on com pr essa
Ne´stor M. Carballeira,* Roberto Colo´n, and Anastacio Emiliano
Department of Chemistry, University of Puerto Rico, P.O. Box 23346, San J uan, Puerto Rico 00931
Received December 30, 1997
The phospholipid fatty acid composition of Amphimedon compressa was reinvestigated and
the 2-methoxyhexadecanoic acid was identified for the first time in nature. Structure
characterization was accomplished by means of gas chromatography-mass spectrometry and
total synthesis from commercially available 2-hydroxyhexadecanoic acid.
Naturally occurring R-methoxy fatty acids have been
identified only from the phospholipids of sponges.1-3
Some examples include normal-chain saturated 2-meth-
oxy fatty acids between 19 and 24 carbons and very
long-chain monounsaturated fatty acids such as (2R,-
21Z)-2-methoxy-21-octacosenoic acid, which was the
first-reported naturally occurring R-methoxy fatty acid
from a phospholipid.2 J ust recently, we identified
2-methoxy-5-hexadecenoic acid and 2-methoxy-6-hexa-
decenoic acid from the phospholipids of several Carib-
bean sponges.3 We also established that the 2-methoxy-
5-hexadecenoic acid can be found more often in the
phospholipids of sponges, but the ∆6 analogue is quite
rare.4 Despite all of these efforts, the parent and
perhaps more classic 2-methoxyhexadecanoic acid has
neither been isolated nor identified from any natural
source. The closest naturally occurring structure known
is a glycerol ether, namely 1-O-(2-methoxyhexadecyl)
glycerol, which was isolated from Greenland shark liver
oil.5 In the present communication we report the first
naturally occurring 2-methoxyhexadecanoic acid, which
we found in a reinvestigation of the sponge Amphime-
don compressa Pallas (order Haplosclerida, family
Nephatidae).4
The main phospholipids from the present A. com-
pressa were also identified as phosphatidylethanola-
mine, phosphatidylserine, and phosphatidylcholine.4 A
reexamination of the fatty acid composition of this A.
compressa resulted in a similar fatty acid composition
to that which we previously reported, which the key
fatty acids are 5,9,23-nonacosatrienoic acid and 5,9,23-
tricontatrienoic acid, together with a series of unusual
monounsaturated fatty acids ranging in length between
C22 and C26.4 The 2-methoxy-5-hexadecenoic acid was
also identified in this fatty acid mixture. Essential for
the characterization were GC retention times of the
corresponding fatty acid methyl esters, MS data on the
methyl esters and their corresponding dimethyl disul-
fide derivatives, and catalytic hydrogenation of the
whole fatty acid methyl ester mixture for easier elucida-
tion of methyl branching.
The GC retention time of methyl ester 1 corresponded
to an equivalent chain-length (ECL) value of 17.22,
implying unusual branching. The MS of 1 displayed a
molecular ion peak at m/z 300 and a strong M+ -59
peak at m/z 241, together with a small peak at m/z 104
(McLafferty rearrangement).4 This information strongly
suggested methyl 2-methoxyhexadecanoate (1) as the
most probable structure. Final structure characteriza-
tion was achieved by total synthesis. Methyl 2-meth-
oxyhexadecanoate (1) is a known synthetic compound,
but its synthesis was achieved through a synthetic
scheme ending with the reaction of methyl 2-bromo-
hexadecanoate with sodium methoxide.6,7 We employed
a simpler synthesis through the double methylation of
commercially available 2-hydroxyhexadecanoic acid
(Matreya, Inc.). Several methods were initially tried for
this transformation, for example, direct double methy-
lation with CH2N2, reaction of the R-hydroxy acid with
NaOH and methyl sulfate, and reaction with MeI in
refluxing K2CO3. None of these procedures was effective
in this double methylation, presumably in part due to
hydrogen bonding of the R-hydroxy to the carbonyl. Our
best results, however, were obtained by reacting 2-hy-
droxyhexadecanoic acid with NaH and MeI in DMSO,
which afforded the desired methyl 2-methoxyhexa-
decanoate (1) in 70-80% yields. Synthetic methyl ester
1 was co-injected in GC with the isolated sample from
A. compressa, and they proved to be identical. Com-
plete NMR data for 1 was also obtained and is presented
in the Experimental Section for comparison.8 R-Meth-
oxy fatty acids from sponges are known to have the R
configuration at the R-carbon, and 1 may well follow this
trend.1
The identification of 2-methoxyhexadecanoic acid in
A. compressa has biosynthetic relevance. For example,
the commonly occurring 2-methoxy-5-hexadecenoic acid
in sponges may well originate from ∆5 desaturation on
the 2-methoxyhexadecanoic acid, which is reported here
for the first time as a naturally occurring fatty acid.
Work is in progress elucidating the origin of unusual
fatty acids in marine invertebrates.
Our main interest was centered on the new com-
pound, 2-methoxyhexadecanoic acid, which was char-
acterized as the methyl ester in 0.5% relative abundance
as compared to the other 30 fatty acids in the mixture.
* To whom correspondence should be addressed. Tel.: (787) 764-
0000 ext. 4791. Fax: (787) 751-0625. E-mail: ncarball@
upracd.upr.clu.edu.
S0163-3864(97)00578-8 CCC: $15.00
© 1998 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/08/1998

676 J ournal of Natural Products, 1998, Vol. 61, No. 5
Notes
Exp er im en ta l Section
dissolved in 1 mL of DMSO (also under a nitrogen
atmosphere) and added dropwise, after which the reac-
tion mixture was stirred at room temperature for 10
min. An excess of MeI was then added, and the reaction
mixture was further stirred for 20 min. After this time
the reaction mixture was diluted with hexane-ether
(1:1) and washed twice with H2O to remove the DMSO.
The organic phase was dried over MgSO4, filtered, and
evaporated in vacuo, affording 0.08 g (0.26 mmol, 73%)
of 1. Spectral data for 1 is presented below.
Meth yl 2-m eth oxyh exa d eca n oa te (1): 1H NMR
(CDCl3, 300 MHz) δ 4.18 (1H, dd, J ) 7.2, 2.9 Hz, H-2),
3.78 (3H, s, -OCH3), 3.37 (3H, s, CO2CH3), 1.6-1.8 (2H,
m, H-3), 1.2-1.4 (24H, m, -CH2-), 0.87 (3H, t, J ) 6.4
Hz, CH3); 13C NMR (CDCl3, 75 MHz) δ 175.8 (s, C-1),
80.7 (q, -OCH3), 58.0 (d, C-2), 52.40 (q, CO2CH3), 32.8
(t), 31.9 (t), 29.7 (t), 29.64 (t), 29.61 (t), 29.5 (t), 29.4 (t),
29.33 (t), 29.29 (t), 24.7 (t), 22.7 (t), 14.1 (q, C-16); GC-
MS (70 eV) m/z 300 [M]+ (2), 242 (17), 241 (100), 208
(1), 207 (2), 153 (1), 138 (1), 125 (6), 123 (2), 111 (19),
109 (3), 104 (4), 99 (2), 97 (39), 95 (8), 85 (11), 83 (42),
81 (11), 71 (43), 69 (37), 67 (13), 58 (10), 57 (34), 55 (39).
Gen er a l Exp er im en ta l P r oced u r es. Fatty acid
methyl esters were analyzed by GC in a Hewlett-
Packard 5890A Series II gas chromatograph equipped
with a fused silica capillary column (30 m × 0.32 mm
i.d.) containing either SE-54 or SPBTM-1 (carrier gas
He). Analyses were performed as previously described.
Samples were also analyzed by GC-MS at 70 eV using
a Hewlett-Packard 5972A MS ChemStation equipped
with a 30 m × 0.25 mm special performance capillary
column (HP-5MS) of polymethyl siloxane cross-linked
with 5% phenyl methylpolysiloxane. NMR data were
collected in a Bruker Avance DPX 300 spectrometer. J
values are given in Hz.
Sa m p le Collection . A. compressa was collected
November 1, 1997, at Cayo Enrique, Lajas, Puerto Rico,
at a depth between 1 and 2 m. The sponge was washed
in seawater, carefully cleaned of all nonsponge debris,
and lyophilized. A voucher specimen is available at the
Department of Chemistry of the University of Puerto
Rico, R´ıo Piedras campus.
Extr a ction a n d Isola tion of P h osp h olip id s. The
sponge (250 g) was carefully cleaned and cut into small
pieces. Extraction with 2 × 250 mL of CHCl3-MeOH
(1:1) yielded the total lipids (6.7 g). The neutral lipids,
glycolipids, and phospholipids (480 mg) were separated
by column chromatography on Si gel (60-200 mesh)
using the procedure of Privett et al.9
P r ep a r a tion a n d Isola tion of F a tty Acid Der iva -
tives. The fatty acyl components of the phospholipids
were obtained as their methyl esters by reaction of the
phospholipids with methanolic HCl followed by column
chromatography.4 The double-bond positions in the
polyunsaturated fatty acids were determined by prepar-
ing the corresponding dimethyl disulfide derivatives.4
Hydrogenations were carried out in 10 mL of MeOH and
catalytic amounts of PtO2.
Ack n ow led gm en t. We thank the NSF-MRCE and
the University of Puerto Rico FIPI programs for finan-
cial support. The technical assistance of Yolanda Marte
is appreciated.
Refer en ces a n d Notes
(1) Ayanoglu, E.; Popov, S.; Kornprobst, J . M.; Aboud-Bichara, A.;
Djerassi, C. Lipids 1983, 18, 830-836.
(2) Ayanoglu, E.; Kornprobst, J . M.; Aboud-Bichara, A.; Djerassi,
C. Tetrahedron Lett. 1983, 24, 1111-1114.
(3) Carballeira, N.; Sepu´lveda, J . Lipids 1992, 27, 72-74.
(4) Carballeira, N.; Negro´n, V.; Reyes, E. J . Nat. Prod. 1992, 55,
333-339.
(5) Hallgren, B.; Sta¨llberg, G. Acta Chem. Scand. 1967, 21, 1519-
1529.
(6) Sta¨llberg, G. Chem. Scr. 1973, 3, 117-124.
(7) Stanacey, N.; Prostenik, M. Croat. Chem. Acta 1957, 29, 107-
113.
(8) Prostenik, M.; Stanacey, N.; Munk-Weinert, M. Croat. Chem.
Acta 1962, 34, 1-6.
(9) Privett, O. S.; Dougherty, K. A.; Erdahl, W. L.; Stolyhwo, A. J .
Am. Oil Chem. Soc. 1973, 50, 516-520.
Meth yl 2-Meth oxyh exa d eca n oa te (1). Into a two-
necked round-bottom flask provided with a magnetic
stirrer and under a nitrogen atmosphere, were placed
0.10 g (0.37 mmol) of 2-hydroxyhexadecanoic acid in 3
mL of DMSO. Separately, two equivalents of NaH were
NP970578V