Algal pheromone biosynthesis: Stereochemical analysis and mechanistic implications in gametes of ectocarpus siliculosus

Article abstract of DOI:10.1021/jo1004372

During sexual reproduction, female gametes or eggs of brown algae release pheromones to attract their male mating partners. The biologically active compounds comprise linear or alicyclic unsaturated hydrocarbons derived from the aliphatic terminus of C₂₀ polyunsaturated fatty acids (PUFAs) by oxidative cleavage. The current study addresses the stereochemical course of the pheromone biosynthesis using female gametes of the marine brown alga E. siliculosus and chiral deuterium-labeled arachidonic acids. The biosynthetic sequence is likely to proceed via an intermediary 9-hydroperoxyarachidonic acid, which is cleaved with loss of the C(16)-H_R into the C ₁₁-hydrocarbon dictyopterene C and 9-oxonona-(5Z,7E)-dienoic acid.

Full text of DOI:10.1021/jo1004372

pubs.acs.org/joc
Algal Pheromone Biosynthesis: Stereochemical Analysis and Mechanistic
Implications in Gametes of Ectocarpus siliculosus
Fabio Rui and Wilhelm Boland*
Max Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry
Hans-Kno€ll-Strasse 8, D-07745 Jena, Germany
boland@ice.mpg.de
Received March 9, 2010
During sexual reproduction, female gametes or eggs of brown algae release pheromones to attract
their male mating partners. The biologically active compounds comprise linear or alicyclic unsatu-
rated hydrocarbons derived from the aliphatic terminus of C20 polyunsaturated fatty acids (PUFAs)
by oxidative cleavage. The current study addresses the stereochemical course of the pheromone
biosynthesis using female gametes of the marine brown alga E. siliculosus and chiral deuterium-
labeled arachidonic acids. The biosynthetic sequence is likely to proceed via an intermediary
9-hydroperoxyarachidonic acid, which is cleaved with loss of the C(16)-HR into the C11-hydrocarbon
dictyopterene C and 9-oxonona-(5Z,7E)-dienoic acid.
Introduction
mediated by eggs or sessile female gametes and flagellated male
gametes, these compounds direct the motile male gametes to the
signaling female. The relevant compounds are linear or alicyc-
lic unsaturated C8-, C11-, or oxygenated C11-hydrocarbons6
(selected examples are shown in Figure 1).
The structurally different C11 hydrocarbons 1-8 derive
from the aliphatic terminus of polyunsaturated fatty acids
(PUFAs) such as arachidonic acid (9) and eicosapentaenoic
acid, both of which are abundant in the female gametes.7 The
mechanistic details of this biosynthetic pathway have been
elucidatedwithcertaindiatoms, astheseunicellularorganisms
can be cultivated easily and produce the same compounds.
Cell homogenates of the diatom Gomphonema parvulum
synthesize hydrocarbons dictyopterene A (7) and hormosir-
ene (2) from arachidonic and eicosapentaenoic acid.8
Brown algae live on marine coasts worldwide.1 They are
distantly related to other eukaryotic groups such as plants
and fungi, and they provide a valuable source of novel
polysaccharides and lipids.2 Their lipid metabolites comprise
not only complex and bioactive oxylipins3,4 but also C11
hydrocarbons deriving from the transformation of C20 poly-
unsaturated fatty acids (PUFAs).5 During sexual reproduction,
(1) van den Hoek, C.; Mann, D. G.; Jahns, H. M.; van den Hoek, C.;
Mann, D. G.; Jahns, H. M. Algae: An Introduction to Phycology; Cambridge
University Press: Cambridge, 1995.
(2) Charrier, B.; Coelho, S. M.; Le Bail, A.; Tonon, T.; Michel, G.; Potin,
P.; Kloareg, B.; Boyen, C.; Peters, A. F.; Cock, J. M. New Phytol. 2008, 177,
319–332.
(3) Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Wise, M. L.; Jiang,
Z. D.; Bernart, M. W.; Hamberg, M. Hydrobiologia 1993, 261, 653–665.
(4) Ritter, A.; Goulitquer, S.; Salaun, J. P.; Tonon, T.; Correa, J. A.;
Potin, P. New Phytol. 2008, 180, 809–821.
€
(5) Stratmann, K.; Boland, W.; Muller, D. G. Angew. Chem., Int. Ed.
Engl. 1992, 31, 1246–1248.
(6) Maier, I. Prog. Phycol. Res. 1995, 11, 51–102.
(7) Schmid, C. E.; Muller, D. G.; Eichenberger, W. J. Plant Physiol. 1994,
143, 570–574.
(8) Pohnert, G.; Boland, W. Tetrahedron 1996, 52, 10073–10082.
3958 J. Org. Chem. 2010, 75, 3958–3964
Published on Web 05/26/2010
DOI: 10.1021/jo1004372
r
2010 American Chemical Society

Rui and Boland
JOCFeatured Article
SCHEME 1. Biosynthesis of C11 Hydrocarbons in the Diatom
G. parvuluma
FIGURE 1. Structures of selected brown algal pheromones.
aHPETE: hydroperoxyeicosatetraenoic acid.
As illustrated in Scheme 1, first a lipoxygenase activates
the fatty acid to the (9S)-hydroperoxide 10 which in turn
suffers an oxidative cleavage to the respective hydrocarbon
and the polar fragment 9-oxonona-5(Z),7(E-)dienoic acid
(11). In the diatom, the abstraction of the pro-R hydrogen
from C(16) of 10 is the rate-determining step of the cleavage
reaction, which generates an enantiomeric mixture of trans-
divinylcyclopropane (dictyopterene A) (7) along with a small
amount of the linear hydrocarbon cystophorene (1). In the
latter case, the oxidative cleavage involves the loss of a
hydrogen atom from C(13).9 Mechanistic studies have also
been conducted with the asteraceae Senecio isatidaeus, which
produces C11 hydrocarbons by decarboxylation of a C12
fatty acid ultimately derived from linolenic acid.10 Previous
studies with female gametes of E. siliculosus established the
origin of the algal pheromones form arachidonic and eico-
sapentaenoic acid, but mechanistic details have not been
addressed.11 The chemotactic system of the brown alga
E. siliculosus is particularly interesting since the genuine phero-
mone pre-ectocarpene (6) is inactivated after a short time by a
Cope rearrangement to ectocarpene (5).12 While the thermo-
labile pre-ectocarpene (6) (t1/2 = 21 min at 18 °C)12 attracts
male gametes down to 5 pmol L-1, the rearranged ectocarpene
(5) requires 2000-fold higher concentrations for attraction.13
To investigate the stereochemical and mechanistic aspects
of algal pheromone biosynthesis, we administered chirally
labeled deuterated arachidonic acids as metabolic probes to
suspensions of female gametes of E. siliculosus and analyzed
their transformation products by GLC-MS. The results
allowed to reconstruct the biosynthetic sequence from the
fatty acid substrate to the genuine pheromone.
Results and Discussion
In diatoms, the C11 hydrocarbons of the type dictyopter-
ene A (7), pre-ectocarpene (6), as well as dictyotene (4), and
ectocarpene (5) are produced from PUFAs by consecutive
action of a lipoxygenase and a lyase. As illustrated in
Scheme 1, the lipoxygenase generates a (9S)-hydroperoxide
that is cleaved by a lyase activity into a hydrocarbon and
9-oxonona-5Z,7E-dienoic acid (11). In diatoms, the mechan-
istic details were obtained by administration of labeled
arachidonic acid to growing cultures of G. parvulum. In the
present study, we followed the previous strategy and first
developed a novel route to chirally labeled (16R)- and
(16S)-[16,19,20-2H3]-arachidonic acids. Two additional deu-
terium atoms at C(19) and at C(20) of the probe were
introduced to warrant a safe detection of the metabolites
even after removal of the deuterium atom at C(16).
As illustrated in Scheme 2, the scaffold of the chiral,
deuterated arachidonic acid (16R)-[2H3]-9 was envisaged to
be constructed by double-Wittig olefination with the bis-ylide
of (Z)-hex-3-enyl-1,6-bis(triphenylphosphonium bromide) 12
and two easily accessible aldehydes.14,15 This strategy allows the
simultaneous generation of a segment with three methylene-
interrupted Z-double bonds which is typical for highly un-
saturated fatty acids. The deuterium-labeled hexanal 13 was
synthesized as outlined in Scheme 3. Jacobsen’s hydrolytic
kinetic resolution of benzylglycidyl ether 1416 provided the
chiral building block (2S)-14 at a gram scale. Regioselective
(9) Hombeck, M.; Pohnert, G.; Boland, W. Chem. Commun. 1999, 243–
244.
(10) Neumann, C.; Boland, W. Eur. J. Biochem. 1990, 191, 453–459.
(11) Stratmann, K.; Boland, W.; Muller, D. G. Tetrahedron 1993, 49,
3755–3766.
(14) Pohnert, G.; Boland, W. Eur. J. Org. Chem. 2000, 2000, 1821–1826.
(15) Pohnert, G.; Adolph, S.; Wichard, T. Chem. Phys. Lipids 2004, 131,
159–166.
(12) Pohnert, G.; Boland, W. Tetrahedron 1997, 53, 13681–13694.
(13) Boland, W.; Pohnert, G.; Maier, I. Angew. Chem., Int. Ed. Engl.
1995, 34, 1602–1604.
(16) Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.; Hansen,
K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. J. Am. Chem. Soc. 2002,
124, 1307–1315.
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SCHEME 2. Synthesis of (16R)-[16,19,20-2H3]-Arachidonic
SCHEME 3. Synthesis of Chiral, Deuterium-Labeled Hexanal 13a
Acid (16R)-[2H3]-9a
aReagents and conditions: (a) (1) KN[Si(CH3)3]2, THF, -78 °C, (2)
oxoester 19, (3) aldehyde 13; (b) LiOH, THF, H2O.
aReagents and conditions: (a) Jacobsen’s (R,R) catalyst, H2O; (b)
CH2dCHCH2MgBr, CuI, THF, -40 °C; (c) Et3N, MsCl, CH2Cl2, -78 °C;
(d) LiAl2H4, THF, -78 °C; (e) Wilkinson’s catalyst,2H2, benzene; (f) Pd-C,
H2, CH2Cl2; (g) IBX, CH2Cl2, reflux.
opening of the epoxide at C(3) with allylmagnesium bromide in
the presence of CuI at low temperatures17 provided (2S)-
1-(benzyloxy)hex-5-en-2-ol 15. The enantiomeric purity of 15
(er=96.3 ( 0.1) was determined by GLC-MS of diastereo-
isomeric carbamates formed by 15 with 1-phenylethyl
isocyanate.18 Stereospecific introduction of the deuterium label
was achieved by a two-step mesylation/reduction procedure
using LiAl2H4 without isolating the reactive mesylate.19,20 The
displacement of the mesylate of a secondary alcohol by a
deuteride from LiAl2H4 proceeds with complete inversion of
configuration at the stereocenter.20 To confirm inversion at the
stereocenter also in the mesylate of the benzyloxy alcohol 15,
the absolute configuration of deuterated hexanol 18 was
determined by 1H NMR after oxidation21 to deuterated
hexanoic acid and esterification with methyl (S)-mandelate.22
If a chiral R-deuterated carboxylic acid is esterified with
(S)-mandelate, the acid pro-S proton resonates at high fields
of the pro-R proton.22 1H NMR of the methyl (S)-mandelate
diester of [2,5,6-2H3]-hexanoic acid displays a major signal at
2.20-2.10 ppm and a small signal at 2.30-2.30 ppm, confirm-
ing the (R)-configuration of 18. The optical purity of 18 cannot
be determined with this method because the procedure for the
oxidation to hexanoic acid promotes enolization of the labeled
aldehyde intermediate.
(2R)-[2,5,6-2H3]-hexanol (18) without concomitant scram-
bling. Oxidation with IBX25 in CH2Cl2 afforded the labeled
hexanal 13 and allowed to isolate the volatile aldehyde in
moderate yield. The second carbonyl component, namely
methyl 8-oxooct-(5Z)-enoate (19), was obtained from com-
mercially available δ-valerolactone 20 by a one-pot methano-
lysis and oxidation (Scheme 4, see the Supporting Infor-
mation). Methyl 5-oxopentanoate 21 was olefinated with the
C3-homologating agent 2-(1,3-dioxan-2-yl)ethyltriphenylpho-
sphonium bromide (22),26 resulting in methyl 7-(1,3-dioxan-
2-yl)hept-(5Z)-enoate (23). Methanolysis gave the correspond-
ing dimethyl acetal 24, and hydrolysis in pentane/formic acid
resulted in the target β,γ-unsaturated 8-oxoester 19 with the
correct stereochemistry (Z/E ratio >95:5, GLC-MS). The
sequential addition14 of carbonyl 19 and 13 to a solution of the
bis-ylide of 12 gave the labeled fatty acid methyl ester 25 after
HPLC purification. The low yield of the double olefination
could be possibly due to the instability of the homoconjugated
ester ylide intermediate formed by the bis-ylide of 12 and β,γ-
unsaturated oxoester 19, as preliminary experiments with non
labeled, saturated carbonyls gave higher yields. Methyl ester 25
was hydrolyzed in small batches shortly before the incubation
experiments in order to avoid autoxidation, and resulted in
(16R)-[16,19,20-2H3]-arachidonic acid (16R)-[ 2H3]-9.
The additional two deuterium atoms were introduced in
the chiral, deuterated benzyl ether 16 by deuteration with
Wilkinson’s catalyst23 to avoid deuterium scrambling.24 The
benzyl group of 1-((2R)-[2,5,6-2H3]-hexyloxy)methylbenzene
(17) was removed by hydrogenolysis with Pd(C) yielding
The metabolic probe (16R)-[2H3]-9 was obtained with
good isotopic and stereoisomeric purity. The isotopic purity
(d2:d3:d4 = 1:98:1) of the fatty acid methyl ester 25 was
determined by GLC-MS comparing the molecular ion
traces of the d1-d5 isotopomers corrected for the contribu-
tion of the natural abundance of 13C.
(17) Huynh, C.; Derguini-Boumechal, F.; Linstrumelle, G. Tetrahedron
Lett. 1979, 20, 1503–1506.
(18) Habel, A.; Spiteller, D.; Boland, W. J. Chromatogr. A 2007, 1165,
182–190.
(19) Abad, J. L.; Serra, M.; Camps, F.; Fabrias, G. J. Org. Chem. 2007,
72, 760–764.
(20) Abad, J. L.; Camps, F.; Fabrias, G. J. Org. Chem. 2000, 65, 8582–
8588.
(21) Thottumkara, A. P.; Bowsher, M. S.; Vinod, T. K. Org. Lett. 2005, 7,
2933–2936.
(22) Parker, D. J. Chem. Soc., Perkin Trans. 2 1983, 83–88.
(23) Osborn, J. A.; Jardine, F. H.; Young, J. F.; Wilkinson, G. J. Chem.
Soc. Inorg. Phys. Theor. 1966, 1711–1732.
Transformation of (16R)- and (16S)-[16,19,20-2H3]-
Arachidonic Acids in Gametes of Ectocarpus siliculosus. The
typical volatiles released from suspensions of female gametes
of E. siliculosus were ectocarpene (>95%), dictyotene
(24) Thyman, J. H. P.; Crombie, L. Synthesis in Lipid Chemistry; The
Royal Society of Chemistry: Cambridge, 1996.
(25) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001–3003.
(26) Stowell, J. C.; Keith, D. R. Synthesis 1979, 132–134.
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SCHEME 4. Reaction Pathways and Kinetic Isotope Effect (KIE) in E. siliculosus
SCHEME 5. Biosynthesis of Dictyotene (4) in Gametes of
E. siliculosus
FIGURE 2. GLC separation of (6R)- and (6S)-dictyotene. GLC-
separation of dictyotene enantiomers was achieved on Hydrodex-β-
6-TBDMS: (a) dictyotene from gametes of E. siliculosus; (b) dictyo-
tene reference from G. parvulum.
(ca. 3-4%), and trace amounts of the linear hydrocarbon
cystophorene (<1%). Dictyotene 4 is a direct metabolite
of arachidonic acid and proved to be of high er (97.9 ( 0.3),
which demonstrates that its biosynthesis from arachidonic
acid proceeds with high stereoselectivity (Figure 2). The
main product ectocarpene (5), a metabolite of the higher
unsaturated eicosapentaenoic acid, was not resolved on
this column.
The overall stereochemical course (Scheme 5) of the
transformation of arachidonic acid into dictyotene (4) and
the linear hydrocarbon cystophorene 1 was investigated by
incubating algal gametes with (16S)- and (16R)-[2H3]-9. The
resulting C11 hydrocarbons were collected by SPME and
analyzed by GLC-MS.
Figure 3 (lane a) illustrates the result for endogenous,
nonlabeled arachidonic acid 9, which yielded the two C11H16
hydrocarbons dictyotene (4) (m/z 150) and a small amount of
the linear hydrocarbon cystophorene (1) (m/z 150; Figure 3,
lane a). Administration of (16R)-[2H3]-9 resulted in the
release of [2H2]-4 as the major metabolite, demonstrating
that a single deuterium atom had been removed from the
terminal segment of the precursor acid (Figure 3, m/z 152,
lane b). Moreover, the genuine product ratio of dictyotene (4)
to cystophorene (1) (Figure 3, lane a) changed dramatically in
favor of cystophorene (1) (Figure 3, lane c). On the contrary,
administration of (16S)-[2H3]-9 resulted in the formation of
[2H3]-4 and did not affect the original product distribution of
dictyotene and cystophorene (not shown). Accordingly, the
product shift was due to the presence of a pro-R deuterium
atom at C(16) of the precursor acid.
These observations are perfectly in line with the stereo-
chemical and mechanistic course of the biosynthesis of
dictyotene (4) in the diatom G. parvulum9 and of ectocarpene
(5) in the higher plant S. isatidaeus.10 In all these organisms,
the C11 hydrocarbons were generated from the aliphatic part
of unsaturated fatty acids via thermolabile cis-divinylcyclo-
propane intermediates (Scheme 5). The latter rearrange in a
highly concerted manner to the thermally stable dictyotenes
(4).8,10,13 Moreover, in diatoms the isotopic substitution of
the abstracted hydrogen atom by a deuterium atom led to a
significant shift in the product spectrum.9 This is consistent
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JOCFeatured Article
FIGURE 3. GLC-MS analysis of labeled C11H16 metabolites.
Lane a: natural ratio of dictyotene (4) and cystophorene (1) in
female gametes of E. siliculosus. Lane b: selective monitoring of the
ion trace m/z 152 representing the major metabolite of (16R)-[2H3]-
9. Lane c: selective monitoring of the ion trace m/z 153 demonstrat-
ing the shift toward the linear hydrocarbon cystophorene (1) after
administration of (16R)-[2H3]-9.
FIGURE 4. Identification of 9-oxononadienoic acid 11 as the polar
metabolite.
TABLE 1. Calculation of the Kinetic Isotope Effect (KIE) on Dictyotene
C (4) Formation
pentafluorobenzylhydroxylamine (PFBHA) to the incuba-
tion medium containing [2H8]-arachidonic acid and female
gametes of E. siliculosus allowed to trap the reactive aldehyde
as the PFB-oxime (Figure 4). After esterification (CH2N2),
the polar metabolite was identified by GLC-MS using an
authentic reference.8 The mass spectrum showed the molec-
ular ion of the O-(2,3,4,5,6)-pentafluorobenzyl oxime (PFB-
oxime) of [2H4]-11 at m/z 381, the PFB fragment ion at m/z
181, and a pyridinium ion at m/z 280, which is characteristic
for R,β,γ,δ-unsaturated aldehydes.29 Since the fragment ions
are shifted by four mass units compared to the unlabeled
reference (m/z 377, 276) the origin from the administered
precursor [2H8]-arachidonic acid is obvious. Using chemical
ionization (CI) in the negative mode confirmed the presence
of [2H4]-11 by intense [M - HF]•- signals of the PFB-oximes
(Supporting Information).
Mechanistic Aspects and Stereochemistry of Algal Phero-
mone Biosynthesis. The identification of 9-oxononadienoic
acid 11 as the second fragment of arachidonic acid en route
to the C11H16 hydrocarbon dictyotene (4) demonstrates the
principal similarity of this transformation in brown algae
and diatoms. In the diatom G. parvulum, arachidonic acid (9)
is first activated by a lipoxygenase to the (9S)-hydroperoxide
10 (Scheme 5). Protonation and loss of water could induce a
Hock-Criegee-type rearrangement30,31 yielding the polar
fragment 11 along with the thermolabile dictyopterene C
(8). The cis-bisalkenylcyclopropane 8 rearranges sponta-
neously in a highly coordinated fashion and with complete
chirality-transfer to the cycloheptadiene 4.32 Since the in-
volved enzymes are still unknown, the initial step of the
oxidative fragmentation of the precursor 10, namely the rate-
limiting removal of the hydrogen atom or the activation of
the hydroperoxy group, remains to be established. The high
relative ratio
(16S)-[2H3]-9/(16R)-[2H3]-9
[2H3]-4/[2H2]-4
KIE (kH/kD)
5.4 ( 0.6
0.039 ( 0.001
0.21 ( 0.02
with a strong primary kinetic isotope effect (KIE) associated
with the removal of the hydrogen isotope. In the present
study, the KIE was calculated from the ratio of the peak
areas (corrected for the natural abundance of 13C) of [2H3]-4
and [2H2]-4 obtained after administration of (16R)-[2H3]-9
(Table 1). The ratio of the two precursors (16S)-[2H3]-9 and
(16R)-[2H3]-9 was calculated from the er (96.3 ( 0.1) of the
metabolic probe (16R)-[2H3]-9.19 The procedure implies that
the secondary isotope effect caused by presence of a (16S)
deuterium atom in the precursor (16S)-[2H3]-9 can be
neglected27 for the abstraction of the C(16) pro-R hydrogen
atom. Moreover, the rate of the subsequent cyclization of
[2H3]-8 to [2H3]-4 is largely unaffected by deuterium sub-
stitution at the double bonds.28
The product shift observed after administration of
(16R)-[2H3]-9 is caused by the rather strong KIE associated
with the abstraction of the hydrogen atom from C(16). If the
pro-R position is occupied by a deuterium atom, abstraction
of a hydrogen atom from C(13) is the preferred reaction (no
KIE) and generates cystophorene (1) instead of dictyotene
(4) (Scheme 5). The order of the KIE (5.4 ( 0.6) is in line
with previous data for the same reaction in the diatom
G. parvulum9 and confirms that the hydrogen abstraction is
generally the rate-limiting step in the enzymatic transformation
of the precursor acid to algal pheromones.
Identification of the Polar Metabolite of the Oxidative
Cleavage of Arachidonic Acid. In the diatom, G. parvulum
9-oxononadienoic acid 11 was identified as the polar frag-
ment of the oxidative cleavage of arachidonic acid en route
to dictyotene (4). In algal gametes or higher plants the
polar fragment has not been observed as yet. Addition of
(29) Spiteller, G.; Kern, W.; Spiteller, P. J. Chromatogr. A 1999, 843, 29–
98.
(30) Gerwick, W. H. Chem. Rev. 1993, 93, 1807–1823.
(31) Gardner, H. W.; Weisleder, D.; Nelson, E. C. J. Org. Chem. 1984, 49,
508–515.
(27) Rickert, K. W.; Klinman, J. P. Biochemistry 1999, 38, 12218–12228.
(28) Ozkan, A. l.; Zora, M. J. Org. Chem. 2003, 68, 9635–9642.
(32) Nubbemeyer, U. Synthesis 2003, 961–1008.
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JOCFeatured Article
er of dictyotene (4) supports an arrangement of the arachi-
donic acid precursor as outlined in Scheme 5.
ensure tightness, was applied through the septum. The mixture
was purged with deuterium for 1 min, warmed to 18 °C, and
stirred. After 45 min, the flask turned an intense dark red color,
and after 8 h the gas balloon was removed. The solution was
concentrated at reduced pressure; the catalyst was precipitated
with hexane and filtered over Celite. The solvent was evaporated
under reduced pressure, and the residue was dissolved in hexane,
filtered through a silica pad, and concentrated under reduced
pressure to give a colorless liquid (1.41 g, 75%).
The U-shaped orientation of the precursor can be easily
adopted by other fatty acids, for example, by eicosa-
5,8,11,14,17-pentaenoic acid or eicosa-5,8,11,14,17,19-hex-
aenoic acid both of which are known to occur in brown
algae.33 Eicosapentaenoic acid is the precursor of ectocar-
pene (5), and the eicosahexaenoic acid is the logic precursor
of desmarestene (6-buta-1,3-dienylcyclohepta-1,4-diene),
which acts as a potent gamete-releasing and gamete-attracting
pheromone in the brown alga Desmarestia aculeata.34 Evolu-
tionary modifications of the active center may have
created environments that favor other conformations of
the precursor acid which could generate the families of
trans-bisalkenylcycloporpanes, cyclopentene, and cyclo-
hexene hydrocarbons35 according to the same principal
mechanism. The simultaneous formation of a pheromone
and 9-oxononadienoic acid (11) from a single precursor
such as arachidonic acid represents an exiting example of
“atom-economy”36 in nature, since dictyotene (4) acts as a
gamete attractant while 9-oxononadienoic acid 11 was
shown to act as a strong deterrent37 and toxin for marine
copepods that feed on algal resources.
1H NMR δ: 7.37-7.27 (m, 5H, aromatics); 4.51 (s, 2H, C(10));
3.47 (d, J = 6.6 Hz, 2H, C(1)); 1.65-1.55 (m, 1H, C(2));
1.40-1.34 (m, 2H, C(3)); 1.32-1.25 (m, 3H, (C(4), C(5));
13
0.90-0.80 (m, 2H, C(6)). C NMR (CDCl3) δ: 138.7 (C(20));
128.3 (C(40)); 127.6 (C(50)); 127.4 (CDJ = 19 Hz; C(5)); 13.6
(
CDJ = 19 Hz; C(6)). MS m/z: 195 (Mþ•, 1); 108 (10); 92 (69); 91
(100); 65 (7). IR (cm-1): 3032, 2924, 2855, 2165 (C-D),
2000-1700, 1602, 1454. HRMS (EI): calcd for C13H17D3O
195.170246, found 195.171005.
(2R)-[2,5,6-2H3]-Hexanal (13). Freshly prepared 2-idoxyben-
zoic acid38 (IBX, 2.36 g, 8.4 mmol) was added to a solution of
labeled hexanol 18 (290 mg, 2.8 mmol) in 15 mL of CH2Cl2 in a
flask with cock. A reflux condenser closed by a balloon filled
with argon was applied, and the suspension was stirred under
reflux. Reaction control measurements were taken by 1H NMR
after precipitation of suspension aliquots (0.5 mL) at -20 °C,
filtration, and evaporation of the solvent. After 15 h, the
suspension was precipitated at -20 °C and filtered over a pad of
Florisil. The solvent was removed at reduced pressure using a
Vigreux column. The colorless liquid obtained contained di-
chloromethane, which was determined to be 36 mol % (31%
w/w) by 1H NMR. The product was used immediately for
olefination (186 mg, 69% w/w, 45%).
Experimental Section
1-((2R)-[2-2H]-Hex-5-enyloxy)methylbenzene (16). Mesyl chlo-
ride (2.52 g, 22.0 mmol) in CH2Cl2 was added dropwise to a
stirred solution of (S)-1-(benzyloxy)hex-5-en-2-ol (15) (2.52 g,
12.2 mmol) and triethylamine (3.70 g, 36.6 mmol) in 50 mL of
CH2Cl2 at -78 °C. After the mixture was allowed to warm to
0 °C and stirred for 30 min, the excess base was neutralized with
2 N HCl. The solution was washed with brine and extracted with
ether, and the combined organic layers were dried (Na2SO4) and
evaporated. The resulting oil was dissolved in 3 mL of dry diethyl
ether in a flame-dried flask, and the solution was cooled to -78 °C
under argon to exclude humidity. After addition of LiAl2H4
(1.54 g, 36.6 mmol), the suspension was stirred for 30 min and
allowed to come slowly to 20 °C overnight. After careful hydrolysis
with water and 1 M sulfuric acid, the mixture was extracted with
diethyl ether. The organic phase was washed with brine, dried
(Na2SO4), and evaporated at reduced pressure to give a colorless
1H NMR δ: 9.76 (d, J = 1.8 Hz, 2H, C(1)); 2.42-2.35 (m, 1H,
C(2)); 1.65-1.69 (m, 2H (C(3)); 1.33-1.22 (m, 3H, C(4), C(5));
0.90-0.82 (m, 3H). 13C NMR (CDCl3) δ: 203.1 (C(1)); 43.5
(t, CDJ = 19 Hz; C(2)); 31.1 (C(4)); 21.9 (CDJ = 19 Hz, C(5));
21.7 (C(3)); 13.4 (CDJ = 19 Hz, C(6)). MS m/z : 103 (Mþ•, 1), 85
(21), 84 (9), 74 (35), 59 (33), 58 (93), 46 (20), 45 (100). HRMS
(EI): calcd for C6H9D3O 103.107645, found 103.108058.
(16R)-[16,19,20-2H3]-Methyl Arachidonate (25). A flame-
dried 50 mL flask with a cock loaded with the (Z)-hex-3-enyl-
1,6-bis(triphenylphosphonium bromide) 12 (927 mg, 1.2 mmol)
was dried under high vacuum to remove traces of water from the
hygroscopic salt. After venting with argon, dry THF (20 mL)
was added through a septum and the suspension was cooled
to -78 °C. A solution of KN[Si(CH3)3]2 (0.91 M, 2.6 mL,
2.4 mmol) in THF was slowly added, and the mixture was
allowed to warm to 0 °C. The orange solution was stirred for
30 min and cooled again to -78 °C. A solution of the oxoester 19
(165 mg, 77% w/w, 1.2 mmol) in 2 mL of THF was added with a
syringe pump over 40 min, and then the mixture was warmed
slowly to 0 °C. After the suspension was cooled again to -78 °C,
a solution of the aldehyde 13 (180 mg, 69% w/w, 1.2 mmol) in
1 mL of THF was added, and the mixture was allowed to warm
to room temperature within 30 min. After the addition of 2 N
HCl and extraction with ether, the organic phase was washed
with brine until a pH of 6 was reached, dried with Na2SO4 in an
ice bath, and evaporated at reduced pressure. The substance was
purified on a silica column (PE/Et2O = 20/1), equipped with a
cooling jacket in darkness. Separation of 25 (Rf 0.32) from
impurities of Ph3PdO and 2,6-di-tert-butyl-4-methylphenol
(Rf 0.36) was incomplete. The mixed fractions were dried and
further purified by semipreparative straight-phase HPLC under
the following conditions: hexane (A)/THF (B) 2% B to 10% B in
20 min, then 90 B % for 6 min; 3 mL/min, UV detection 210 nm.
1
liquid (1.87 g, 80%). H NMR (CDCl3) δ: 7.38-7.28 (m, 5H;
aromatics); 5.86-5.76 (m, 1H, C(5)); 5.12 (m, 1H, C(6)); 4.95 (m,
1H, C(6)); 4.51 (s, 2H, C(10)); 3.48 (d, J = 8 Hz; 2H, C(1));
2.12-2.04 (m, 2H, C(4)); 1.62 (m, 1H, C(2)); 1.49 (m, 2H, C(3)).
13C NMR (CDCl3) δ: 138.8 (C(5)); 138.7(C(20)); (128.4, (C(40));
(127.6 (C(50)); 127.5 (C(30); 114.5, (C(6)); 72.9 (C(10)); 70.2 (C(1));
33.6 (C(4)); 28.9 (t, CDJ = 19 Hz, C(2)); 25.4, (C (3)). MS m/z: 191
(Mþ•, 1), 162 (2), 107 (16), 91 (100), 82 (11), 65 (8). IR (cm-1):
3070, 3031, 2925, 2156 (C-D), 2000-1700, 1640, 1605, 1452.
HRMS (EI): calcd for C13H17DO 191.142042, found 191.142712.
1-((2R)-[2,5,6-2H3]-Hexyloxy)methylbenzene (17). Dry benzene
was degassed using the freeze-thaw method, and then 40 mL was
transferred with a syringe in a flask with a septum and cooled to
0 °C; the headspace was purged with argon to exclude oxygen.
After Wilkinson’s catalyst Rh[P(Ph)3]3Cl (445 mg, 0.48 mmol,
0.05 equiv) and 19 (1.84 g, 9.6 mmol) were added to the solvent,
a balloon filled with deuterium, connected with a Pasteur pipet to
€
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4537–4538.
J. Org. Chem. Vol. 75, No. 12, 2010 3963

Rui and Boland
JOCFeatured Article
The pure product obtained was a colorless liquid (31 mg, 8%).
The isotopic purity was calculated from the molecular ion m/z
321 of the ester 25 and the molecular ions m/z 320 and 322,
respectively, of the [2H2]- and [2H4]-analogues. Extracted ion
chromatograms were obtained by GLC-MS with sector field
analyzer, and the peak areas were corrected for the contribution
of the natural isotopic abundance of 13C. The resulting areas at
m/z 320, 321, 322, attributed, respectively, to d2-, d3-, and d4-
methyl arachidonate, were used for calculating isotopic purity.
1H NMR δ: 5.45-5.30 (m, 8H, olefinic protons, C(5/6/8/9/
11/12/14/15)), 3.67 (s, 3H, C(10)), 2.90-2.75 (broad, 6H, C(7/10/
13)), 2.35 (t, J = 7.6 Hz, 2H, C(2)), 2.14-2.08 (m, 2H, C(4)),
2.07-2.01 (m, 1H, C(16)), 1.71 (quintet, J = 7.6 Hz, 2H, C(3)),
1.4-1.2 (broad, 5H, C(17/18/19)), 0.9-0.8 (broad, 2H, C(20)).
13C NMR δ: 174.03 (C(1)), 130.45 (C(15)), 128.92 (C(5),
128.87 (C(6)), 128.58 (C(12)), 128.20(C(8)), 128.16 (C(14)),
127.86 (C(11)), 127.55 (C(9)), 51.45 (C(10)), 33.44 (C(2)),
31.35 (C(18)), 29.20 (C(17)), 26.86 (t, CDJ = 19 Hz, C(16)),
26.55 (C(4)), 25.63, 25.62, 25.60 (C(7), (C(10)), (C(13)), 24.78
(C(3)), 22.08 (t, CDJ = 19 Hz, C(19)), 13.63 (t, CDJ = 19 Hz,
C(20)). MSm/z: 321 (Mþ•, 5), 180 (27), 153 (35), 120 (22), 119 (28),
106 (52), 106 (52), 105 (42), 93 (56), 91 (69), 80 (85), 79 (100). IR
(cm-1): 3012, 2923, 2854, 2168 (C-D), 1741, 1656, 1438, 1365,
1156. HRMS (EI): calcd for C21H31D3O2 321.274711, found
321.275711. Isotopic purity: d2:d3:d4 =1:98:1; the sum of d0, d1,
and d5 was less than 1%.
(Na2SO4), and the solvent was evaporated. After the residue
was dissolved in 40 μL of hexane, GLC-MS analysis and
external calibration using methyl arachidonate were carried
out. The amount of (16R)-[2H3]-9 in the cell suspension was
determined as ca. 22 μg.
Incubation with [2H8]-9 and Analysis of Oxylipins. Diatom
cells were harvested (see the Supporting Information) and
incubated with a solution containing 6 μg/μL of [5,6,8,9,
11,12,14,15-2H8]-arachidonic acid ([2H8]-9). After SPME, a
water solution of PFBHA (0.75 mL; 25 mmol/L) containing
2,6-di-tert-butyl-4-methylphenol (BHT, 10 μg/mL) was added
to the homogenate and mixed. After 30 min, it was carefully
acidified to pH 3 with diluted H2SO4 and extracted three times
with 0.5 mL of dichloromethane containing BHT (10 μg/mL) in
Eppendorf tubes. After centrifugation, the combined lower
phases were dried (Na2SO4), transferred to a GLC vial, con-
centrated, and treated with diazomethane. After evaporation of
the solvent, the residue was dissolved in dichloromethane,
transferred in a microvial, concentrated, and dissolved in
40 μL of the same solvent. For the incubation of E. siliculosus,
2 mL aliquots of gamete suspension were transferred into 4 mL
screw-cap vials and incubated with 30 μg of [2H8]-9 dissolved in
1 μL of DMSO. After gentle agitation, the suspension was left
for 5 h in a growth chamber at 16 °C to allow complete cell
settling, and at the fifth hour SPME was carried out for 60 min.
After volatile sampling, 0.75 mL of a water solution of PFBHA
(25 mM) containing BHT (10 μg/mL) was added, and the
suspension was sonicated in an ice bath with high power for
30 s with a 0.5 s pulse. The homogenate was acidified, extracted,
and derivatized as described for G. parvulum (see above). After
concentration, the residue was dissolved in a microvial with
40 mL of dichloromethane.
Incubation with (16R)-[2H3]-9 and Analysis of Volatiles. Sus-
pensions of female gametes of E. siliculosus with typical con-
centration 1 ꢀ 107 cells/mL were prepared (see the Supporting
Information). Aliquots of 2 mL were transferred into 4 mL
screw-cap vials and incubated with 30 μg of labeled arachidonic
acid (16R)-[2H3]-9 dissolved in 1 μL of DMSO. After gentle
agitation, the suspension was left for 5 h in a growth chamber at
16 °C to allow complete cell settling, and at the fifth hour solid-
phase microextraction (SPME) was carried out for 60 min. For
the analysis of total fatty acids after a test incubation with
(16R)-[2H3]-9, a modified protocol for the extraction and trans-
esterification of fatty acid was used.39 The gamete suspension
was centrifuged and the pellet was transferred into a sealed
hydrolysis vial and homogenized in 0.5 mL of methanol. Then
0.5 mL of a mixture of acetyl chloride/methanol 1:9 and 0.5 mL
of hexane were added together with 2 μg of d27-myristic acid as
an internal standard. Heating at 100 °C for 10 min caused the
formation of a single phase. After cooling on ice and addition of
1 mL of water, the upper phase was collected and dried
Acknowledgment. We thank Dr. Chuanghai Xia for kindly
€
providing the intermediate 12, Prof. D. G. Muller for the gift of
the strain of E. siliculosus used for the investigations, and
Daniela Schmid for support in algal cultivation techniques
and fruitful discussions. The Max Planck Society is acknowl-
edged for funding.
Supporting Information Available: Synthetic procedures
and characterization data for all compounds (except 13, 16,
17, and 25, reported here), copies of NMR spectra for all
compounds, cultivation of algae, and additional graphics. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(39) Rodriguez-Ruiz, J.; Belarbi, E. H.; Sanchez, J. L. G.; Alonso, D. L.
Biotechnol. Tech. 1998, 12, 689–691.
3964 J. Org. Chem. Vol. 75, No. 12, 2010