Composition of the cloacal gland secretion of tuatara, Sphenodon punctatus

Article abstract of DOI:10.1002/cbdv.200800265

The lipophilic content of the cloacal gland secretion of the tuatara (Sphenodon punctatus) was investigated. GC/EI-MS Analysis of CH₂Cl₂ extracts of the secretions revealed triacylglycerols as major glandular constituents. Twelve major medium-chain fatty acids were found to be conjugated to glycerol in different combinations, resulting in complex mixtures. These acids were identified by transesterification and subsequent derivatization of natural samples, and their structures were verified by synthesis. The natural glycerides contain predominantly three of the following acids: octanoic (A), (E)- and (Z)-oct-4-enoic (B and C, resp.), (4E,6Z)-octa-4,6-dienoic (tuataric acid;D), (R)-2,6-dimethylheptanoic (E), (R)-2,6-dimethylhept-5-enoic (F), (Z)-dec-4-enoic (G), (4Z,7Z)-deca-4,7-dienoic (H), (R)-3,7-dimethyloct-6-enoic (I), (R)-4,8-dimethylnon-7-enoic (J), (2R,6S)-2,6,10-trimethylundec-9-enoic (K), and (2R,5E)-2,6,10-trimethylundeca-5,9-dienoic acids (L). Several additional acids, occurring in trace amounts only, were tentatively identified by MS. The elucidation of the absolute configuration of the acids was performed by GC on chiral phases. Individual tuatara show specific mixtures of glycerides with up to 100 components. The individual mixtures may permit individual recognition because the bouquets seem to be stable over years.

Full text of DOI:10.1002/cbdv.200800265

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Composition of the Cloacal Gland Secretion of Tuatara, Sphenodon punctatus
by Birte Flachsbartha), Matthias Fritzschea), Paul J. Weldonb), and Stefan Schulz*a)
a) Institut fꢀr Organische Chemie, Technische Universitꢁt Braunschweig, Hagenring 30,
D-38106 Braunschweig (phone: þ495313917353; e-mail: stefan.schulz@tu-bs.de)
b) Conservation and Research Center, Smithsonian Institution, 1500 Remount Road, Front Royal,
VA 22630, USA
The lipophilic content of the cloacal gland secretion of the tuatara (Sphenodon punctatus) was
investigated. GC/EI-MS Analysis of CH2Cl2 extracts of the secretions revealed triacylglycerols as major
glandular constituents. Twelve major medium-chain fatty acids were found to be conjugated to glycerol in
different combinations, resulting in complex mixtures. These acids were identified by transesterification
and subsequent derivatization of natural samples, and their structures were verified by synthesis. The
natural glycerides contain predominantly three of the following acids: octanoic (A), (E)- and (Z)-oct-4-
enoic (B and C, resp.), (4E,6Z)-octa-4,6-dienoic (tuataric acid; D), (R)-2,6-dimethylheptanoic (E), (R)-
2,6-dimethylhept-5-enoic (F), (Z)-dec-4-enoic (G), (4Z,7Z)-deca-4,7-dienoic (H), (R)-3,7-dimethyloct-
6-enoic (I), (R)-4,8-dimethylnon-7-enoic (J), (2R,6S)-2,6,10-trimethylundec-9-enoic (K), and (2R,5E)-
2,6,10-trimethylundeca-5,9-dienoic acids (L). Several additional acids, occurring in trace amounts only,
were tentatively identified by MS. The elucidation of the absolute configuration of the acids was
performed by GC on chiral phases. Individual tuatara show specific mixtures of glycerides with up to 100
components. The individual mixtures may permit individual recognition because the bouquets seem to be
stable over years.
Introduction. – Many extant reptiles, including snakes, lizards, and crocodylians,
possess exocrine skin glands that open into or near the cloaca [1–3]. These organs
generally are thought to produce pheromones, e.g., sexual attractants or predator
deterrents [4].
Tuatara, Sphenodon punctatus and S. guntheri, the sole extant representatives of the
order Rhynchocephalia, are long-lived (> 80 yrs), nocturnal, lizard-like animals that
inhabit ca. 30 islands near New Zealand (Fig. 1). These reptiles possess a prominent
skin gland, called the cloacal gland, that opens on each side of the margin of the vent.
This gland is divided by connective tissue septa into lobules packed with cells that stain
positively with periodic acid–Schiff, thus denoting carbohydrates likely associated with
glycoproteins [1][5]. Analyses by gel electrophoresis and MALDI-MS of adult male
and female S. punctatus revealed that the cloacal gland secretion consists chiefly of a ca.
50 kDa glycoprotein [6].
Histochemical studies of cloacal gland epithelial cells reveal that lipids are present
as minor inclusions [1][5]. We report here the results of GC/EI-MS analysis of lipids
from the cloacal gland of S. punctatus. Our study demonstrates triacylglycerols as major
lipoidal components of the cloacal gland secretions, and it elucidates the detailed
structures of the fatty acid components, including novel compounds. Previous studies,
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. a) Sphenodon punctatus from Stephens Island, New Zealand (Photo by J. Sartore). b) Ventral
view of male S. punctatus showing the white secretion of the cloacal gland being expressed after manual
pressure was applied to the base of the tail.
primarily by TLC [7], have indicated triacylglycerols in epidermal and skin gland
extracts of non-avian reptiles. However, the fatty-acid substituents of these compounds
have been examined in only a few studies of the epidermal lipids of snakes [8].
Results and Discussion. – Chromatograms of the cloacal gland secretions of S.
punctatus showed a large number of compounds within a relatively narrow retention-
time range and exhibiting similar mass spectra (Fig. 2,a).
Some characteristics of these spectra are small molecular-ion peaks between m/z
450 and 600 and up to three intensive peak groups between m/z 319 and 487, as well as
peaks at m/z 41, 55, and 69, indicating unsaturations. Since many of the observed ion
peaks occur in different spectra, we assumed that the corresponding compounds are
composed of a small array of building blocks. Therefore, we hypothesized that the
compounds were triacylglycerols. Transesterification of the natural samples with
MeONa confirmed this, because methyl esters of fatty acids were formed, together with
glycerol. We found sixteen major methyl esters of acids A–P, four of which are derived
from the common palmitic, stearic, oleic, and linoleic acids (M–P, resp.). A gas
chromatogram of a transesterified extract is shown in Fig. 2,b. The acids M–P, which
are common tetrapod skin products, are known as components of internal tissue as well
as from eggs of S. punctatus [9][10]. The methyl esters of octanoic (A), (E)-oct-4-enoic
(B), (Z)-oct-4-enoic (C), 2,6-dimethylheptanoic (E), (Z)-dec-4-enoic (G), and
citronellic acids (I) were readily identified via mass-spectral libraries, and their
identities confirmed by comparison with synthetic samples.
Because several esters obviously were unsaturated, a transesterified natural sample
was hydrogenated. The number of peaks decreased, indicating that the acids A–L were
derived from only six skeletons, differing only in the number of C¼C bonds. In addition
to the methyl esters of A and E, methyl decanoate, methyl 3,7-dimethyloctanoate,
methyl 4,8-dimethylnonanoate, and methyl 2,6,10-trimethylundecanoate were identi-
fied, thus elucidating all skeletons of the parent acids.
The C¼C bonds were located by dimethyl disulfide (DMDS) derivatization of
transesterified samples [11], which, in combination with the mass spectra of the natural

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
3
Fig. 2. a) Gas chromatogram of a natural extract of cloacal gland secretion of Sphenodon punctatus.
b) Methyl esters derived from cloacal secretion after transesterification with MeONa. R: Nonanoic acid, S:
decanoic acid, T: dodecanoic acid.
methyl esters, allowed us to determine the positions of the C¼C bonds. Target
compounds were then synthesized and compared with the methyl esters of the natural
acids to confirm the location and configuration of the C¼C bonds. In addition, the
absolute configurations were determined by synthesis and analysis using chiral GC.
Below, the results for each unknown acid are presented in detail.
Methyl Esters of B and C (methyl-B and methyl-C, resp.). Mass spectra of the
DMDS adducts showed characteristic ions at m/z 103 (C4H8SCH3), 147 ([Mꢀ103]þ ),
115 ([Mꢀ103ꢀCH3OH]þ ), and 87 ([Mꢀ103ꢀCH3OHꢀCO]þ ), thus indicating the
C(4)¼C(5) bond. Since the formation of the DMDS adducts is highly stereoselective
[11], the presence of two DMDS derivatives indicated that both (E)- and (Z)-oct-4-
enoic acids occur naturally. Methyl (E)-oct-4-enoate (2) was synthesized by Ireland–
Claisen rearrangement of hex-1-en-3-ol (1) and 1,1,1-trimethoxyethane with propanoic

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
acid as catalyst (Scheme 1) [12]. Compound 2 proved to be identical to the minor
diastereoisomer of the natural extract, methyl-B, while methyl-C is, therefore, methyl
(Z)-oct-4-enoate.
Scheme 1
a) MeC(OMe)3, cat. EtCO2H, toluene; 75%.
Methyl Ester of D (methyl-D). The methyl ester of D eluted after methyl-B and
methyl-C on an apolar phase, thus suggesting a conjugated system of C¼C bonds. This
was supported by the formation of a DMDS derivative expected for conjugated dienes.
Therefore, we assumed that an additional C¼C bond could be located at C(2) or C(6).
While the mass spectra of synthetic methyl octa-2,4-dienoate did not match those of
methyl-D, those of methyl octa-4,6-dienoate did. All four double-bond isomers of
methyl octa-4,6-dienoate were synthesized according to Scheme 2 to determine the
configuration of the natural acid. Bromination of (2E,4E)-hexa-2,4-dien-1-ol (3) with
Br3P gave bromide 4 [13]. Alkylation of dimethyl malonate using this bromide,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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followed by Krapcho de(methoxycarbonylation), furnished methyl (4E,6E)-octa-4,6-
dienoate (6) [14]. The two isomers, methyl (4Z,6E)-octa-4,6-dienoate (11) and methyl
(4Z,6Z)-octa-4,6-dienoate (12), were available starting from pent-4-ynoic acid (7).
After reaction with CH2N2, the methyl ester 8 was used in a Sonogashira coupling with
(E)-1-bromoprop-1-ene or (Z)-1-bromoprop-1-ene, respectively [15][16]. Partial
catalytic hydrogenation with P-2 nickel furnished the desired methyl dienoates 11
and 12 [17]. The fourth double-bond isomer, methyl (4E,6Z)-octa-4,6-dienoate (17),
required a five-step synthesis, starting from pent-4-yn-1-ol (13). After protection of the
OH group as TBDMS ether [18][19], hydroboration of the CꢁC bond of 14 with
benzo[d][1,3,2]dioxaborole, followed by Suzuki coupling with (Z)-1-bromoprop-1-ene,
yielded the protected dienol 15 [20]. After deprotection [19–21], the alcohol 16 was
oxidized using PDC [22], followed by esterification with CH2N2. All four diaster-
eoisomers of methyl octa-4,6-dienoate were separated by GC on an apolar phase.
Compound D proved to be (4E,6Z)-octa-4,6-dienoic acid. Nevertheless, trace amounts
of the (4E,6E)-, (4Z,6Z)-, and (4Z,6E)-diastereoisomers were also present (see
Table 2). Formation of the respective methyl esters during the transesterification
process can be dismissed because treatment of pure 17 under the same conditions did
not furnish isomerization products.
Scheme 2
a) Br3P, CH2Cl2; 76%. b) 1. Dimethyl malonate, NaH, THF; 2. 4; 27%. c) LiCl, H2O, DMSO; 73%. d)
CH2N2, Et2O; 100%. e) (E)- or (Z)-1-Bromoprop-1-ene, [PdCl2(PhCN)2], CuI, piperidine; 85%. f) 1.
AcONi·4 H2O, NaBH4, EtOH; 2. (CH2NH2)2, 9, 10; 20–52%. g) (t-Bu)Me2SiCl (TBDMSCl), 1H-
imidazole, DMF; 74%. h) 1. 1,3,2-Benzodioxaborole; 2. (Z)-1-bromoprop-1-ene, [Pd(PPh3)4], EtONa,
toluene; 27%. i) Bu4NF, THF; 58%. j) pyridinium dichromate (PDC), DMF; 28%. k) CH2N2, Et2O;
35%.
The configuration of the C¼C bonds was confirmed by GC/IR. In vapor-phase IR
spectra, (Z)-double bonds show an absorbance at 3010–3030 cmꢀ1, while (E)-double

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
bonds can be identified by a band at 965–985 cmꢀ1 [23]. GC/IR Analysis showed that
the four isomers of methyl octa-4,6-dienoate are distinguished by the intensities of the
absorption of the (E)-double bond. The (all-Z)-isomer 12 shows no absorption in this
range, the (4Z,6E)-isomer 11 (which absorbs at 980 cmꢀ1) and the (4E,6Z)-isomer 17
(979 cmꢀ1) show nearly the same intensity relative to the absorption of the C¼O bond,
while the intensity of the (all-E)-isomer 6 (absorption at 983 cmꢀ1) is doubled. All four
compounds additionally show bands in the region around 3020 cmꢀ1. The (all-E)-
isomer 6 absorbs at 3019 cmꢀ1. The (4Z,6E)-isomer 11 shows an absorption at
3023 cmꢀ1, and the (4E,6Z)-isomer 17 at 3027 cmꢀ1. The relatively broad absorption
band at 3026 cmꢀ1 for the (all-Z)-isomer 12 can be explained by overlapping
absorptions of both (Z)-double bonds. The major natural dienoate showed an IR
spectrum consistent with the (4E,6Z)-configuration, thus confirming our double-bond
geometry assignment of the natural acid D.
Methyl Esters of E and F (methyl-E and methyl-F, resp.). The determination of the
absolute configuration of both acids required enantiomerically enriched samples.
Therefore, both methyl (R)-2,6-dimethylhept-5-enoate (21) and methyl (R)-2,6-
dimethylheptanoate (22) were obtained from commercially available (ꢀ)-(R)-b-
citronellene (18) according to Scheme 3. Diene 18 was mono-epoxidized at the higher
substituted C¼C bond with meta-chloroperbenzoic acid (mCPBA) to give compound
19. Ozonolysis and removal of the epoxide protecting group [24] furnished aldehyde 20,
which was oxidized using silver oxide [25]. The resulting acid F was esterified with
CH2N2 leading to 21. Hydrogenation of 21 using Pd/C as catalyst yielded 22. This
synthesis confirmed the identity of natural acids E and F, and provided pure
enantiomers needed for the determination of the absolute configuration of the natural
acids (see below).
Scheme 3
a) meta-Chloroperbenzoic acid (mCPBA), CHCl3; 93%. b) 1. O3, CH2Cl2; 2. Zn, AcONa·3 H2O, NaI,
AcOH; 85%. c) 1. AgNO3, NaOH, H2O; 2. 20; 42%. d) CH2N2, Et2O; 80%. e) H2, Pd/C, Pentane; 52%.
Methyl Esters of G and H (methyl-G and methyl-H, resp.). The mass spectrum of
the DMDS adduct of methyl-G revealed the location of the double bond at C(4),
showing characteristic ions at m/z 131 ([C6H12SCH3]þ ), 147 ([CH3OCOC3H5SCH3]þ ),
and 115 ([147ꢀCH3OH]). The IR spectrum suggested a (Z)-configuration of the C¼C
bond (no absorption at 965–985 cmꢀ1, but at 3014 cmꢀ1, characteristic for (Z)-double

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
7
bonds). This proposal was confirmed by synthesis of methyl (Z)-dec-4-enoate. The
ester was obtained from (Z)-dec-4-enal by oxidation with silver oxide, followed by
esterification with MeOH using BF3 ·Et2O as catalyst [26].
Structure elucidation of compound H was more difficult. The DMDS adduct
formed from the corresponding ester methyl-H contained a cyclic thioether with
unknown ring size, so methyl deca-4,6-dienoate was synthesized, but proved not to be
identical to the target compound. The IR spectrum pointed to a (Z,Z)-geometry of the
C¼C bonds. Therefore, methyl (4Z,7Z)-deca-4,7-dienoate (26) was synthesized in
three steps (Scheme 4). Transformation of 1-bromopent-2-yne (23) with NaI [27] gave
iodide 24 that was coupled with the copper acetylide of 8 [28]. The resulting diyne ester
25 was then partially hydrogenated using P-2 nickel to obtain the desired methyl ester
26. Both synthetic methyl esters were identical to those of the natural acids G and H.
Scheme 4
a) NaI, Acetone; 88%. b) 8, CuI, Bu4NCl, Na2CO3, DMF; 39%. c) 1. AcONi·4 H2O, NaBH4, EtOH;
2. (CH2NH2)2, 25; 20%.
Methyl Ester of J (methyl-J). The synthesis of (S)-4,8-dimethylnon-7-enoic acid
(30) started from (ꢀ)-(S)-citronellol (27), which was tosylated with TsCl, converted to
the nitrile 29 using NaCN, and hydrolyzed with NaOH to yield the acid 30 (Scheme 5),
as previously reported for (R)-30 by Mori and Tamada [29]. The methyl ester of this
compound proved to have the same constitution as methyl-J and could be used for the
determination of the absolute configuration of the natural acid (see below).
Scheme 5
a) TsCl, Pyridin; 72%. b) NaCN, DMSO; 89%. c) NaOH, 60% aq. EtOH; 94%. d) CH2N2, Et2O; 76%.
Acid K and Its Methyl Ester (methyl-K). Racemic methyl 2,6,10-trimethylundec-9-
enoate (35) was obtained by the addition of the Grignard reagent of citronellyl
bromide (33), obtained from 32 [30], to methyl methacrylate [31]. The identity of K

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
was thus confirmed. For the determination of the configuration of its stereogenic
centers, several stereoisomers were synthesized as depicted in Scheme 6 according to
Evans methodology. By using methyl acrylate instead of methyl methacrylate, methyl
(6RS)-6,10-dimethylundec-9-enoate (36) was obtained. The chiral Evans auxiliary (S)-
4-benzyloxazolidin-2-one was then introduced after saponification, guiding the
following diastereoselective methylation at C(2) [32]. Alkaline hydrolysis of 44
afforded 47, a mixture of (2S,6S)- and (2S,6R)-2,6,10-trimethylundec-9-enoic acids.
Similarly, the use of (R)-4-benzyloxazolidin-2-one furnished the respective mixture of
(2R,6S)- and (2R,6R)-2,6,10-trimethylundec-9-enoic acids (48). The same synthetic
strategy, starting from (S)-citronellyl bromide (34), furnished pure (2R,6R)-2,6,10-
trimethylundec-9-enoic acid (49). The synthesized acids were used to determine the
absolute configuration of the natural compounds (see below).
Scheme 6
a) PPh3, Br2, Pyridin, CH2Cl2; 99%. b) 1. Mg, THF; 2. 4-(Dimethylamino)pyridine (DMAP), CuBr,
Me2S, Me3SiCl (TMSCl), methyl methacrylate or methyl acrylate, THF; 34–57%. c) NaOH, H2O; 69–
94%. d) Et3N, Pivaloyl chloride, (S)- or (R)-4-benzyloxazolidin-2-one, BuLi, THF; 76–94%. e) Sodium
hexamethyldisilazane (¼sodium bis(trimethylsilyl)amide; NaHMDS), MeI, THF; 55–78%. f) LiOH,
H2O, THF; 42–69%. g) CH2N2, Et2O; not estimated.
Methyl Ester of L (methyl-L). Methyl (S)-2,6,10-trimethylundeca-5,9-dienoate (65)
was synthesized via Wittig olefination using methyl 5-oxopentanoate (53) and (1,5-

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
9
dimethylhex-4-enyl)triphenylphosphonium bromide (Scheme 7), which was generated
in situ from (5-methylhex-4-enyl)triphenylphosphonium bromide (59). The carbonyl
compound 53 was derived from tetrahydro-2H-pyran-2-one (52) through methanolysis,
followed immediately by oxidation of the alcohol with PCC [33]. The phosphonium salt
59 was prepared in five steps. First, 1-bromo-3-methylbut-2-ene (54) was transformed,
according to standard methodology, to ethyl 5-methylhex-4-enoate (56). The ester 56
was reduced with LiAlH4, brominated with Br2/PPh3, and transformed to the Wittig salt
59 [34]. After the Wittig reaction and hydrolysis, again Evans methodology was used to
introduce stereoselectively the Me group at C(2) [32]. After hydrolysis and
methylation, 65 was formed. The product was a mixture of (E)- and (Z)-isomers.
GC/MS and NMR analyses as well as coinjection revealed that the naturally occurring
compound L is (E)-2,6,10-trimethylundeca-5,9-dienoic acid.
Scheme 7
a) 1. H2SO4, MeOH; 2. Pyridinium chlorochromate (PCC), CH2Cl2; 49%. b) Diethyl malonate, NaH,
THF; 86%. c) LiCl, H2O, DMSO; 51%. d) LiAlH4, EtO2; 88%. e) Ph3P, Br2, Pyridine, CH2Cl2; not
estimated. f) PPh3; 61%. g) 1. BuLi, MeI; 2. BuLi, 53; 41%. h) NaOH, H2O; 89%. i) Et3N, Pivaloyl
chloride, (S)-4-benzyl-2-oxazolidinone, BuLi, THF; 88%. j) NaHMDS, MeI, THF; 80%. k) KOH, H2O,
THF; 22%. l) CH2N2, Et2O; 77%.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Configuration of Stereogenic Centers. The determination of the configuration of the
stereogenic centers of the methyl-branched fatty acids was performed by chiral GC on
three different stationary phases. Some compounds could be separated as free acids,
while others were separable as methyl esters. The separation results are summarized in
Table 1.
Table 1. GC Retention Times (tR ) of Enantiomers in Chiral Separations. Values indicated in italics denote
naturally occurring stereoisomers.
Compound
tR [min]
(R) (S) (2S,6RS) (2R,6RS) (2RS,6R) (2RS,6S)
Methyl 2,6-dimethylheptanoatea)
35.81 36.47
39.96 40.52
47.65 50.80
54.79 55.04
Methyl 2,6-dimethylhept-5-enoatea)
Methyl 3,7-dimethyloct-6-enoateb)
4,8-Dimethylnon-7-enoic acida)
2,6,10-Trimethylundec-9-enoic acida)
Methyl 2,6,10-trimethylundec-9-enoatec)
Methyl 2,6,10-trimethylundeca-5,9-dienoateb) 34.18 34.81
36.37
36.77
214.23
214.29
a) Hydrodex-6-TBDMS (6-O-TBDMS-2,3-di-O-methyl-b-cyclodextrin, 35 mꢂ0.25 mm). b) 3-O-Dec-
anoyl-2,6-di-O-methyl-b-cyclodextrin (15 mꢂ0.32 mm). c) Lipodex G (6-O-methyl-2,3-di-O-pentyl-g-
cyclodextrin, 50 mꢂ0.25mm).
Generally, the difference of the retention times of the stereoisomers of monofunc-
tional aliphatic compounds decreases with increasing distance between functional (acid
or ester) group and the aliphatic stereogenic center. Therefore, the assignment of the
configuration at C(6) of K was a major problem. This was solved using two different
phases (Fig. 3). Each phase allowed separation of the four stereoisomers into two
peaks. The free acid K was separated on a Hydrodex-6-TBDMS-b-cyclodextrin phase
(2,3-di-O-methyl-6-O-TBDMS-b-cyclodextrin) into the (2R,6RS)- and (2S,6RS)-
isomers. Because natural K eluted as single peak, its configuration was either
(2R,6R) or (2R,6S). The ester methyl-K was separated on a Lipodex G phase.
Surprisingly, C(2)-epimers did not separate, in contrast to the C(6)-epimers. The
compound methyl (2R,6RS)-2,6,10-trimethylundec-9-enoate (50) was separated into
two peaks. Because natural methyl-K eluted as second peak, natural K could be either
(2R,6S)- or (2S,6R)-configurated. Combination of both results confirmed that the
natural acid K occurs only as the (2R,6S)-enantiomer.
On the basis of these results, the major glycerol-bound fatty acids of the cloacal
gland are octanoic (A), (E)- and (Z)-oct-4-enoic (B and C, resp.), (4E,6Z)-octa-4,6-
dienoic (D), (R)-2,6-dimethylheptanoic (E), (R)-2,6-dimethylhept-5-enoic (F), (Z)-
dec-4-enoic (G), (4Z,7Z)-deca-4,7-dienoic (H), (R)-3,7-dimethyloct-6-enoic (I), (R)-
4,8-dimethylnon-7-enoic (J), (2R,6S)-2,6,10-trimethylundec-9-enoic (K), and
(2R,5E)-2,6,10-trimethylundeca-5,9-dienoic acids (L). Several additional acids, occur-
ring only in trace amounts, were tentatively identified by MS. They are listed in Table 2.
Several of these acids or their methyl esters are known from nature. While A is
widespread, C occurs in guava (Psidium guajava) [35], pawpaw (Asimina triloba) [36],
yellow mombin fruits (Spondias mombin) [37], and in baga (Annona glabra) [38]. The

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
11
Fig. 3. Determination of the absolute configuration of both stereogenic centers of natural 2,6,10-
trimethylundec-9-enoic acid (K) by chiral GC on Hydrodex-6-TBDMS (1508, 2 min, then 18/min to 1908)
(a–e) and of the methyl ester of K on Lipodex G (908, 210 min, then 208/min to 2008) (f–j) stationary
phases. a) Natural sample after saponification. b) Synthetic rac-2,6,10-trimethylundec-9-enoic acid (38).
c) Coinjection of compounds tested in a and b. d) Synthetic (2R,6RS)-2,6,10-trimethylundec-9-enoic acid
(48). e) Coinjection of compounds tested in b and d. f) Natural sample after transesterification. g)
Synthetic methyl (2R,6RS)-2,6,10-trimethylundec-9-enoate (50). h) Coinjection of compounds tested in
f and g. i) Synthetic methyl (2R,6R)-2,6,10-trimethylundec-9-enoate (51). j) Coinjection of compounds
tested in g and i.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 2. Fatty Acids from Cloacal Gland Triacylglycerols of Sphenodon punctatus, Identified as Methyl
Esters after Transesterification of Natural Samples. The relative amounts of three different individuals are
given (SPU-2: Big Guy, male; SPU-3: Gans, male; SPU-4: female).
Entry
Compound
Ia)
Sampleb)
SPU-2
SPU-3
SPU-4
1
2
3
4
5
6
7
8
9
4-Methylpentanoic acid
Heptanoic acid
(E)- Oct-4-enoic acid (B)
(Z)- Oct-4-enoic acid (C)
2,6-Dimethylheptanoic acid (E)
Octanoic acid (A)
2,6-Dimethylhept-5-enoic acid (F)
(4Z,6E)- Octa-4,6-dienoic acid
(4E,6E)- Octa-4,6-dienoic acid
(4E,6Z)- Octa-4,6-dienoic acid (D)
(4Z,6Z)- Octa-4,6-dienoic acid
Nonanoic acid (R)
3,7-Dimethyloct-6-enoic acid (I)
Benzenepropanoic acid
(Z)- Dec-4-enoic acid (G)
(4Z,7Z)- Deca-4,7-dienoic acid (H)
Decanoic acid (S)
893
nd
0.87
nd
0.69
1028
1115
1118
1124
1131
1153
1177
1180
1184
1188
1224
1258
1279
1304
1305
1317
1366
1520
1559
1586
1600
1712
1727
1906
1928
2030
2061
2098
2105
2112
2132
2265
2302
2306
2338
2429
2513
2538
2740
2812
2943
nd
0.05
10.86c)
3.12c)
9.11d)
4.94
2.69
16.69
0.05
0.19
6.54
0.66
nd
nd
nd
19.40
nd
nd
nd
nd
nd
25.83
1.25
nd
nd
nd
4.30
nd
nd
3.38
2.64
nd
0.62
traces
nd
nd
nd
nd
nd
20.08
4.27
0.33
2.02
18.56
0.03
0.06
9.57e)
5.10e)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
0.16
0.37
3.88
0.16
11.99f)
2.67
0.65
1.58
0.27
nd
0.39
33.87
0.12
0.13
0.12
1.24
nd
0.09
0.55
0.39
0.09
0.19
0.26
nd
0.41
0.10
11.08
0.36
5.25g)
0.48
17.39
0.18
0.06
17.51
0.99
0.08
0.40
0.84
4.34
nd
4,8-Dimethylnon-7-enoic acid (J)
Dodecanoic acid (T)
6,10-Dimethylundec-9-enoic acidh)
2,6,10-Trimethylundec-9-enoic acid (K)
(E)-2,6,10-Trimethylundeca-5,9-dienoic acid (L)
3,7,11-Trimethyldodeca-6,10-dienoic acidh)
Tetradecanoic acid
Hexadecenoic acid
Palmitic acid (M)
Heptadecanoic acid
2,6,10,14-Tetramethylpentadeca-5,9,13-trienoic acidh)
Linoleic acid (N)
Oleic acid (O)
Octadecenoic acid
Stearic acid (P)
Icosatetraenoic acid
Icosadienoic acid
Icosenoic acid
Icosanoic acid
Henicosanoic acid
Docosenoic acid
Docosanoic acid
Tetracosanoic acid
Squalene
nd
2.20
1.75
0.67
0.82
0.72
0.25
0.15
0.10
nd
nd
nd
0.08
nd
0.10
nd
0.03
0.01
0.02
0.02
0.01
0.58
nd
nd
nd
nd
nd
Hexacosanoic acid
a) GC Retention index of methyl ester. b) nd: Not detected. c) Integration includes Entries 3 and 4
because of incomplete separation. d) Integration includes Entries 3, 4, and 5 because of incomplete
separation. e) Integration includes Entries 9 and 10 because of incomplete separation. f) Integration
g
includes coeluting unesterified octa-4,6-dienoic acid. ) Integration includes Entries 15 and 16 because of
incomplete separation. h) Tentative identifications by interpretation of tR and MS data.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
13
methyl esters of A and B occur in pineapple [39]. The norterpenoid E has been found
in peppermint oil [40], while its unsaturated analog F (with unknown configuration)
was found in the skin extracts of the opisthobranch mollusc, Melibe leonina [41], and in
the sex-attracting secretion of the dung beetle, Kheper lamarcki [42]. Compound (S)-F
is the major volatile constituent of the sex-attracting secretion of another dung beetle,
K. subaeneus [24]. Citronellic acid I is widespread in nature. The monounsaturated acid
G was found in the thrips, Holurothrips morikawai [43]. Methyl-G occurs in secretions
of the sternal glands from male cotton stainers (Dysdercus cingulatus) [44]; in Asian
pears (Pyrus serotina) and flowers of Michelia champaca, it is accompanied by methyl-
A [45][46], and in carabao mangoes (Mangifera indica) by methyl-H [47]. The
norsesquiterpene K was reported from roots of the black pepper (Piper nigrum) [48].
Methyl-L is a male-produced sex pheromone component of the stink bugs, Chlorochroa
ligata and C. uhleri [49].
The two acids D and J have not been reported from nature before, to the best of our
knowledge. While J is a degraded sesquiterpene acid, compound D, for which we
propose the name tuataric acid, shows a unique conjugated double-bond system.
It should be noted that the fatty-acid composition as well as the amounts of single
acids vary from one natural sample to another. Seven of the eleven analyzed samples
contain all twelve acids A–L (see Table 2), while the other samples lack certain acids.
Synthesis of Triacylglycerols. Model compounds were needed for the analysis of
intact triacylglycerols. Therefore, three triacylglycerols 66–68 made up of the acids A,
F, and I were synthesized as shown in Scheme 8. The positions of the acids on the
glycerol were verified by 2D-NMR spectroscopy.

14
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 8
a) Acid, N,N’-dicyclohexylcarbodiimide (DCC), DMAP, CH2Cl2; 89–93%. b) BCl3, CH2Cl2; 47–48%.
c) TBDMSCl, 1H-imidazole, THF; 62–68%. d) Acid, DCC, DMAP, CH2Cl2; 37–49%. e) 1. HCl,
MeOH, CH2Cl2; 2. Acid, DCC, DMAP, CH2Cl2; 53–76%.
Initially, 4-(hydroxymethyl)-2,2-dimethyl-1,3-dioxolane (69) was esterified with
one acid. The acetal group was cleaved, and the primary OH group was protected as
TBDMS ether. The second acid was then added to the secondary alcohol. After
removal of the silyl protecting group, the last OH group was esterified with the third
fatty acid.
Identification of Triacylglycerols. The identification of natural triacylglycerols
proved to be difficult because complex mixtures of glycerides with up to three different
acids are produced by the tuatara (Fig. 4). Triacylglycerols can be identified by their EI
mass spectra (Figs. 5 and 6) [50]. The Mþ peak is usually very small, but three groups of
characteristic peaks can be detected. The loss of one acyloxy group furnishes fragments
x ([MꢀRCOO]þ ), accompanied by [MꢀRCOOꢀH]þ in varying relative intensities.
Fragment y is formed by sequential loss of a neutral acylꢀH fragment and an
additional acyloxy group ([Mꢀ(RCOꢀH)ꢀRCOO]þ ). The third diagnostic group z
are acyl ions, often accompanied by [RCOꢀH]þ in the case of unsaturated fatty acids.
The identification is difficult, because the intensities of the fragments x, y, and z can
vary widely, depending on the presence of C¼C bonds, position on the glycerol, and
branched methyl groups near the ester group. In addition, side-chain fragmentation

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
15
induced by the C¼C bonds present can hamper identification. Information about the
masses of the triacylglycerols can also be obtained by CI-MS using NH3 as reactant gas.
This method leads to an abundant [Mþ18]þ ion (Fig. 5,b).
Investigation of the compounds 66–68 showed that the position of the acids on the
glycerol backbone cannot be derived from the EI or CI mass spectra.
Analysis of the Natural Glycerides. A big problem for the identification of the
natural triacylglycerols was the incomplete separation of the sometimes more than 100
glycerides present in the secretions. Many triacylglycerols coelute and make
identification by the described method difficult because of overlapping spectra.
Tuatara individuals contain specific mixtures of glycerides, with differences in the
quantity of the acids present. The gas chromatograms of secretions of three individuals
are shown in Fig. 4. Many of the glycerides contain three different acids, resulting in
rather complex mixtures. In a first attempt, we tried to identify the composition of the
major peaks using the diagnostic fragments x, y, and z, and results from CI-MS
investigations. The results are shown in Table 3. The composition of the glycerides is
given by a three letter code, e.g., A/D/E indicating that the three acids A, D, and E are
present in this glyceride. Acids B and C as well as G and I cannot be differentiated by
this method, because they have identical molecular masses. No information about the
position and absolute configuration of the glyceride was obtained. Further detailed
analysis using silver-ion micro-chromatography or argentation HPLC leading to
fractions differing in the number of C¼C bonds [51] will be reported elsewhere.
The results indicate that esterification with glycerol occurs combinatorially with all
acids occurring in a given individual. The acids present may differ among individuals
qualitatively and quantitatively, resulting in individual profiles of the triacylglycerol
mixtures. Samples from one individual taken two years apart exhibited nearly identical
glyceride patterns, thus suggesting remarkable stability of the bouquet. However,
whether the triacylglycerol mixture of the cloacal glands contributes to chemical
recognition of individuals is unclear, as is the more general issue of the use of chemical
cues in the social interactions of tuatara [52]. Besides communication, other functions
are possible. For example, many terpenoid compounds as well as certain middle-chain
fatty acids similar to those identified exhibit antimicrobial activity [53].
We thank the Fonds der Chemischen Industrie for financial support. W. Card, R. Hartdegen (Dallas
Zoo, Texas), K. Bekker, B. Flanagan, J. Herman, G. Lipps, A. Odum (Toledo Zoo, Ohio), R. Goellner,
and P. Taylor (St. Louis, Missouri) permitted access to tuatara in their collections. M. Wanner (Saint
Louis Zoo, Missouri), J. Sartore, and J. Greff (Tonal Vision LLC, Baltimore, Maryland) assisted in
obtaining figures.
Experimental Part
General. Chemicals were purchased from Acros (Belgium), Alfa Aesar (Germany), Fluka
(Switzerland), Merck (Germany), Sigma-Aldrich (Germany), or Strem (France). Solvents were purified
by distillation and dried according to standard methods. Reaction progress was monitored by TLC with
0.2-mm pre-coated plastic sheets (Polygram Sil G/UV254; Marcherey-Nagel). Compounds were detected
by the use of a molybdatophosphoric acid soln. (10% in EtOH), followed by heat-gun treatment or by
UV inspection (254 nm). Column chromatography (CC): Kieselgel 60 (Merck); solvent mixtures given in
volume ratios. Optical rotations: Dr. Kernchen Propol Digital Automatic Polarimeter. NMR Spectra:
Bruker DRX-400 spectrometer at 400 (1H) or 100 MHz (13C); chemical shifts d in ppm relative to Me4Si
as internal standard, coupling constants J in Hz.

16
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 4. Gas chromatograms of cloacal gland secretion of Sphenodon punctatus. a) SPU-2 (male) 1. b)
SPU-3 (male). c) SPU-4 (female).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
17
Fig. 4 (cont.)
GC/EI-MS. Experiments were carried out on a HP 6890 GC system connected to a HP 5973 mass-
selective detector (Hewlett-Packard) fitted with a BPX-5 fused-silica cap. column (25 mꢂ0.22 mm i.d.,
0.25-mm film thickness; SGE). Conditions were as follows: He carrier gas, 1 ml minꢀ1; injection volume,
1 ml; transfer line, 3008; electron energy, 70 eV. Identification of compounds was performed by
comparison of mass spectra to the Wiley-7 Library and the Essential Oils Library (Massfinder), or by
comparison with synthetic standards.
GC/CI-MS. Experiments were carried out on a HP 5890A series GC system (Hewlett-Packard)
connected to a TSQ 700 triple quadrupole mass spectrometer (Finnigan) fitted with a ZB-1 anal. cap.
column (30 mꢂ0.32 mm i.d., 0.25-mm film thickness; Phenomenex). He was used as the carrier gas at
1.6 ml mꢀ1. The injection volume was 1 ml. The mass spectra were recorded in the positive-ion mode using
NH3 as a reagent gas.
GC/IR. Experiments were carried out on a HP 6890 series GC system connected to a HP 5965B IR
detector (Hewlett-Packard) fitted with a HP-5 fused-silica cap. column (30 mꢂ0.32 mm i.d., 0.25-mm
film thickness; Hewlett-Packard), He served as carrier gas at 2 ml mꢀ1
.
GC on Chiral Stationary Phase. Separations of enantiomers were carried out on a 8000Top GC
(Thermoquest) fitted with a chiral fused-silica cap. column and a FID. Conditions were as follows: H2
carrier gas, 1 ml minꢀ1; injection volume, 1 ml. Three different stationary phases were used: Hydrodex-6-
TBDMS (2,3-Di-O-methyl-6-O-TBDMS-b-cyclodextrin, 35 mꢂ0.25 mm i.d., 0.25-mm film thickness;
Macherey-Nagel), 2,6-Di-O-methyl-3-O-decanoyl-b-cyclodextrin (15 mꢂ0.32 mm i.d., 0.25-mm film
thickness), and Lipodex G (2,3-Di-O-pentyl-6-O-methyl-g-cyclodextrin, 50 mꢂ0.25 mm i.d.; Macherey-
Nagel).
Sample Collection. Cloacal gland secretions were obtained from four male and two female S.
punctatus originating from Stephens Island, New Zealand, and maintained at the Dallas Zoo, Texas, and
Saint Louis Zoo, Missouri. Animals were housed singly or in pairs, and were fed crickets and mice. All

18
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 5. Mass spectra of synthetic glycerides 66–68. a) EI-MS and b) CI-MS of 66. c) EI-MS and d) CI-MS
of 67. e) EI-MS and f) CI-MS of 68.
secretion donors were wild caught, except for one female that was hatched in captivity. Subjects ranged in
age from 13 to 44 years old (snout-vent lengths 23–27 cm). Samples were obtained in May and July;
several individuals were sampled repeatedly over two years.
Secretions were collected by placing manual pressure on the base of the tail, causing the gland ducts
to evert at the lateral margins of the vent. Additional pressure applied to the gland resulted in the
expression of a white secretion, which was taken up on the tip of a pipette. The pipette tip was broken off
into glass vials, several milliliters of CH2Cl2 were added to each vial, and the samples were cooled on dry
ice and stored cool until analysis.
Transesterification. The natural sample (5 ml), abs. CH2Cl2 (50 ml), and MeONa (100 ml, 0.5m in
MeOH) were heated to 508 for 30 min. After cooling, glacial AcOH (10 ml) was added, and the mixture

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
19
Fig. 6. Mass spectra of natural glycerides a) BC/F/F, b) D/F/GI, c) D/F/H, and d) BC/L/L

20
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 3. Composition of Glycerides in the Natural Secretion of Sphenodon punctatus. The capital letters
refer to the acids A–L. Acids with identical mass cannot be differentiated (B and C, G and I). No
semiquantitative data are given because large peak overlap makes quantification impossible.
Entry
Triacylglyceride
Mr [g/mol]
Ia)
Sample
SPU-2
SPU-3
SPU-4
1
2
3
4
5
6
7
8
9
A/BC/BC
F/F/F
466
506
480
494
496
480
498
480
494
482
494
478
480
492
464
494
522
492
524
508
510
462
476
478
490
506
520
492
508
538
606
460
474
492
494
506
508
522
524
492
506
504
506
508
518
506
2726
2726
2738
2738
2818
2851
2858
2902
2902
2905
2924
2952
2952
2952
2954
2954
2956
2970
2974
3013
3013
3017
3017
3017
3017
3063
3063
3076
3076
3076
3082
3084
3084
3101
3101
3101
3101
3123
3123
3130
3130
3140
3140
3140
3140
3144
ꢂ
ꢂ
A/BC/F
BC/E/F
BC/E/E
BC/BC/E
A/E/E
A/BC/F
BC/E/F
A/BC/E
D/E/E
BC/BC/F
A/BC/F
BC/F/F
BC/BC/BC
A/F/F
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
E/F/GI
D/E/F
ꢂ
ꢂ
ꢂ
E/E/GI
BC/E/GI
A/E/GI
BC/BC/D
BC/D/F
A/D/F
D/F/F
BC/F/GI
F/F/GI
BC/BC/GI
BC/E/GI
E/E/J
E/J/K
BC/D/D
D/D/F
BC/BC/G
A/BC/G
BC/F/G
A/F/G
BC/E/J
A/E/J
BC/BC/GI
BC/F/GI
BC/F/H
BC/F/GI?
A/F/GI
F/F/H
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
D/E/GI
ꢂ

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
21
Table 3 (cont.)
Entry
Triacylglyceride
Mr [g/mol]
Ia)
Sample
SPU-2
SPU-3
SPU-4
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
F/G/G
D/E/G
F/F/K
BC/BC/J
A/BC/J?
A/A/J
F/F/J
BC/F/J
A/F/J
BC/D/GI
BC/D/J
D/F/GI
BC/F/G
D/E/J
BC/D/H
D/F/H
F/GI/J
534
560
576
506
508
510
534
520
522
490
504
504
506
520
488
502
532
506
506
550
534
532
580
488
534
536
520
564
566
516
518
522
518
534
536
564
518
562
518
562
548
590
516
518
546
560
562
532
3147
3147
3147
3165
3165
3165
3165
3169
3169
3198
3198
3198
3198
3198
3214
3214
3217
3234
3234
3234
3238
3248
3252
3262
3275
3275
3296
3296
3296
3313
3313
3313
3341
3341
3341
3341
3358
3358
3377
3377
3384
3384
3394
3394
3394
3394
3394
3406
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
A/D/J
BC/BC/J?
E/GI/J
F/GI/GI
F/GI/H
E/E/K
D/D/GI
BC/GI/J
A/GI/J
BC/GI/GI
BC/E/K
A/E/K
D/GI/H?
BC/GI/H
A/G/G
D/GI/GI
BC/GI/J
A/GI/J
E/J/J
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
D/GI/GI
D/E/K
D/G/G
BC/F/K
BC/J/J
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
A/J/L
D/GI/H
D/GI/GI
BC/BC/L
BC/F/K
A/F/L
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
D/GI/J
ꢂ

22
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 3 (cont.)
Entry
Triacylglyceride
Mr [g/mol]
Ia)
Sample
SPU-2
SPU-3
SPU-4
95
96
97
98
E/GI/K
D/E/L
BC/D/K
D/F/K
E/GI/L
D/GI/J
BC/GI/K
A/GI/K
F/GI/K
BC/D/L
D/F/L
F/GI/L
GI/J/J
BC/GI/L
A/GI/L
E/J/K
BC/GI/K
A/GI/K
D/D/L
BC/J/K
A/J/K
BC/GI/L
A/GI/L
F/H/L
592
560
546
560
590
532
576
578
590
544
558
588
576
574
576
606
576
578
542
590
592
574
576
586
572
574
604
574
602
588
590
590
588
572
570
574
618
586
602
600
632
632
634
632
630
632
628
630
3406
3407
3413
3413
3435
3452
3452
3452
3452
3466
3466
3494
3496
3497
3497
3508
3518
3518
3523
3558
3558
3565
3565
3565
3576
3576
3583
3586
3593
3600
3600
3602
3625
3634
3645
3645
3662
3666
3666
3676
3722
3728
3728
3733
3764
3772
3797
3799
ꢂ
ꢂ
ꢂ
ꢂ
99
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
BC/H/L
A/H/L
G/G/K
D/GI/K
F/J/L
BC/J/L
A/J/L
J/J/J
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
D/J/K
D/GI/L
D/H/L
BC/GI/L
GI/J/K
D/J/L
GI/GI/L
GI/H/L
BC/K/K
BC/K/K
A/K/K
A/K/L?
BC/K/L
J/J/K
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
BC/L/L
D/K/K
ꢂ
ꢂ

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
23
Table 3 (cont.)
Entry
Triacylglyceride
Mr [g/mol]
Ia)
Sample
SPU-2
SPU-3
SPU-4
143
144
145
146
147
148
149
150
A/L/L
630
628
602
660
628
626
656
654
3808
3808
3822
3832
3835
3879
3909
3986
ꢂ
ꢂ
ꢂ
BC/L/L
GI/GI/L
GI/K/K
D/K/L
D/L/L
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
GI/L/L
H/L/L
a) GC Retention index.
was diluted with H2O (250 ml) and extracted with pentane (2ꢂ350 ml). The combined org. phases were
dried (NaSO4), and the solvent was allowed to evaporate at r.t. to a volume of ca. 50 ml [54].
Saponification. The natural sample (20 ml) was placed in a vial, and the solvent was evaporated.
NaOH Soln. (200 ml, 1m in H2O) was added, and the mixture was heated to 1108 overnight. After cooling,
the mixture was diluted with H2O (200 ml) and extracted with CH2Cl2 (350 ml) to remove impurities. The
aq. phase was acidified with conc. HCl and extracted with CH2Cl2 (2ꢂ350 ml). The combined org. phases
were dried (NaSO4), and the solvent was evaporated to a volume of ca. 50 ml.
Silylation. The natural sample (20 ml) and 2,2,2-trifluoro-N-methyl-N-(trimethylsilyl)acetamide
(MSTFA) (50 ml) were heated to 508 for 30–60 min. Solvent and excess reagent were removed in a
stream of N2 at 508, and the residue was taken up in CH2Cl2.
DMDS Adducts. The transesterified natural sample (20 ml) was placed in a vial, and the solvent was
allowed to evaporate at r.t. Dimethyl disulfide (DMDS) (170 ml) and I2 soln. (35 ml, 5% in Et2O) were
added, and the mixture was heated to 1108 overnight. After cooling, the mixture was diluted with pentane
(200 ml). Excess I2 was reduced by shaking with aq. sodium thiosulfate soln. After separation of the
phases, the aq. phase was extracted with pentane (300 ml). The combined org. phases were dried (NaCl),
and the solvent was removed in a stream of N2. The residue was taken up in pentane (100 ml) [11].
Methyl (E)-Oct-4-enoate (2). 1,1,1-Trimethoxyethane (5.00 ml, 39 mmol) and propanoic acid (50 ml,
0.67 mmol) were added to a soln. of hex-1-en-3-ol (1; 1.00 g, 10 mmol) in abs. toluene (20 ml), and the
mixture was heated to reflux for 48 h. After removal of solvent and excess 1,1,1-trimethoxyethane in
vacuo, the residue was purified by CC (SiO2; pentane/Et2O 9 :1) to afford pure 2 (1.17 g, 75%). Colorless
1
liquid [12]. H-NMR (400 MHz, CDCl3): 5.50–5.35 (m, HꢀC(4), HꢀC(5)); 3.67 (s, MeO); 2.37 (m,
2 HꢀC(3)); 2.32 (t, J¼6.4, 2 HꢀC(2)); 1.95 (q, J¼7.2, 2 HꢀC(6)); 1.35 (m, 2 HꢀC(7)); 0.88 (t, J¼7.3,
Me). 13C-NMR (100 MHz, CDCl3): 173.7 (s, C(1)); 131.6 (d); 128.1 (d); 51.4 (q, MeO); 34.6 (t); 34.2 (t);
27.9 (t); 22.5 (t); 13.5 (q, Me(8)). EI-MS (70 eV): 156 (8, Mþ ), 125 (30), 124 (74), 113 (9), 96 (69), 85
(48), 82 (70), 81 (39), 74 (100), 67 (83), 59 (41), 55 (95), 54 (37), 43 (30), 41 (64), 39 (45).
(3R)-6,7-Epoxy-3,7-dimethyloct-1-ene (19). (ꢀ)-b-Citronellene (18) was epoxidized according to the
method of Burger et al. [24]. Yield: 6.45 g (93%). The product was a 1:1 mixture of the (6R)- and (6S)-
1
diastereoisomers. [a]2D2 ¼ ꢀ5.3 (c¼4.5, Et2O). H-NMR (400 MHz, CDCl3): 5.74–5.63 (m, HꢀC(2));
5.00–4.92 (m, 2 HꢀC(1)); 2.72–2.69 (m, HꢀC(6)); 2.21–2.13 (m, HꢀC(3)); 1.58–1.32 (m, 2 HꢀC(4),
2 HꢀC(5)); 1.31 (d, J¼0.7, Me); 1.26 (d, J¼0.9, Me); 1.02 (d, J¼6.8, MeꢀC(3)). 13C-NMR (100 MHz,
CDCl3): 144.1/144.0 (d, C(2)); 113.1/112.9 (t, C(1)); 64.5/64.4 (d, C(6)); 37.7/37.5 (d, C(3)); 33.3/33.1 (t,
C(4)); 26.7/26.5 (t, C(5)); 24.9 (q); 20.3/20.1 (q, MeꢀC(3)); 18.7/18.6 (q). Diastereoisomer with GC tR of
9.01 min: EI-MS (70 eV): 139 (1), 111 (2), 95 (7), 85 (7), 81 (100), 67 (33), 59 (38), 55 (49), 43 (21), 41
(40), 39 (28). Diastereoisomer with GC tR of 9.07 min: EI-MS (70 eV): 139 (2), 111 (2), 95 (7), 85 (7), 81
(100), 67 (33), 59 (37), 55 (47), 43 (19), 41 (38), 39 (26).

24
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
(R)-2,6-Dimethylhept-5-enal (20). Compound 20 was prepared from 19 by ozonolysis, followed by
reductive workup, as described by Burger et al. [24]. Yield: 593 mg (85%). [a]2D2 ¼ ꢀ16.6 (c¼2.0, Et2O).
1H-NMR (400 MHz, CDCl3): 9.62 (d, J¼2.0, 1 HꢀC(1)); 5.08 (tquint., 3J¼7.2, 4J¼1.4, 1 HꢀC(5)); 2.39–
4
2.30 (m, HꢀC(2)); 2.09–1.99 (m, 2 HꢀC(4)); 1.81–1.72 (m, 1 HꢀC(3)); 1.69 (d, J¼1.3, 3 HꢀC(7));
1.60 (d, J¼0.7, MeꢀC(6)); 1.44–1.36 (m, 1 HꢀC(3)); 1.09 (d, J¼7.0, MeꢀC(2)). 13C-NMR (100 MHz,
CDCl3): 205.2 (d, C(1)); 132.7 (s, C(6)); 123.4 (d, C(5)); 45.8 (d, C(2)); 30.7 (t, C(3)); 25.7 (q, C(7)); 25.3
(t, C(4)); 17.7 (q, MeꢀC(6)); 13.3 (q, MeꢀC(2)). EI-MS (70 eV): 140 (5, Mþ ), 82 (100), 69 (35), 67 (63),
55 (25), 53 (11), 41 (63), 39 (24).
(R)-2,6-Dimethylhept-5-enoic acid (F). According to the methods of Pearl and Bailey et al. [25], 20
(491 mg, 3.50 mmol) was added dropwise to a freshly prepared suspension of silver oxide (1.14 g,
4.92 mmol) in NaOH soln. (8.4 ml, 1m in H2O). The mixture was heated to 608 for 3 h. After cooling, the
precipitated silver and excess silver oxide was filtered off. The filter residue was washed with aq. NaOH
soln. and H2O. The combined aq. layers were extracted with Et2O, acidified with conc. HCl, and extracted
consecutively with CH2Cl2 and Et2O. The combined org. layers were washed with brine, dried (MgSO4),
and the solvent was removed. Purification of the residue by CC (SiO2; pentane/Et2O 9 :1) furnished pure
F (227 mg, 42%). Colorless liquid. [a]D22 ¼ ꢀ10.8 (c¼2.9, Et2O). 1H-NMR (400 MHz, CDCl3): 5.09
3
4
(tquint., J¼7.2, J¼1.4, HꢀC(5)); 2.51–2.39 (m, HꢀC(2)); 2.06–2.00 (m, 2 HꢀC(4)); 1.79–1.65 (m,
1 HꢀC(3)); 1.68 (d, 4J¼1.1, 3 HꢀC(7)); 1.60 (d, J¼0.6, MeꢀC(6)); 1.50–1.41 (m, 1 HꢀC(3)); 1.18 (d,
J¼7.0, MeꢀC(2)). 13C-NMR (100 MHz, CDCl3): 182.5 (s, C(1)); 132.3 (s, C(6)); 123.5 (d, C(5)); 38.8 (d,
C(2)); 33.5 (t, C(3)); 25.65 (q, C(7)); 25.59 (t, C(4)); 17.6 (q, MeꢀC(6)); 16.8 (q, MeꢀC(2)).
Preparation of Methyl Esters. A freshly prepared soln. of CH2N2 in Et2O was added dropwise to a
soln. of the corresponding acid (i.e., D, F, I, 7, or 64) in Et2O (1.3m), until the soln. remained pale yellow,
and no more N2 was evolved. The mixture was washed with aq. NaOH soln. (1m) and H2O, dried
(MgSO4), and the solvent was removed to afford the esters (i.e., methyl-I, 8, 17, 21, or 65, resp.) as
colorless liquids.
Methyl (R)-3,7-Dimethyloct-6-enoate (methyl-I). Yield: 84 mg (77%). [a]2D2 ¼ þ8.4 (c¼4.3, Et2O).
3
4
1H-NMR (400 MHz, CDCl3): 5.09 (tquint., J¼7.1, J¼1.4, HꢀC(6)); 3.67 (s, MeO); 2.32 (dd, J¼14.7,
J¼5.9, 1 HꢀC(2)); 2.12 (dd, J¼14.7, J¼8.2, 1 HꢀC(2)); 2.02–1.92 (m, HꢀC(3), 2 HꢀC(5)); 1.68 (d,
J¼1.2, 3 HꢀC(8)); 1.60 (d, J¼0.5, MeꢀC(7)); 1.39–1.17 (m, 2 HꢀC(4)); 0.94 (d, J¼6.7, MeꢀC(3)).
13C-NMR (100 MHz, CDCl3): 173.7 (s, C(1)); 131.5 (s, C(7)); 124.3 (d, C(6)); 51.3 (q, MeO); 41.6 (t,
C(2)); 36.8 (t, C(4)); 30.0 (d, C(3)); 25.7 (q, C(8)); 25.4 (t, C(5)); 19.6 (q, MeꢀC(3)); 17.6 (q, MeꢀC(7)).
EI-MS (70 eV): 184 (15, Mþ ), 152 (71), 137 (10), 129 (7), 110 (86), 95 (87), 82 (44), 74 (31), 69 (100), 59
(32), 55 (54), 41 (90), 39 (32).
1
Methyl Pent-4-ynoate (8). Yield: 2.05 g (95%). H-NMR (400 MHz, CDCl3): 3.71 (s, MeO); 2.59–
2.48 (m, 2 HꢀC(2), 2 HꢀC(3)); 1.99 (t, 4J¼2.5, HꢀC(5)). 13C-NMR (100 MHz, CDCl3): 172.1 (s, C(1));
82.4 (s, C(4)); 68.9 (d, C(5)); 51.7 (q, MeO); 33.0 (t, C(2)); 14.2 (t, C(3)). EI-MS (70 eV): 112 (2, Mþ ),
111 (18), 97 (10), 84 (29), 81 (39), 69 (9), 59 (15), 53 (100), 43 (34), 39 (23).
1
Methyl (4E,6Z)-Octa-4,6-dienoate (17). Yield: 11 mg (35%). H-NMR (400 MHz, CDCl3): 6.41–
6.34 (m, HꢀC(5)); 5.99–5.93 (m, HꢀC(6)); 5.67–5.60 (m, HꢀC(4)); 5.42 (dq, J¼7.1, J¼10.8,
3
4
HꢀC(7)); 3.68 (s, MeO); 2.46–2.35 (m, 2 HꢀC(2), 2 HꢀC(3)); 1.73 (dd, J¼7.1, J¼1.7, 3 HꢀC(8)).
13C-NMR (100 MHz, CDCl3): 173.5 (s, C(1)); 131.5 (d, C(4)); 129.1 (d, C(6)); 126.5 (d, C(5)); 125.0 (d,
C(7)); 51.6 (q, MeO); 33.9 (t, C(2)); 28.1 (t, C(3)); 13.3 (q, C(8)). EI-MS (70 eV): 154 (59, Mþ ), 139 (1),
123 (11), 111 (4), 94 (77), 81 (71), 79 (100), 74 (6), 67 (23), 55 (17), 53 (21), 41 (25), 39 (25).
Methyl (R)-2,6-Dimethylhept-5-enoate (21). Yield: 175 mg (80%). [a]2D2 ¼ ꢀ7.1 (c¼1.5, Et2O).
1H-NMR (400 MHz, CDCl3): 5.08 (tquint., 3J¼7.2, 4J¼1.4, HꢀC(5)); 3.67 (s, MeO); 2.49–2.41 (m,
HꢀC(2)); 2.01–1.95 (m, 2 HꢀC(4)); 1.79–1.66 (m, 1 HꢀC(3)); 1.68 (d, 4J¼1.2, 3 HꢀC(7)); 1.59 (d, J¼
0.6, MeꢀC(6)); 1.48–1.36 (m, 1 HꢀC(3)); 1.15 (d, J¼7.0, MeꢀC(2)). 13C-NMR (100 MHz, CDCl3):
177.3 (s, C(1)); 132.2 (s, C(6)); 123.6 (d, C(5)); 51.4 (q, MeO); 38.9 (d, C(2)); 33.8 (t, C(3)); 25.70 (q,
C(7)); 25.69 (t, C(4)); 17.6 (q, MeꢀC(6)); 17.1 (q, MeꢀC(2)). EI-MS (70 eV): 170 (18, Mþ ), 139 (9), 110
(9), 95 (9), 88 (100), 83 (37), 69 (39), 67 (20), 57 (31), 55 (22), 41 (53), 39 (19).
Methyl (2S)-2,6,10-Trimethylundeca-5,9-dienoate (65). Yield: 24 mg (77%). The product was a (E)/
(Z)-mixture. [a]2D2 ¼ þ38.2 (c¼1.4, Et2O). 1H-NMR (400 MHz, CDCl3): 5.12–5.06 (m, HꢀC(5),
HꢀC(9)); 3.67 (s)/3.66 (s) (MeO); 2.50–2.41 (m, HꢀC(2)); 2.09–1.96 (m, 2 HꢀC(4), 2 HꢀC(7),

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
25
2 HꢀC(8)); 1.79–1.65 (m, 3 HꢀC(11), MeꢀC(10)); 1.61–1.58 (m, MeꢀC(6)); 1.48–1.37 (m,
2 HꢀC(3)); 1.150 (d, J¼7.0)/1.147 (d, J¼7.0) ( Me ꢀC(2)). 13C-NMR (100 MHz, CDCl3): 177.3/177.2
(s, C(1)); 135.9 (s); 135.8 (s); 131.6 (s); 131.4 (s); 124.31 (d); 124.29 (d); 124.26 (d); 123.5 (d); 51.4 (q,
MeO); 39.7 (t, C(7) (E)); 39.0/38.9 (d, C(2)); 34.1 (t); 33.8 (t); 31.9 (t, C(7) (Z)); 26.65 (t); 26.57 (t); 25.69
(q); 25.67 (q); 25.56 (t); 25.47 (t); 23.4 (q, MeꢀC(6) (Z)); 17.7 (q); 17.6 (q); 17.1 (q); 17.0 (q); 15.9 (q,
MeꢀC(6) (E)). (Z)-Isomer (GC tR 16.86 min): EI-MS (70 eV): 238 (6, Mþ ), 223 (2), 206 (2), 195 (45),
182 (2), 169 (3), 163 (20), 151 (4), 137 (11), 123 (16), 109 (100), 95 (19), 88 (36), 81 (31), 69 (95), 67
(36), 55 (19), 53 (17), 41 (66). (E)-isomer (GC tR 17.17 min): EI-MS (70 eV): 238 (2, Mþ ), 223 (1), 206
(1), 195 (56), 182 (1), 169 (4), 163 (26), 151 (3), 137 (14), 123 (17), 109 (100), 95 (16), 88 (27), 81 (30),
69 (81), 67 (36), 55 (17), 53 (16), 41 (60).
(R)-2,6-Dimethylheptanoate (22). A cat. amount of Pd on activated charcoal was suspended in
pentane (4.0 ml). Compound 21 (100 mg, 0.59 mmol) was added, and the mixture was stirred in an
autoclave under H2 for 3 h. After filtration, the solvent was removed to afford 22 (82 mg, 82%). Colorless
liquid. [a]2D2 ¼ ꢀ7.5 (c¼0.7, Et2O). 1H-NMR (400 MHz, CDCl3): 3.67 (s, MeO); 2.44 (sext., J¼7.0,
HꢀC(2)); 1.68–1.57 (m, 1 HꢀC(3)); 1.56–1.47 (m, 1 HꢀC(6)); 1.43–1.32 (m, 1 HꢀC(3)); 1.32–1.23
(m, 2 HꢀC(4)); 1.19–1.13 (m, 2 HꢀC(5)); 1.14 (d, J¼7.0, MeꢀC(2)); 0.86 (d, J¼6.6, 3 HꢀC(7),
MeꢀC(6)). 13C-NMR (100 MHz, CDCl3): 177.4 (s, C(1)); 51.4 (q, MeO); 39.5 (d, C(2)); 38.8 (t, C(5));
34.0 (t, C(3)); 27.8 (d, C(6)); 25.0 (t, C(4)); 22.6 (q); 22.5 (q); 17.1 (q, MeꢀC(2)). EI-MS (70 eV): 172 (1,
Mþ ), 157 (3), 141 (5), 129 (8), 115 (4), 101 (35), 97 (8), 88 (100), 82 (2), 69 (13), 59 (15), 57 (23), 55
(21), 43 (22), 41 (32), 39 (11).
(2E,4E)-1-Bromohexa-2,4-diene (4). The bromide 4 was prepared as described by Kim et al. [13] by
bromination of the corresponding alcohol 3 using PBr3 in CH2Cl2. Yield: 6.08 g (76%). EI-MS (70 eV):
162 (6, Mþ with 81Br), 160 (6, Mþ with 79Br), 81 (100), 79 (26), 66 (7), 65 (7), 53 (23), 41 (30), 39 (19).
Dimethyl 2-[(2E,4E)-Hexa-2,4-dienyl]propanedioate (5). According to the method of Cane [14],
dimethyl malonate (4.62 g, 35.0 mmol) was treated with NaH (1.16 g, 38.5 mmol, 80% in mineral oil) in
abs. THF (40 ml) at 08 for 30 min. Compound 4 (5.64 g, 35 mmol) was added to the suspension, and the
mixture was stirred at r.t. for 2 h. After addition of sat. aq. NH4Cl soln., the mixture was extracted with
Et2O. The combined org. layers were dried (MgSO4), and the solvent was removed in vacuo. Purification
of the raw product by CC (SiO2; pentane/Et2O 9 :1) afforded pure 5 (2.02 g, 27%). Colorless liquid.
1H-NMR (400 MHz, CDCl3): 6.10–5.95 (m, HꢀC(3’), HꢀC(4’)); 5.67–5.58 (m, HꢀC(2’)); 5.49–5.42
(m, HꢀC(5’)); 3.73 (s, 2 MeO); 3.43 (t, J¼7.6, HꢀC(2)); 2.64 (t, J¼7.4, 2 HꢀC(1’)); 1.72 (d, J¼6.7,
3 HꢀC(6’)). 13C-NMR (100 MHz, CDCl3): 169.2 (s, C(1)); 133.3 (d, C(3’)); 131.0 (d, C(4’)); 128.7 (d,
C(5’)); 125.9 (d, C(2’)); 52.4 (q, MeO); 51.8 (d, C(2)); 31.8 (t, C(1’)); 17.9 (q, C(6’)). EI-MS (70 eV): 212
(16, Mþ ), 152 (24), 137 (10), 121 (10), 109 (5), 93 (100), 91 (37), 81 (37), 79 (40), 77 (40), 74 (1), 65 (14),
59 (44), 53 (18), 41 (21), 39 (25).
Methyl (4E,6E)-Octa-4,6-dienoate (6). The diester 5 was monodecarboxylated as described by Cane
[14]. A mixture of 5 (1.49 g, 7 mmol), LiCl (0.89 g, 21 mmol), H2O (0.13 g, 7 mmol), and DMSO (13 ml)
was heated under reflux for 5 h. Sat. aq. NH4Cl soln. was added, and the mixture was extracted with Et2O.
The combined org. layers were dried (MgSO4), and the solvent was removed in vacuo to afford 6 (0.79 g,
1
73%). Colorless liquid. H-NMR (400 MHz, CDCl3): 6.09–5.93 (m, 2 H); 5.75–5.47 (m, 2 H); 3.67 (s,
MeO); 2.51–2.30 (m, 2 HꢀC(2), 2 HꢀC(3)); 1.73 (d, J¼6.5, 3 HꢀC(8)). 13C-NMR (100 MHz, CDCl3):
173.5 (s, C(1)); 131.4 (d, C(4)); 131.2 (d, C(5)); 129.1 (d, C(6)); 127.8 (d, C(7)); 51.5 (q, MeO); 33.8 (t,
C(2)); 27.7 (t, C(3)); 17.9 (q, C(8)). EI-MS (70 eV): 154 (25, Mþ ), 123 (5), 111 (3), 94 (49), 81 (58), 79
(100), 77 (42), 74 (5), 67 (31), 59 (27), 55 (21), 53 (29), 51 (21), 42 (36), 41 (41), 39 (52).
Preparation of Methyl Oct-6-en-4-ynoates. According to the method of Alami et al. [16], 8 (2.0 equiv.)
was added to a suspension of (E)- or (Z)-1-bromoprop-1-ene (1.0 equiv.), freshly prepared
PdCl2(PhCN)2 (see [15]; 0.05 equiv.), and CuI (0.1 equiv.) in piperidine (3 ml per mmol bromide)
and stirred at r.t. for 3.5 h. After addition of sat. aq. NH4Cl soln., the mixture was extracted with Et2O.
The combined org. layers were washed with HCl (0.2m), sat. aq. NaHCO3 soln., and twice with H2O,
dried (MgSO4), and the solvent was removed. Purification of the raw products by CC (SiO2; pentane/
Et2O 19 :1) afforded pure 9 and 10, resp., as colorless liquids.
1
(E)-Methyl Oct-6-en-4-ynoate (9). Yield: 0.52 g (85%). H-NMR (400 MHz, CDCl3): 6.06 (dq, J¼
15.7, J¼6.8, HꢀC(7)); 5.44 (dq, J¼15.8, J¼1.9, HꢀC(6)); 3.70 (s, MeO); 2.65–2.49 (m, 2 HꢀC(2),

26
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
2 HꢀC(3)); 1.75 (dd, J¼6.8, J¼1.9, 3 HꢀC(8)). 13C-NMR (100 MHz, CDCl3): 172.4 (s, C(1)); 138.7 (d,
C(7)); 110.7 (d, C(6)); 86.0 (s, C(4)); 79.8 (s, C(5)); 51.7 (q, MeO); 33.5 (t, C(2)); 18.4 (t, C(3)); 15.2 (q,
C(8)). EI-MS (70 eV): 152 (17, Mþ ), 137 (18), 124 (59), 121 (21), 109 (100), 91 (89), 79 (46), 77 (92), 74
(6), 65 (27), 63 (19), 59 (12), 53 (20), 51 (29), 41 (13), 39 (33).
(Z)-Methyl Oct-6-en-4-ynoate (10). Yield: 0.56 g (91%). 1H-NMR (400 MHz, CDCl3): 5.91 (dq, J¼
10.6, J¼6.8, HꢀC(7)); 5.43 (dq, J¼10.6, J¼1.9, HꢀC(6)); 3.70 (s, MeO); 2.76–2.52 (m, 2 HꢀC(2),
2 HꢀC(3)); 1.83 (dd, J¼6.8, J¼1.7, 3 HꢀC(8)). 13C-NMR (100 MHz, CDCl3): 172.4 (s, C(1)); 137.6 (d,
C(7)); 110.0 (d, C(6)); 92.4 (s, C(4)); 77.8 (s, C(5)); 51.7 (q, MeO); 33.6 (t, C(2)); 15.6 (q, C(8)); 15.4 (t,
C(3)). EI-MS (70 eV): 152 (42, Mþ ), 137 (22), 124 (64), 121 (22), 109 (100), 91 (92), 79 (53), 77 (91), 74
(7), 65 (31), 63 (22), 59 (12), 53 (22), 51 (31), 41 (13), 39 (34).
Preparation of Methyl Octadienoates and Methyl Decadienoate. The dienoates 11, 12, and 26 were
prepared by hydrogenation of compound 9, 10, and 25, resp., using P-2 nickel as catalyst, according to a
procedure of Brown et al. [17]. The freshly prepared catalyst (1.0 equiv.) was placed under H2.
Ethylenediamine (2.0 equiv.), and the ester 9, 10, or 25 (8.0 equiv. based on the number of triple bonds),
resp., were added consecutively. The mixture was filtered after completion of the reaction, diluted with
H2O, and extracted with Et2O. The combined org. layers were washed with H2O, dried (MgSO4), and the
solvent was removed. Purification of the raw products by CC (SiO2; pentane/Et2O 19 :1) afforded pure
11, 12, or 26, resp., as colorless liquids.
Methyl (4Z,6E)-Octa-4,6-dienoate (11). Yield: 87 mg (20%). 1H-NMR (400 MHz, CDCl3): 6.37–
6.29 (m, HꢀC(6)); 6.04–5.95 (m, HꢀC(5)); 5.75–5.66 (HꢀC(7)); 5.28–5.21 (m, HꢀC(4)); 3.67 (s,
MeO); 2.52–2.46 (m, 2 HꢀC(3)); 2.42–2.36 (m, 2 HꢀC(2)); 1.78 (dd, 3J¼6.8, 4J¼1.6, 3 HꢀC(8)).
13C-NMR (100 MHz, CDCl3): 173.5 (s, C(1)); 130.2 (d, C(7)); 129.9 (d, C(5)); 126.7 (d, C(4)); 126.6 (d,
C(6)); 51.5 (q, MeO); 34.1 (t, C(2)); 23.2 (t, C(3)); 18.3 (q, C(8)). EI-MS (70 eV): 154 (54, Mþ ), 123
(13), 111 (4), 94 (79), 81 (75), 79 (100), 74 (7), 67 (27), 59 (10), 55 (21), 53 (26), 41 (31), 39 (31).
1
Methyl (4Z,6Z)-Octa-4,6-dienoate (12). Yield: 225 mg (52%). H-NMR (400 MHz, CDCl3): 6.41–
6.24 (m, HꢀC(5), HꢀC(6)); 5.62–5.52 (m, HꢀC(7)); 5.46–5.37 (m, HꢀC(4)); 3.67 (s, MeO); 2.53–2.46
3
4
(m, 2 HꢀC(3)); 2.43–2.36 (m, 2 HꢀC(2)); 1.75 (dd, J¼6.7, J¼1.7, 3 HꢀC(8)). 13C-NMR (100 MHz,
CDCl3): 173.4 (s, C(1)); 128.8 (d, C(4)); 127.0 (d, C(7)); 124.6 (d, C(5)); 124.0 (d, C(6)); 51.5 (q, MeO);
34.0 (t, C(2)); 23.0 (t, C(3)); 13.1 (q, C(8)). EI-MS (70 eV): 154 (41, Mþ ), 123 (10), 111 (3), 94 (74), 81
(67), 79 (100), 74 (6), 67 (25), 59 (10), 55 (20), 53 (25), 41 (30), 39 (31).
1
Methyl (4Z,7Z)-Deca-4,7-dienoate (26). Yield: 0.05 g (20%). H-NMR (400 MHz, CDCl3): 5.44–
5.26 (m, HꢀC(4), HꢀC(5), HꢀC(7), HꢀC(8)); 3.68 (s, MeO); 2.80 (t, J¼7.0, 2 HꢀC(6)); 2.43–2.29
(m, 2 HꢀC(2), 2 HꢀC(3)); 2.11–2.01 (m, 2 HꢀC(9)); 0.97 (t, J¼7.5, 3 HꢀC(10)). 13C-NMR (100 MHz,
CDCl3): 173.6 (s, C(1)); 132.1 (d); 129.7 (d); 127.5 (d); 126.9 (d); 51.5 (q, MeO); 34.0 (t, C(2)); 25.4 (t);
22.7 (t); 20.5 (t); 14.2 (q, C(10)). EI-MS (70 eV): 182 (2, Mþ ), 150 (32), 122 (6), 108 (43), 93 (38), 79
(100), 74 (5), 67 (10), 59 (15), 55 (23), 41 (39), 39 (31).
(tert-Butyl)(dimethyl)(pent-4-ynyloxy)silane (14). According to the method of Akita et al. [18], a
soln. of pent-4-yn-1-ol (13; 2.94 g, 35.00 mmol), 1H-imidazole (4.92 g, 71.00 mmol), and TBDMSCl
(7.97 g, 53.00 mmol) in abs. DMF (20 ml) was stirred at r.t. for 1 h. After addition of brine, the mixture
was extracted with AcOEt/hexane 1:1. The combined org. layers were washed with brine, dried
(MgSO4), and the solvent was removed. Purification of the residue by CC (SiO2; pentane/Et2O 30 :1)
furnished pure 14 (5.11 g, 74%). Colorless liquid. 1H-NMR (400 MHz, CDCl3): 3.70 (t, J¼6.0,
2 HꢀC(1)); 2.27 (dt, 3J¼7.1, 4J¼2.6, 2 HꢀC(3)); 1.92 (t, 4J¼2.7, HꢀC(5)); 1.72 (tt, J¼6.1, J¼7.1,
2 HꢀC(2)); 0.90 (s, t-Bu); 0.06 (s, 2 Me). 13C-NMR (100 MHz, CDCl3): 84.2 (s, C(4)); 68.2 (d, C(5)); 61.4
(t, C(1)); 31.5 (t, C(2)); 25.9 (q, Me3C); 18.3 (s, Me3C); 14.8 (t, C(3)); ꢀ5.4 (q, 2 Me). EI-MS (70 eV):
183 (1), 155 (3), 141 (100), 123 (3), 111 (29), 99 (8), 83 (10), 75 (85), 67 (3), 59 (12), 45 (9), 41 (9).
(tert-Butyl)(dimethyl)[(4E,6Z)-octa-4,6-dienyloxy]silane (15). The protected dienol 15 was pre-
pared from 14 in two steps by a modified published procedure [20]. Instead of (E)-1-bromoprop-1-ene,
1
the (Z)-isomer was used. Yield: 1.48 g (27%). H-NMR (400 MHz, CDCl3): 6.38–6.30 (m, HꢀC(5));
6.00–5.94 (m, HꢀC(6)); 5.66 (dt, J¼7.0, J¼15.1, HꢀC(4)); 5.37 (dq, J¼7.1, J¼10.7, HꢀC(7)); 3.62 (t,
J¼6.4, 2 HꢀC(1)); 2.20–2.14 (m, 2 HꢀC(3)); 1.73 (dd, 3J¼7.1, 4J¼1.7, 3 HꢀC(8)); 1.66–1.58 (m,
2 HꢀC(2)); 0.90 (s, t-Bu); 0.05 (s, 2 Me). 13C-NMR (100 MHz, CDCl3): 133.8 (d, C(4)); 129.5 (d, C(6));
125.7 (d, C(5)); 124.0 (d, C(7)); 62.5 (t, C(1)); 32.5 (t, C(2)); 29.1 (t, C(3)); 26.0 (q, Me3C); 18.3 (s, Me3C);

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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13.2 (q, C(8)); ꢀ5.3 (q, 2 Me). EI-MS (70 eV): 240 (0.1, Mþ ), 225 (3), 183 (73), 165 (2), 153 (25), 141
(14), 127 (8), 115 (8), 108 (36), 101 (31), 93 (65), 89 (54), 79 (30), 75 (100), 67 (9), 59 (23), 53 (8), 45
(8), 41 (21).
(4E,6Z)Octa-4,6-dien-1-ol (16). The deprotection of 15 was achieved as described by Hong et al.
[20]. Yield: 0.43 g (58%). 1H-NMR (400 MHz, CDCl3): 6.41–6.33 (m, HꢀC(5)); 6.00–5.94 (m,
HꢀC(6)); 5.67 (dt, J¼7.0, J¼15.1, HꢀC(4)); 5.45–5.36 (m, HꢀC(7)); 3.67 (t, J¼6.5, 2 HꢀC(1)); 2.24–
2.17 (m, 2 HꢀC(3)); 1.74 (dd, 3J¼7.1, 4J¼1.6, 3 HꢀC(8)); 1.72–1.65 (m, 2 HꢀC(2)). 13C-NMR
(100 MHz, CDCl3): 133.3 (d, C(4)); 129.3 (d, C(6)); 126.0 (d, C(5)); 124.4 (d, C(7)); 62.5 (t, C(1)); 32.3 (t,
C(2)); 29.2 (t, C(3)); 13.3 (q, C(8)).
(4E,6Z)-Octa-4,6-dienoic acid (Tuataric acid, D). The acid D was prepared by standard oxidation of
16 with PDC in abs. DMF [22]. Yield: 87 mg (28%). 1H-NMR (400 MHz, CDCl3): 6.43–6.36 (m,
HꢀC(5)); 6.00–5.93 (m, HꢀC(6)); 5.69–5.61 (m, HꢀC(4)); 5.43 (dq, J¼7.1, J¼10.8, HꢀC(7)); 2.50–
2.40 (m, 2 HꢀC(2), 2 HꢀC(3)); 1.74 (dd, 3J¼7.1, 4J¼1.7, 3 HꢀC(8)). 13C-NMR (100 MHz, CDCl3):
178.9 (s, C(1)); 131.2 (d, C(4)); 129.0 (d, C(6)); 126.7 (d, C(5)); 125.2 (d, C(7)); 33.8 (t, C(2)); 27.8 (t,
C(3)); 13.3 (q, C(8)).
Methyl (Z)-Dec-4-enoate (methyl-G). (Z)-Dec-4-enoic acid (G) was prepared from (Z)-dec-4-enal
by oxidation with silver oxide as described by Pearl [25]. The raw product was methylated according to
the methods of Taber and Hoerrner, and Marshall et al. [26] to furnish methyl-G. Yield: 598 mg (63%).
1H-NMR (400 MHz, CDCl3): 5.40 (m, HꢀC(4), HꢀC(5)); 3.67 (s, MeO); 2.35 (m, 2 HꢀC(2),
2 HꢀC(3)); 2.04 (q, J¼6.5, 2 HꢀC(6)); 1.40–1.20 (m, 2 HꢀC(7), 2 HꢀC(8), 2 HꢀC(9)); 0.89 (t, J¼
6.8, 3 HꢀC(10)). 13C-NMR (100 MHz, CDCl3): 173.5 (s, C(1)); 131.6 (d); 127.2 (d); 51.5 (q, MeO); 34.1
(t, C(2)); 31.5 (t); 29.3 (t); 27.1 (t); 22.8 (t); 22.5 (t); 14.0 (q, C(10)). EI-MS (70 eV): 184 (6, Mþ ), 152
(57), 135 (20), 123 (20), 110 (72), 96 (48), 85 (44), 84 (39), 81 (49), 74 (100), 69 (52), 68 (40), 67 (69), 59
(34), 55 (66), 54 (35), 43 (33), 41 (74), 39 (37).
1-Iodopent-2-yne (24). Similarly to the method of Burnell et al. [27], NaI (8.09 g, 54.0 mmol) was
added to a soln. of 1-bromopent-2-yne (23; 4.41 g, 30.0 mmol) in abs. acetone (18 ml), and the mixture
was heated to reflux for 5 h. After cooling, the mixture was diluted with H2O and extracted with Et2O.
The combined org. layers were washed with diluted aq. NaHSO3 soln. and brine, dried (MgSO4), and the
1
solvent was removed to afford 24 (5.11 g, 88%). Colorless liquid. H-NMR (400 MHz, CDCl3): 3.71 (t,
5J¼2.4, 2 HꢀC(1)); 2.21 (tq, 3J¼7.5, 5J¼2.4, 2 HꢀC(4)); 1.12 (t, 3J¼7.5, 3 HꢀC(5)). 13C-NMR
(100 MHz, CDCl3): 87.9 (s, C(3)); 76.4 (s, C(2)); 13.4 (q, C(5)); 12.8 (t, C(4)); ꢀ16.8 (t, C(1)). EI-MS
(70 eV): 194 (25, Mþ ); 127 (74), 67 (100), 52 (27), 51 (29), 50 (26), 41 (71), 39 (59).
Methyl (4Z,7Z)-Deca-4,7-diynoate (25). The diynoate 25 was prepared from 8 and 24 as described by
Jeffery et al. [28]. Yield: 0.62 g (63%). 1H-NMR (400 MHz, CDCl3): 3.70 (s, MeO); 3.13–3.08 (quint., J¼
2.3, 2 HꢀC(6)); 2.55–2.46 (m, 2 HꢀC(2), 2 HꢀC(3)); 2.17 (tq, J¼2.4, J¼7.5, 2 HꢀC(9)); 1.12 (t, J¼7.5,
3 HꢀC(10)). 13C-NMR (100 MHz, CDCl3): 172.4 (s, C(1)); 81.9 (s); 78.2 (s); 75.4 (s); 73.4 (s); 51.7 (q,
MeO); 33.3 (t, C(2)); 14.6 (t); 13.8 (q, C(10)); 12.3 (t); 9.6 (t, C(6)). EI-MS (70 eV): 178 (1, Mþ ), 177 (6),
163 (21), 147 (15), 135 (31), 121 (32), 119 (33), 117 (35), 103 (37), 91 (100), 77 (62), 74 (10), 65 (29), 63
(29), 59 (18), 51 (31), 41 (27), 39 (33).
(S)-3,7-Dimethyloct-6-enyl Toluenesulfonate (28). The toluenesulfonate 28 was prepared as
described by Mori and Tamada [29], using (ꢀ)-(S)-3,7-Dimethyloct-6-en-1-ol (27) instead of the (R)-
1
3
isomer. Yield: 1.57 g (72%). [a]2D2 ¼ þ175.4 (c¼2.6, Et2O). H-NMR (400 MHz, CDCl3): 7.79 (dt, J¼
8.4, 4J¼1.9, 2 arom. H); 7.34 (d, 3J¼8.5, 2 arom. H); 5.02 (tquint., 3J¼7.1, 4J¼1.4, HꢀC(6)); 4.11–4.02
(m, 2 HꢀC(1)); 2.45 (s, Me of Ts); 2.04–1.05 (m, 2 HꢀC(2), 2 HꢀC(3), 2 HꢀC(4), 2 HꢀC(5)); 1.67 (d,
4J¼1.3, 3 HꢀC(8)); 1.57 (d, 4J¼0.4, MeꢀC(7)); 0.82 (d, 3J¼6.5, MeꢀC(3)). 13C-NMR (100 MHz,
CDCl3): 144.6 (s, C(4) of Ts); 133.2 (s, arom. C); 131.4 (s, arom. C); 129.8 (2 d, arom. C); 127.8 (2 d, arom.
C); 124.3 (d, C(6)); 69.0 (t, C(1)); 36.7 (t); 35.6 (t); 28.8 (d, C(3)); 25.7 (q, C(8)); 25.2 (t); 21.6 (q, Me of
Ts); 19.0 (q, MeꢀC(3)); 17.6 (q, MeꢀC(7)). EI-MS (70 eV): 173 (10), 155 (18), 138 (54), 123 (75), 109
(46), 95 (92), 91 (95), 82 (81), 81 (100), 69 (69), 68 (39), 67 (61), 65 (36), 55 (44), 41 (89), 39 (20).
(S)-4,8-Dimethylnon-7-enenitrile (29). The preparation of 29 was carried out according to the
procedure of Mori and Tamada [29]. Yield: 587 mg (89%). [a]2D2 ¼ ꢀ5.2 (c¼2.6, Et2O). 1H-NMR
3
4
(400 MHz, CDCl3): 5.08 (tquint., J¼7.1, J¼1.4, HꢀC(7)); 2.41–2.27 (m, 2 HꢀC(2)); 2.07–1.91 (m,
2 HꢀC(6)); 1.75–1.64 (m, 1 HꢀC(3)); 1.63–1.54 (m, HꢀC(4)); 1.53–1.43 (m, 1 HꢀC(3)); 1.39–1.14

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
4
4
3
(m, 2 HꢀC(5)); 1.69 (d, J¼1.2, 3 HꢀC(9)); 1.61 (d, J¼0.3, MeꢀC(8)); 0.92 (d, J¼6.6, MeꢀC(4)).
13C-NMR (100 MHz, CDCl3): 131.6 (s, C(8)); 124.1 (d, C(7)); 119.9 (s, C(1)); 36.2 (t, C(5)); 32.2 (t, C(3));
31.5 (d, C(4)); 25.6 (q, C(9)); 25.2 (t, C(6)); 18.6 (q, MeꢀC(4)); 17.6 (q, MeꢀC(8)); 14.8 (t, C(2)). EI-MS
(70 eV): 165 (8, Mþ ), 150 (47), 136 (10), 122 (33), 108 (35), 94 (8), 83 (11), 69 (100), 55 (53), 41 (81), 39
(24).
(S)-4,8-Dimethylnon-7-enoic Acid (30). Hydrolysis of 29 according to the method of Mori and
Tamada [29] furnished 30. Yield: 435 mg (94%). [a]2D2 ¼ þ0.7 (c¼2.6, Et2O). 1H-NMR (400 MHz,
CDCl3): 10.77 (br. s, OH); 5.08 (tquint., 3J¼7.1, 4J¼1.4, HꢀC(7)); 2.43–2.28 (m, 2 HꢀC(2)); 2.06–1.90
(m, 2 HꢀC(6)); 1.75–1.64 (m, 1 H, HꢀC(3)); 1.68 (d, J¼1.1, 3 HꢀC(9)); 1.60 (d, J¼0.4, MeꢀC(8));
1.52–1.40 (m, 1 HꢀC(3), HꢀC(4)); 1.38–1.12 (m, 2 HꢀC(5)); 0.90 (d, J¼6.4, MeꢀC(4)). 13C-NMR
(100 MHz, CDCl3): 180.7 (s, C(1)); 131.3 (s, C(8)); 124.6 (d, C(7)); 36.7 (t, C(5)); 32.0 (d, C(4)); 31.9 (t,
C(2)); 31.5 (t, C(3)); 25.7 (q, C(9)); 25.4 (t, C(6)); 19.1 (q, MeꢀC(4)); 17.6 (q, MeꢀC(8)).
Preparation of Alkyl Bromides. The bromides 33 and 58 were prepared from the corresponding
alcohols 32 and 57, resp., according to a modified published procedure [30]. Br2 (2.0 equiv.) was added to
a soln. of Ph3P (2.0 equiv., 1.15m in abs. CH2Cl2) at 08 in such a way, that only a pale yellow color persisted.
After stirring for 20 min at 08, 32 or 57 (1.0 equiv.), dissolved in pyridine (0.15 ml/mmol) und abs. CH2Cl2
(0.25 ml/mmol), was added. The mixture was stirred for 2 h, while the temp. was allowed to increase to
r.t. The mixture was filtered and diluted with pentane, which caused the precipitation of Ph3PO. The
mixture was repeatedly concentrated and taken up in pentane again. To remove the solid, the raw
product was purified by CC (SiO2; pentane/Et2O 9 :1), affording pure 33 or 58, resp., as colorless liquids.
3,7-Dimethyloct-6-enyl Bromide (33). Yield: 4.06 g (62%). 1H-NMR (400 MHz, CDCl3): 5.09
(tquint., 3J¼7.1, 4J¼1.4, HꢀC(6)); 3.51–3.37 (m, 2 HꢀC(1)); 2.06–1.58 (m, 2 HꢀC(2), 2 HꢀC(3),
HꢀC(5)); 1.69 (d, J¼1.3, 3 HꢀC(8)); 1.61 (d, J¼0.5, MeꢀC(7)); 1.39–1.13 (m, 2 HꢀC(4)); 0.90 (d, J¼
6.5, MeꢀC(3)). 13C-NMR (100 MHz, CDCl3): 131.5 (s, C(7)); 124.4 (d, C(6)); 40.0 (t); 36.6 (t); 32.1 (t);
31.4 (d, C(3)); 25.7 (q, C(8)); 25.3 (t, C(5)); 18.8 (q, MeꢀC(3)); 17.7 (q, MeꢀC(7)). EI-MS (70 eV): 220
(9, Mþ with 81Br), 218 (9, Mþ with 79Br), 164 (5), 162 (5), 150 (4), 148 (4), 139 (2), 123 (2), 111 (3), 97
(11), 83 (61), 69 (100), 55 (50), 41 (54).
5-Methylhex-4-enyl Bromide (58). Yield: not estimated, because the solvent could not be removed
completely due to the low b.p. of the product. 1H-NMR (400 MHz, CDCl3): 5.07 (tquint., 3J¼7.3, 4J¼1.4,
HꢀC(4)); 3.40 (t, J¼6.7, 2 HꢀC(1)); 2.17–2.11 (m, 2 HꢀC(3)); 1.92–1.85 (m, 2 HꢀC(2)); 1.70 (d, J¼
1.2, 3 HꢀC(6)); 1.63 (d, J¼0.7, MeꢀC(5)). 13C-NMR (100 MHz, CDCl3): 133.2 (s, C(5)); 122.5 (d, C(4));
33.6 (t); 32.9 (t); 26.5 (t, C(3)); 25.7 (q, C(6)); 17.8 (q, MeꢀC(5)). EI-MS (70 eV): 178 (16, Mþ with
81Br), 176 (17, Mþ with 79Br), 97 (37), 81 (17), 69 (100), 55 (27), 53 (15), 41 (68), 39 (25).
Preparation of Methyl 2,6,10-Trimethylundecenoate and Methyl 6,10-Dimethylundecenoates. Methyl
2,6,10-trimethylundec-9-enoate (35) as well as the methyl 6,10-dimethylundec-9-enoates 36 and 37 were
prepared according to a modified published procedure [31][55]. The alkylmagnesium bromide, freshly
prepared from alkyl bromide 33 or 34, was cooled to ꢀ788 and DMAP (2.3 equiv.), CuBr (0.05 equiv.)
and Me2S (0.05 equiv.) were added. A soln. of methyl methacrylate or methyl acrylate, resp., (1.0 equiv.)
and TMSCl (1.2 equiv.) in abs. THF (0.5 ml/mmol ester) was added dropwise over a period of 30 min, and
the mixture was stirred for 2 h at ꢀ788. After warming to r.t., the mixture was diluted with HCl (2m) and
extracted with Et2O. The combined org. layers were washed with sat. aq. NaHCO3 soln. and brine, dried
(MgSO4), and the solvent was removed. Purification of the raw products by CC (SiO2; pentane/Et2O
30 :1) afforded pure 35, 36, or 37, resp., as colorless liquids.
Methyl 2,6,10-Trimethylundec-9-enoate (35). 1H-NMR (400 MHz, CDCl3): 5.09 (tquint., 3J¼7.1, 4J¼
1.4, HꢀC(9)); 3.67 (s, MeO); 2.44 (sext., J¼6.9, HꢀC(2)); 2.01–1.89 (m, 2 HꢀC(8)); 1.68 (s,
3 HꢀC(11)); 1.60 (s, MeꢀC(10)); 1.43–1.04 (m, 2 HꢀC(3), 2 HꢀC(4), 2 HꢀC(5), 2 HꢀC(6),
2 HꢀC(7)); 1.14 (d, J¼7.0, MeꢀC(2)); 0.86 (d, J¼6.5, MeꢀC(6)). 13C-NMR (100 MHz, CDCl3): 177.4
(s, C(1)); 131.0 (s, C(10)); 125.0 (d, C(9)); 51.4 (q, MeO); 39.5 (d, C(2)); 37.1 (t); 36.8 (t); 34.1 (t, C(3));
32.3 (d, C(6)); 25.7 (q, C(11)); 25.5 (t, C(4)); 24.6 (t, C(8)); 19.5 (q, MeꢀC(6)); 17.6 (q, MeꢀC(10)); 14.0
(q, MeꢀC(2)). EI-MS (70 eV): 240 (19, Mþ ), 208 (6), 180 (4), 152 (21), 138 (6), 124 (22), 111 (12), 110
(11), 109 (13), 101 (23), 97 (51), 88 (77), 83 (26), 69 (100), 59 (20), 55 (54), 41 (68).
Methyl 6,10-Dimethylundec-9-enoate (36). Yield: 1.65 g (57%). [a]2D2 ¼ þ10.0 (c¼2.5, Et2O).
1H-NMR (400 MHz, CDCl3): 5.09 (tquint., 3J¼7.1, 4J¼1.4, HꢀC(9)); 3.66 (s, MeO); 2.31 (t, J¼7.5,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
29
2 HꢀC(2)); 2.02–1.89 (m, 2 HꢀC(8)); 1.68 (d, J¼1.1, 3 HꢀC(11)); 1.60 (d, J¼0.5, MeꢀC(10)); 1.44–
1.06 (m, 2 HꢀC(3), 2 HꢀC(4), 2 HꢀC(5), HꢀC(6), 2 HꢀC(7)); 0.86 (d, J¼6.5, MeꢀC(6)). 13C-NMR
(100 MHz, CDCl3): 174.3 (s, C(1)); 131.0 (s, C(10)); 124.9 (d, C(9)); 51.4 (q, MeO); 37.0 (t); 36.5 (t); 34.1
(t, C(2)); 32.2 (d, C(6)); 26.6 (t, C(4)); 25.7 (q, C(11)); 25.5 (t, C(8)); 25.2 (t, C(3)); 19.5 (q, MeꢀC(6));
17.6 (q, MeꢀC(10)). EI-MS (70 eV): 226 (28, Mþ ), 194 (4), 177 (6), 156 (10), 152 (28), 137 (19), 124
(29), 111 (28), 110 (25), 109 (20), 97 (16), 96 (22), 95 (17), 87 (17), 83 (50), 82 (42), 69 (100), 59 (17), 55
(61), 41 (61).
Methyl (R)-6,10-Dimethylundec-9-enoate (37). Yield: 978 mg (34%). Spectroscopic data corre-
spond to those of 36.
Preparation of Carboxylic Acids. The acids 38, 39, 40, and 61 were prepared from the corresponding
methyl esters 35, 36, 37, or 60, resp., by standard methods [55].
rac-2,6,10-Trimethylundec-9-enoic Acid (38). Yield: 0.33 g (69%). 1H-NMR (400 MHz, CDCl3): 5.09
(tsept., 3J¼7.1, 4J¼1.4, HꢀC(9)); 2.50–2.42 (m, HꢀC(2)); 2.00–1.89 (m, 2 HꢀC(8)); 1.71–1.63 (m,
HꢀC(3)); 1.68 (d, 4J¼0.9, 3 HꢀC(11)); 1.60 (s, MeꢀC(10)); 1.44–1.21 (m, 1 HꢀC(3), 2 HꢀC(4),
2 HꢀC(5), HꢀC(6)); 1.18 (d, J¼7.1, Me ꢀC(2)); 1.15–1.08 (m, 2 HꢀC(7)); 0.86 (d, J¼6.5, MeꢀC(6)).
13C-NMR (100 MHz, CDCl3): 183.4 (s, C(1)); 131.0 (s, C(10)); 125.0 (d, C(9)); 39.42/39.38 (d, C(2));
37.08/37.07 (t, C(7)); 36.8 (t, C(5)); 33.84/33.79 (t, C(3)); 32.2 (d, C(6)); 25.7 (q, C(11)); 25.5 (t, C(8));
24.58/24.54 (t, C(4)); 19.5 (q, MeꢀC(6)); 17.6 (q, MeꢀC(10)); 16.9/16.8 (q, MeꢀC(2)).
1
6,10-Dimethylundec-9-enoic acid (39). Yield: 1.06 g (94%). [a]2D2 ¼ ꢀ3.3 (c¼2.5, Et2O). H-NMR
3
4
(400 MHz, CDCl3): 5.09 (tquint., J¼7.1, J¼1.4, HꢀC(9)); 2.35 (t, J¼7.5, 2 HꢀC(2)); 2.05–1.88 (m,
2 HꢀC(8)); 1.68 (d, J¼1.0, 3 HꢀC(11)); 1.60 (s, MeꢀC(10)); 1.46–1.07 (m, 2 HꢀC(3), 2 HꢀC(4),
2 HꢀC(5), HꢀC(6), 2 HꢀC(7)); 0.86 (d, J¼6.5, MeꢀC(6)). 13C-NMR (100 MHz, CDCl3): 180.3 (s,
C(1)); 131.0 (s, C(10)); 124.9 (d, C(9)); 37.0 (t); 36.5 (t); 34.1 (t, C(2)); 32.2 (d, C(6)); 26.5 (t, C(4)); 25.7
(q, C(11)); 25.5 (t, C(8)); 25.0 (t, C(3)); 19.5 (q, MeꢀC(6)); 17.6 (q, MeꢀC(10)).
(R)-6,10-Dimethylundec-9-enoic Acid (40). Yield: 762 mg (90%). Spectroscopic data correspond to
those of 39.
6,10-Dimethylundeca-5,9-dienoic Acid (61). Yield: 0.47 g (89%). 1H-NMR (400 MHz, CDCl3): 5.13–
5.06 (m, HꢀC(5), HꢀC(9)); 2.34 (t, J¼7.5, 2 HꢀC(2)); 2.10–1.95 (m, 2 HꢀC(4), 2 HꢀC(7),
2 HꢀC(8)); 1.72–1.59 (m, 2 HꢀC(3), 3 HꢀC(11), MeꢀC(6), MeꢀC(10)). 13C-NMR (100 MHz,
CDCl3): 180.24/180.17 (s, C(1)); 136.44/136.36 (s, C(6)); 131.6/131.4 (s, C(10)); 124.3 (d); 124.2 (d); 124.0
(d); 123.2 (d); 39.7 (t, C(7) (E)); 33.6 (t); 33.4 (t); 31.9 (t, C(7) (Z)); 27.11 (t); 27.06 (t); 26.62 (t); 26.55
(t); 25.7 (q, C(11)); 25.0 (t); 24.7 (t); 23.4 (q, MeꢀC(6) (Z)); 17.7/17.6 (q, MeꢀC(10)); 16.0 (q, MeꢀC(6)
(E)).
Preparation of 3-(6,10-Dimethylundec-9-enoyl)- and 3-(6,10-Dimethylundeca-5,9-dienoyl)-4-benzy-
loxazolidin-2-ones. The enantioselective alkylation was carried out as described by Goutopoulos et al.
[32]. Pivaloyl chloride (1.2 equiv.) was added to a soln. of the acid 38, 39, 40, or 61 (1.0 equiv.), and Et3N
(1.2 equiv.) in abs. THF (5.5 ml/mmol acid) at ꢀ788. The resulting white suspension was stirred for
10 min at ꢀ788 and 30 min at 08. After cooling to ꢀ788, a freshly prepared soln. of lithiated (S)- or (R)-
4-benzyloxazolidin-2-one (1.2 equiv.) was added. The mixture was stirred for 3 h, while the temp. was
allowed to increase slowly to 08 and for 30 min at 08. After hydrolysis with sat. aq. NH4Cl soln., the
mixture was extracted with Et2O. The combined org. layers were washed with H2O, dried (MgSO4), and
the solvent was removed. Purification of the raw products by CC (SiO2; pentane/Et2O 5 :1) afforded pure
41, 42, 43, or 62 as colorless liquids.
(4S)-3-[(6RS)-6,10-Dimethylundec-9-enoyl]-4-(phenylmethyl)oxazolidin-2-one (41). Yield: 863 mg
(76%). [a]2D2 ¼ þ37.6 (c¼1.7, Et2O). 1H-NMR (400 MHz, CDCl3): 7.35–7.19 (m, 5 arom. H); 5.10
3
4
(tquint., J¼7.1, J¼1.4, HꢀC(9’)); 4.70–4.65 (m, HꢀC(4)); 4.22–4.14 (m, PhCH2); 3.30 (dd, J¼13.4,
J¼3.3, 1 HꢀC(5)); 3.02–2.85 (m, 2 HꢀC(2’)); 2.77 (dd, J¼13.4, J¼9.6, 1 HꢀC(5)); 2.04–1.89 (m,
2 HꢀC(8’)); 1.68 (d, J¼1.1, 3 HꢀC(11’)); 1.60 (d, J¼0.3, MeꢀC(10’)); 1.47–1.09 (m, 2 HꢀC(3’),
2 HꢀC(4’), 2 HꢀC(5’), HꢀC(6), 2 HꢀC(7)); 0.87 (d, J¼6.5, MeꢀC(6’)). 13C-NMR (100 MHz, CDCl3):
173.4 (s, C(1’)); 153.4 (s, C(1) of Ph); 135.3 (s, C(4)); 131.0 (s, C(10)); 129.4 (2 d, arom. C); 128.9 (2 d,
arom. C); 127.3 (d, arom. C); 125.0 (d, C(9’)); 66.1 (t, PhCH2); 55.1 (d, C(2)); 37.9 (t, C(5)); 37.1 (t); 36.7
(t); 35.5 (t); 32.3 (d, C(6’)); 26.6 (t); 25.7 (q, C(11’)); 25.5 (t); 24.6 (t); 19.5 (q, MeꢀC(6’)); 17.6 (q,

30
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
MeꢀC(10’)). EI-MS (70 eV): 371 (8, Mþ ), 258 (3), 245 (5), 232 (32), 219 (36), 195 (9), 178 (100), 152
(5), 134 (25), 117 (59), 109 (20), 95 (18), 91 (41), 83 (13), 82 (13), 81 (14), 69 (98), 55 (53), 41 (55).
(4R)-3-((6RS)-6,10-Dimethylundec-9-enoyl)-4-(phenylmethyl)oxazolidin-2-one (42). Yield: 1.43 g
(77%). Spectroscopic data correspond to those of 41. [a]2D2 ¼ ꢀ36.8 (c¼2.4, Et2O).
(4R)-3-[(6R)-6,10-Dimethylundec-9-enoyl]-4-(phenylmethyl)oxazolidin-2-one (43). Yield: 1.15 g
(94%). Spectroscopic data correspond to those of 41. [a]2D2 ¼ ꢀ43.4 (c¼2.2, Et2O).
(4S)-3-(6,10-Dimethylundeca-5,9-dienoyl)-4-(phenylmethyl)oxazolidin-2-one (62). Yield: 534 mg
(88%). The product was a mixture of (E)- and (Z)-isomers. [a]2D2 ¼ þ43.8 (c¼3.3, Et2O). 1H-NMR
(400 MHz, CDCl3): 7.35–7.19 (m, 5 arom. H); 5.17–5.07 (m, HꢀC(5’), HꢀC(9’)); 4.70–4.64 (m,
HꢀC(4)); 4.22–4.13 (m, PhCH2); 3.30 (dd, J¼13.3, J¼3.3, 1 HꢀC(5)); 3.01–2.86 (m, 2 HꢀC(2’));
2.80–2.72 (m, 1 HꢀC(5)); 2.12–1.96 (m, 2 HꢀC(4), 2 HꢀC(7), 2 HꢀC(8)); 1.80–1.45 (m, 2 HꢀC(3),
3 HꢀC(11), MeꢀC(6’), MeꢀC(10’)). 13C-NMR (100 MHz, CDCl3): 173.4/173.3 (s, C(1’)); 153.4 (s, C(1)
of Ph); 136.24/136.17 (s, C(6’)); 135.3 (s, arom. C); 131.6/131.3 (s, C(10’)); 129.4 (2 d, arom. C); 128.9 (2 d,
arom. C); 127.3 (d, arom. C); 124.3 (d); 124.25 (d); 124.23 (d); 123.4 (d); 66.1 (t, PhCH2); 55.1 (d, C(2));
39.7 (t, C(7’) (E)); 37.9 (t, C(5)); 35.1 (t); 35.0 (t); 32.0 (t, C(7’) (Z)); 27.2 (t); 27.1 (t); 26.7 (t); 26.6 (t);
25.7 (q, C(11’)); 24.5 (t); 24.4 (t); 23.4 (q, MeꢀC(6’) (Z)); 17.7/17.6 (q, MeꢀC(10’)); 16.0 (q, MeꢀC(6’)
(E)). Diastereoisomer with GC tR of 28.26 min: EI-MS (70 eV): 369 (4, Mþ ), 326 (12), 308 (6), 232 (23),
219 (15), 192 (16), 178 (92), 160 (5), 149 (71), 134 (32), 128 (10), 123 (28), 117 (60), 109 (41), 95 (25), 91
(53), 81 (29), 69 (100), 55 (30), 41 (78). Diastereoisomer with GC tR retention time 28.56 min: EI-MS
(70 eV): 369 (3, Mþ ), 326 (12), 308 (6), 232 (24), 219 (12), 192 (12), 178 (100), 160 (4), 149 (82), 134
(28), 128 (9), 123 (36), 117 (68), 109 (37), 95 (30), 91 (53), 81 (30), 69 (92), 55 (29), 41 (71).
Preparation of 3-(2,6,10-trimethylundec-9-enoyl)- and 3-(2,6,10-Trimethylundeca-5,9-dienoyl)-4-
(phenylmethyl)oxazolidin-2-ones. A soln. of 41, 42, 43, or 62 (1.0 equiv., 4.8m in abs. THF) was added
to a soln. of NaHMDS (1.1 equiv., 1.0m in abs. THF) at ꢀ788. The mixture was stirred for 30 min at ꢀ788.
MeI (5.0 equiv.) was added, and the mixture was stirred for 4 h at ꢀ788. After addition of sat. aq. NH4Cl
soln., the mixture was extracted with Et2O. The combined org. layers were washed with brine, dried
(MgSO4), and the solvent was removed. Purification of the raw products by CC (SiO2; pentane/Et2O
2 :1) afforded pure 44, 45, 46, or 63 as colorless liquids.
(4S)-3-[(2S,6RS)-2,6,10-Trimethylundec-9-enoyl)-4-(phenylmethyl)oxazolidin-2-one (44). Yield:
421 mg (55%). [a]2D2 ¼ þ71.0 (c¼2.2, Et2O). H-NMR (400 MHz, CDCl3): 7.35–7.20 (m, 5 arom. H);
1
5.09 (t, J¼7.1, H ꢀC(9’)); 4.70–4.65 (m, HꢀC(4)); 4.22–4.14 (m, PhCH2); 3.71 (sext., J¼6.8, HꢀC(2’));
3.27 (dd, J¼13.3, J¼3.3, 1 HꢀC(5)); 2.77 (dd, J¼13.3, J¼9.6, 1 HꢀC(5)); 2.04–1.83 (m, 2 HꢀC(8’));
1.68 (d, J¼0.8, 3 HꢀC(11’)); 1.60 (d, J¼0.3, MeꢀC(10’)); 1.45–1.07 (m, 2 HꢀC(3), 2 HꢀC(4),
2 HꢀC(5), HꢀC(6), 2 HꢀC(7)); 1.22 (d, J¼6.8, MeꢀC(2’)); 0.85 (d, J¼6.4, MeꢀC(6’)). 13C-NMR
(100 MHz, CDCl3): 177.3 (s, C(1’)); 153.0 (s, PhCH2); 135.3 (s, C(4)); 131.0 (s, C(10)); 129.4 (2 d, arom.
C); 128.9 (2 d, arom. C); 127.3 (d, arom. C); 124.9 (d, C(9’)); 66.0 (t, PhCH2); 55.3 (d, C(4)); 37.9 (t,
C(5)); 37.72/37.70 (d, C(2’)); 37.1 (t); 36.9 (t); 33.68 (t); 33.66 (t); 32.3/32.2 (d, C(6’)); 25.7 (q, C(11’));
25.53 (t); 25.51 (t); 24.6 (t); 19.5 (q, MeꢀC(6’)); 17.6 (q, MeꢀC(10’)); 17.4/17.3 (q, MeꢀC(2’)). EI-MS
(70 eV): 385 (1, Mþ ), 246 (5), 233 (100), 209 (6), 191 (5), 178 (56), 165 (4), 151 (4), 142 (4), 134 (16),
117 (27), 109 (22), 97 (9), 95 (12), 91 (23), 83 (9), 82 (9), 81 (10), 69 (70), 55 (31), 41 (32).
(4R)-3-[(2R,6RS)-2,6,10-Trimethylundec-9-enoyl]-4-(phenylmethyl)oxazolidin-2-one (45). Yield:
0.96 g (78%). Spectroscopic data correspond to those of 44. [a]2D2 ¼ ꢀ73.7 (c¼2.8, Et2O).
(4R)-3-[(2R,6R)-2,6,10-Trimethylundec-9-enoyl]-4-(phenylmethyl)oxazolidin-2-one (46). Yield:
800 mg (77%). Spectroscopic data correspond to those of 44. [a]2D2 ¼ ꢀ70.6 (c¼1.3, Et2O).
(4S)-3-[(2S)-2,6,10-Trimethylundeca-5,9-dienoyl]-4-(phenylmethyl)oxazolidin-2-one (63). Yield:
322 mg (80%). The product was a mixture of (E)- and (Z)-isomers. [a]2D2 ¼ þ83.4 (c¼1.9, Et2O).
1H-NMR (400 MHz, CDCl3): 7.35–7.20 (m, 5 arom. H); 5.13–5.06 (m, HꢀC(5’), HꢀC(9’)); 4.70–4.63
(m, HꢀC(4)); 4.36–4.13 (m, PhCH2); 3.76–3.67 (m, HꢀC(2’)); 3.26 (dd, J¼13.3, J¼3.3, 1 HꢀC(5));
2.77 (dd, J¼13.3, J¼9.6, 1 HꢀC(5)); 2.08–1.93 (m, 2 HꢀC(4’), 2 HꢀC(7’), 2 HꢀC(8’)); 1.87–1.39 (m,
2 HꢀC(3’), HꢀC(11’), MeꢀC(6’), MeꢀC(10’)); 1.232 (d, J¼6.8)/1.227 (d, J¼6.9) (MeꢀC(2’)).
13C-NMR (100 MHz, CDCl3): 177.2/177.1 (s, C(1’)); 153.0 (s, PhCH2); 135.8/135.7 (s, C(6’)); 135.3 (s,
arom. C); 131.6/131.3 (s, C(10’)); 129.4 (2 d, arom. C); 128.9 (2 d, arom. C); 127.3 (d, arom. C); 124.4 (d);
124.3 (d); 124.2 (d); 123.5 (d); 66.0 (t, PhCH2); 55.3 (d, C(4)); 39.7 (t, C(7’) (E)); 37.9 (t, C(5)); 37.4/37.3