Odd-numbered very-long-chain polyunsaturated fatty acids from the dinoflagellate Amphidinium carterae identified by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

Article abstract of DOI:10.1016/j.phytochem.2008.08.021

A method is described for the enrichment of odd very-long-chain polyunsaturated fatty acids (VLCPUFAs) by means of RP-HPLC and argentation TLC from total fatty acids of the dinoflagellate A. carterae and their identification as picolinyl esters by means of microbore liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The combination of argentation TLC and LC-MS/APCI was used to identify rare and unusual odd VLCPUFAs up to nonacosahexaenoic acid. Two acids, (allZ)-nonacosa-11,14,17,20,23-pentaenoic acid (29:5n-6) and (allZ)-nonacosa-11,14,17,20,23,26-hexaenoic acid (29:6n-3), were synthesized for the first time to unambiguously confirm their structure. Possible biosynthetic pathways for odd VLCPUFAs are also proposed.

Full text of DOI:10.1016/j.phytochem.2008.08.021

Phytochemistry 69 (2008) 2849–2855
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Odd-numbered very-long-chain polyunsaturated fatty acids from the dinoflagellate
Amphidinium carterae identified by atmospheric pressure chemical ionization
liquid chromatography–mass spectrometry
a,
Tomáš Rezanka , Linda Nedbalová b,c, Karel Sigler a
*
ˇ
a
ˇ
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague, Czech Republic
Charles University in Prague, Faculty of Science, Department of Ecology, Vinicná 7, 128 44 Prague 2, Czech Republic
Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Trebon, Czech Republic
b
c
ˇ
ˇ
ˇ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2008
Received in revised form 27 August 2008
Available online 7 October 2008
A method is described for the enrichment of odd very-long-chain polyunsaturated fatty acids (VLCPUFAs)
by means of RP-HPLC and argentation TLC from total fatty acids of the dinoflagellate A. carterae and their
identification as picolinyl esters by means of microbore liquid chromatography-mass spectrometry with
atmospheric pressure chemical ionization (LC–MS/APCI). The combination of argentation TLC and LC–MS/
APCI was used to identify rare and unusual odd VLCPUFAs up to nonacosahexaenoic acid. Two acids,
(allZ)-nonacosa-11,14,17,20,23-pentaenoic acid (29:5n-6) and (allZ)-nonacosa-11,14,17,20,23,26-hexae-
noic acid (29:6n-3), were synthesized for the first time to unambiguously confirm their structure. Possi-
ble biosynthetic pathways for odd VLCPUFAs are also proposed.
Keywords:
Amphidinium carterae
Marine dinoflagellate
Odd very-long-chain polyunsaturated fatty
acids
Ó 2008 Elsevier Ltd. All rights reserved.
Liquid chromatography–mass spectrometry
– atmospheric pressure chemical ionization
1. Introduction
of odd-chain VLCPUFAs can be seen to be one to two orders of mag-
nitude lower than that of even-chain VLCPUFAs of the same chain
In nature, very-long-chain polyunsaturated fatty acids (VLCPU-
FAs, i.e. fatty acids with three and more double bonds and with
chain lengths equal to and/or greater than 20 carbons) are found
infrequently; for a review of these compounds in animal and plant
kingdom see Rezanka (1989). In general, classical, i.e. methylene-
interrupted VLCPUFAs can be said to have been found mainly in
mammals, with several exceptions (Poulos et al., 1986). Among
lower animals and plants, VLCPUFAs have been found in a range
of organisms (e.g., Cladonia lichens, slime-molds, Chlorella kessleri,
the marine dinoflagellate Gymnodinium sp., algal classes Chloro-
phyceae and Prasinophyceae) (Rezanka and Podojil, 1984; Dembit-
sky et al., 1991; Dunstan et al., 1992; Rezanka, 1993, 2002;
Mansour et al., 1999a,b, 2003, 2005) and in invertebrates (crusta-
cean Bathynella natans) (Rezanka and Dembitsky, 2004).
length; for instance, the amount of 30:5n-6 was 13.4% of total FAs
while 31:5n-6 amounted to only 0.21% and 32:5n-6 to 3.9%
(Girland et al., 2007).
Even though fatty acids with even numbers of carbon atoms
predominated, a significant amount of odd-numbered fatty acids
was detected in boar spermatozoa. These included 25:3n-6 and
27:3n-6, the series of 23:4n-6 to 31:4n-6, 29:5n-6 and 31:5n-6
(Poulos et al., 1986).
Aveldano et al. (1993) proposed a hypothesis for the biosynthe-
sis of odd-chain VLCPUFAs. They used radioactive precursors,
(1-14C-labeled n-6 tetraenoic fatty acids (20:4, 24:4, and 32:4)
and [U-14C] acetate), and determined the presence of both major
even-chain FAs and minor odd-chain acids, i.e. one trienoic
(23:3n-6), three tetraenoic (C23-C27) and four pentaenoic (C23
-
In the rat, most of the VLCPUFAs are present in sphingomyelin
and ceramide and belong to the n-6 series and have an even num-
ber of carbon atoms in their chains, but odd-chain VLCPUFAs also
occurred in low amounts. They were mostly represented by two
tetraenoic acids (27:4n-6 and 29:4n-6) amounting to 0.20% of total
FAs. In addition, a series of odd-chains 25:5n-6 to 31:5n-6 was
present in amounts from 0.02 to 0.36 of total FAs. The proportion
C29) acids. The chromatogram shown in this study indicates the
probable presence of two higher homologues, i.e. 31:5 and 33:5.
The authors discuss various possible pathways for the biosynthesis
including
a-oxidation of even-chain VLCPUFAs. They speculated
that -oxidation is an important mechanism for the shortening
a
of fatty acids and that odd-chain PUFAs are metabolic
intermediates.
Identification of odd-chain VLCPUFAs cannot be performed eas-
ily by GC or GC-MS, since the minor n-6 pentaenes with odd-num-
bered chains tend to coelute with the corresponding hexaenes of
* Corresponding author. Tel.: +420 241 062 300; fax: +420 241 062 347.
E-mail address: rezanka@biomed.cas.cz (T. Rezanka).
ˇ
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.08.021

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T. Rezanka et al. / Phytochemistry 69 (2008) 2849–2855
2850
the n-3 series with even-numbered chains having one carbon less,
e.g., 27:5n-6 with 26:6n-3, 29:5n-6 with 28:6n-3, and so on. Some-
thing similar occurs between the n-6 tetraenes with odd-chains
and the n-3 pentaenes with even chains having one carbon less,
e.g., 21:4n-6 with 20:5n-3, and so on. The problem was addressed
by resolving the methyl esters according to unsaturation by argen-
tation TLC. Two tetraenoic (25:4n-6 and 27:4n-6) and a series of
pentaenoic acids from C25 to C33 were found, all being in the n-
6 series; their proportions were minor, about 0.01 to 0.1% of total
FAs (Furland et al., 2003).
The occurrence of odd-chain VLCPUFAs, e.g., 23:5n-3, and trace
25:5n-3 has been detected in blubbers of seals caught both in the
sea and in freshwater lakes of northern Europe. This finding consti-
tutes an exception since mammals usually possess only odd-chain
n-6 VLCPUFAs, not n-3 acids. This finding can be ascribed to the
food chain sequence plankton-fish-mammal (Kakela et al., 1995).
Japanese authors (Shimizu et al., 1991; Shirasaka et al., 1995;
Zhang et al., 2006) have reported on the identification of odd-chain
PUFAs (C17 and C19) in fungi of the genera Mortierella and Sapro-
legnia cultivated on odd-chain carbon sources such as n-heptadec-
ane or fatty acids with 13, 15, or 17 carbons. A scheme of the
biosynthesis of 17:3n-5 and its successors 19:3n-8, 19:4n-5, and
19:5n-2 has been proposed.
time (RP-HPLC), and fragmentation in mass spectrum. By both
methods we succeeded in identifying two series of odd-chain
VLCPUFAs. As with even chain VLCPUFA, odd-chain acids were
found to include both n-6 and n-3 series. Since their amounts are
one to two orders of magnitude lower than even-chain VPCPUFA,
their identification was performed using the SIM (single ion mon-
itoring) mode of protonated molecular ions, see Fig. 1.
Ionization and detection of VLCPUFA ions by LC–MS/APCI deliv-
ered a significant benefit in detection capability when compared to
other detection techniques. The selectivity of SIM gives a signifi-
cantly lower detection limit due to the lack of chemical interfer-
ences at protonated molecular ion compared to total ion current.
In SIM mode the detection limit for picolinyl esters of VLCPUFAs
was 0.0001% of total FAs. The capability of VLCPUFAs identification
afforded by the Ag+-TLC and LC–MS/APCI system is visibly demon-
strated in the co-elution of 27:4n-6 and 21:1n-9. While the total
ion chromatogram indicates a broad peak centered at 2494 s, the
two separate components (27:4n-6 and 21:1n-9) are clearly re-
solved by individual mass chromatograms (Fig. 2). Unfortunately,
no further information on double bond positions is available from
the direct LC–MS/APCI approach and picolinyl esters were there-
fore used for distinguishing them.
As seen in Table 1, only the odd tetraenoic, pentaenoic and
hexaenoic acids were identified. If odd VLCPUFAs with 7 and 8
double bonds are present, their proportion in total FAs is below
the detection limit. The absence of heptaenoic and octaenoic acids
is comparable with the absence of, e.g., hexaenoic acids in mam-
malian materials, in which even-chain FAs were identified up to
hexaenoic while odd-chain FAs only up to pentaenoic (Poulos
et al., 1986; Aveldano et al., 1993). On the basis of our results we
cannot determine whether this is due to a ‘‘faulty‘‘ metabolism
or simply to their minor proportions that could not be detected,
even though our detection range spanned 6 orders of magnitude.
To confirm the structure of odd-chain VLCPUFAs, two of the
minor FAs were prepared for the first time by total synthesis using
methods described in our previous paper (Rezanka et al., 2008).
Briefly, retrosynthetic analysis revealed a suitably protected 11-
dodecynol as an ideal starting material. Thus, commercially avail-
able 10-bromo-decanol was protected by treatment with 3,4-dihy-
dro-2H-pyran in the presence of catalytic amounts of pyridinium
p-toluene sulfonate to afford tetrahydropyranyl acetal in 80% iso-
lated yield. Reaction of this acetal with lithium acetylide ethylene-
diamine complex gave 79% of 3 (Ettmayer et al., 2004) that was
allowed to react with all-(Z)-1-bromoheptadeca-2,5,8,11,14-pent-
aene (4) or (all-Z)-1- bromoheptadeca-2,5,8,11-tetraene (5) which
were synthesized as previously described (Rezanka et al., 2008) in
the presence EtMgBr solution and copper bromide to yield the 20-
((all-Z)-nonacosa-14,17,20,23,26-pentaen-11-ynyloxy)tetrahydro-
2H-pyran (6) or 20-((all-Z)-nonacosa-14,17,20,23-tetraen-11-ynyl-
oxy)tetrahydro-2H-pyran (7), respectively. Our previously de-
scribed methods, i.e. partial reduction, hydrolysis and oxidation,
were used to prepare from these two compounds (all-Z)-
nonacosa-11,14,17,20,23,26-hexaenoic (1) and (all-Z)-nonacosa-
11,14,17,20,23-pentaenoic (2) acids, respectively.
Another study dealing with the biosynthesis of odd-chain
VLCPUFAs (Nakano et al., 2000) demonstrated that rat liver cells
converted exogenous C19 odd-chain-polyunsaturated fatty acids
into the corresponding C21- and C23-PUFAs, e.g., from 19:3n-8 to
21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8; from 19:4n-5 to 21:4n-
5, 21:5n-5, 23:4n-5, and 23:5n-5; and from 19:5n-2 to 21:5n-2,
21:6n-2, 23:5n-2, and 23:6n-2. It presumed that these C19 PUFAs
were converted through a route mimicking that of docosahexae-
noic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3).
The VLCPUFAs from trienes to hexaenes were found in the lipids
of Amphidinium carterae to contain a series of minor odd-chain
homologues. By separating these acids first by degree of unsatura-
tion (by Ag+-TLC) and then by chain length by (RP-HPLC), we were
able to recognize unusual odd-chain polyenoic fatty acids of di-
verse chain lengths. Their identity was confirmed by APCI mass
spectra and also by comparison with synthesized standards. On re-
verse phase columns, the odd-chain PUFAs of dinoflagellate con-
formed to the chromatographical behavior expected of
a
complete homologous series, covering the whole range of chain
lengths from 21 to 31 carbon atoms.
2. Results and discussion
As described in our previous paper on fatty acids present in the
dinoflagellate A. carterae (Rezanka et al., 2008), the VLCPUFAs that
have so far been identified are solely even-chain ones, up to C36
(36:7n-6 and 36:8n-3). According to the literature odd-chain
VLCPUFAs are contained in the lipids isolated from different
sources in amounts one to two orders of magnitude lower than
even-chain FAs; we therefore attempted to enrich their fraction
by using the previously described and tested method (Van Pelt
et al., 1999; Rezanka and Sigler, 2007; Rezanka et al., 2008).
LC–MS/APCI analysis proved a superior method for the identifi-
cation of PUFA chain length and degree of unsaturation compared
to GC–MS. The correlation of retention time with unsaturation for
the chain series demonstrated the powerful ability of the LC–MS/
APCI system to directly identify PUFAs from the presence of the
protonated molecular ion. Close correlations based on both chain
length and degrees of unsaturation were demonstrated for the ser-
ies of PUFAs including VLCPUFAs. Any potential ambiguity for fatty
acids of the same molecular weight (e.g., 27:1 and 28:8) is re-
moved by such observation of retention factor (Ag+-TLC), retention
As already described by Rezanka et al. (2008), the APCI mass
spectra of two peaks had characteristic ions consistent with syn-
thesized (all-Z)-nonacosa-11,14,17,20,23,26-hexaenoic (1) and
(all-Z)-nonacosa-11,14,17,20,23-pentaenoic (2) picolinyl esters,
see Figs.
4 and 5. Picolinyl esters have prominent ions at
m/z = 92, 108, 151 (the McLafferty ion) and 164, which are all frag-
ments about the pyridine ring. Furthermore, the pseudomolecular
ion [M + 54]+ was also characteristic, but it arises by the addition of
acetonitrile from the mobile phase. The molecular ion is easily dis-
tinguished and it is always odd-numbered, because of the presence
of the nitrogen atom, but most other ions are even-numbered.
From the molecular ion, there is the loss of a methyl group,

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T. Rezanka et al. / Phytochemistry 69 (2008) 2849–2855
2851
600
80
m/z = 574
33:6n-3
m/z = 574
33:6n-3
500
0
80
31:6n-3
m/z = 546
0
400
80
29:6n-3
m/z = 518
0
300
80
27:6n-6
27:6n-3
m/z = 490
m/z = 462
200
0
80
25:6n-3
0
100
80
23:6n-3
m/z = 434
0
0
900
1200
1500
1800
2100
2400
2700
3000
time(s)
Fig. 1. LC–MS/APCI SIM chromatograms of FAMEs from total FAs of A. carterae after Ag+-TLC; fraction with six double bonds is shown. The SIM traces depict molecular ion
masses, see m/z values.
followed by a series of ions 14 Da apart for the loss of successive
methylene groups. When a double bond is reached, there is a
gap of 26 Da. This gap can sometimes be difficult to locate pre-
cisely, and a fragmentation at the adjacent methylene group on
the carboxyl side giving a gap of 40 is often easier to locate, espe-
cially with polyenes, see below. The greater complexity of the mass
spectra of PUFA picolinyl esters can make interpretation rather
difficult.
The gap of 26 amu between m/z = 488 and 462 locates the ter-
minal double bond rather easily, and that between m/z = 448 and
422 locates a double bond in position 22 of the picolinyl ester 1.
This pattern is repeated up to the first double bond. The gap of
40 amu for the double bond and an associated methylene group,
in this instance from m/z = 262 to 302 to 342 to 382 to 422 to
462 to 502, (see Fig. 3), is also very useful.
for saturated FAMEs, i.e. m/z 74, 87 and weak ions at [M]+, together
with diagnostic ions at [M-MeO]+ and [M-C3H7]+.
Further, the PUFAs were not conjugated, as shown by the UV
spectra (see experimental). The double bond stereochemistry was
established by FTIR. All double bonds were Z because the IR spec-
trum of the PUFAs exhibited absorption at 723 cmꢀ1 and no
absorption in the 960–980 cmꢀ1 regions (Doumenq et al., 1990).
The presence of odd-chain VLCPUFA in the alga poses several
important questions, the most fundamental of them being the
manner of their biosynthesis. Saturated or monoenoic odd-num-
bered acids are formed by de novo synthesis through successive
additions of two-carbon units to an initial three-carbon unit, or
by one-carbon shortening of even-numbered fatty acids by a-oxi-
dation (Diedrich and Henschell, 1990). A completely different situ-
ation is found with PUFA, especially with VLCPUFA. They are
assumed to be formed either (1) from odd-numbered saturated
or monoenoic acids via analogous pathways - ‘‘mimic routes” as
The structures of other PUFAs, including the synthesized com-
pounds, e.g., (all-Z)-nonacosa-11,14,17,20,23-pentaenoic picolinyl
ester (2), were identified in an analogous manner, as illustrated
in Fig. 4.
even-numbered PUFA (Nakano et al., 2000), (2) by
a-oxidation of
even-numbered PUFA (Aveldano et al., 1993) or (3)
a
-ketoacid
We also used other methods for the full confirmation of the
above structures, i.e. hydrogenation of the compounds from the
lower spots after Ag+-TLC. Only peaks corresponding to saturated
straight-chain FAMEs were observed by means of GC-MS. The EI
mass spectra of these peaks contained fragment ions characteristic
elongation without participation of fatty acid synthase-mediated
reactions or -independent thioesterases (Kroumova et al., 1994).
Another obvious possibility is a mixed biosynthesis, which involves
a marriage of routes 1 and 2. We used the data from our analyses,
especially the data in Table 1 and Fig. 5 to suggest a tentative

ˇ
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T. Rezanka et al. / Phytochemistry 69 (2008) 2849–2855
Table 1
Total VLCPUFAs after Ag+-TLC; only acids with three and more double bonds are given
m/z 494
27:4n-6
80
Peak no.
FA
%
Peak no.
FA
%
1
2
3
4
5
6
7
8
21:5n3
23:6n3
20:4n3
22:5n3
20:4n6
24:6n3
22:5n6
26:7n3
28:8n3
21:4n3
23:5n3
25:6n3
23:5n6
22:4n3
24:5n3
26:6n3
24:5n6
28:7n3
26:6n6
21:3n3
28:7n6
23:4n3
25:5n3
23:4n6
27:6n3
25:5n6
27:6n6
22:3n3
24:4n3
26:5n3
24:4n6
28:6n3
26:5n6
30:7n3
0.43
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
28:6n6
23:3n3
30:7n6
25:4n3
27:5n3
25:4n6
29:6n3
24:3n3
26:4n3
24:3n6
28:5n3
26:4n6
32:7n3
32:7n6
27:4n3
25:3n6
27:4n6
31:6n3
29:5n6
28:4n3
26:3n6
28:4n6
34:7n3
34:7n6
27:3n3
36:8n3
27:3n6
31:5n3
33:6n3
28:3n3
28:3n6
36:7n3
36:7n6
3.43
0.31
1.91
8.13
12.04
5.61
3.63
1.40
15.77
0.06
0.48
0.62
0.20
1.34
2.92
1.47
1.59
1.66
0.45
0.11
7.32
0.05
0.12
0.11
0.09
0.02
0.20
2.04
0.89
1.27
0.76
1.72
0.38
1.24
0.20
1.74
0.04
0.05
0.07
0.10
1.21
0.83
0.19
1.02
1.15
1.98
1.47
0.05
0.30
0.04
0.06
0.07
1.08
1.53
0.96
2.06
1.66
0.03
0.61
0.01
0.08
tr
9
0
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
m/z 416
21:1n-9
80
0
TIC
80
0.51
0.45
0.49
0.19
The sum of odd FA is 0.61% of total FA, whereas the sum of n-3 odd FA is 0.45% of
total FA, tr - traces, less than 0.01%.
0
2470
2490
time(s)
2510
HPLC equipment consisted of a 1090 system, PV5 ternary pump
and automatic injector (HP 1090 series, Hewlett Packard, USA) and
Fig. 2. LC–MS/APCI SIM and TIC chromatograms of total FAMEs from A. carterae.
Only compounds with retention times between 2470 s and 2510 s are shown.
two Hichrom columns HIRPB-250AM 250 ꢁ 2.1 mm ID, 5
lm
phase particle, in series. This setup provided us with a high-effi-
ciency column – approximately ꢂ26000 plates/250 mm. A quadru-
pole mass spectrometer system Navigator (Finnigan MAT, San Jose,
CA, USA) was used for the analysis. The instrument was fitted with
an atmospheric pressure chemical ionization source (vaporizer
temperature 400 °C, capillary heater temperature 220 °C, corona
pathway for the biosynthesis of VLCPUFAs in A. carterae. We spec-
ulate that a-oxidation is an important mechanism in the shorten-
ing of fatty acids and that odd-chain VLCPUFA are normal
metabolic products that, however, occur in concentrations an order
of magnitude lower than even-numbered ones.
current
5 lA, sheath gas – high-purity nitrogen, pressure
3. Experimental
0.38 MPa, and auxiliary gas (also nitrogen) flow rate 15 ml/min.
Positively charged ions with m/z 50–700 were scanned with a scan
time of 0.5 s. The whole HPLC flow (0.37 ml/min) was introduced
into the APCI source without any splitting. Fatty acid picolinyl es-
ters were separated using a gradient solvent program with aceto-
nitrile (ACN), dichloromethane (DCM) and propionitrile (EtCN) as
follows: initial ACN/EtCN/DCM (60:30:10, vol/vol/vol); linear from
5 min to 50 min ACN/EtCN/DCM 30:40:30, vol/vol/vol); held until
60.5 min; the composition was returned to the initial conditions
over 8 min. A peak threshold of 0.3% intensity was applied to the
mass spectra. Data acquisition and analyses were performed using
PC with MassLab 2.0 for Windows XP applications/operating
software.
3.1. Instrumental methods
UV spectra were measured by a Cary 118 (Varian) apparatus in
MeOH in the range 200–350 nm. A Perkin–Elmer Model 1310
(PerkinElmer, Norwalk, CT) infrared spectrophotometer was used
for scanning infrared spectroscopy of methyl esters as a neat film.
NMR spectra were recorded on a Bruker AMX 500 spectrometer
(Bruker Analytik, Karlsruhe, Germany) at 500.1 MHz (1H) and
125.7 MHz (13C) in CDCl3. The fatty acids and other compounds
were purchased from Sigma-Aldrich (Prague, Czech Republic).
GC–MS of a FAME mixture was done on a Finnigan 1020-B in EI
mode. The hydrogenated sample was injected onto
a
25 m ꢁ 0.25 mm ꢁ 0.1
l
m
Ultra-1 capillary column (Supelco,
3.2. Standards, cultivation, isolation and synthesis
Czech Republic) (injector and detector temperatures of 300 °C) un-
der a temperature program of: 5 min at 50 °C, increasing at 10 °C
minꢀ1 to 320 °C and 15 min at 320 °C. Helium was the carrier gas
at a flow of 0.52 ml minꢀ1. All spectra were scanned within the
range m/z 50–700.
Standards of fatty acids were prepared as described below. All
solvents were double distilled and degassed before use.
The strain of the dinoflagellate A. carterae was obtained from
the Culture Collection of Algae and Protozoa in Scotland (strain

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T. Rezanka et al. / Phytochemistry 69 (2008) 2849–2855
2853
100
75
50
25
0
518
N
262
COO
302
93
109
342
234
382
557
151
164
1
462
422
408
422
382
368
571
192
206
342
328
462
448
55
220
248
302
288
67
262
178
488
502
50
100
150
200
250
300
350
m/z
400
450
500
550
600
650
700
Fig. 3. Mass spectrum of picolinyl ester of 29:6n-3 acid. For explanation of values see the text.
CCAP 1102/3, isolated from a ditch in Essex, England). The alga was
cultivated in liquid K minimum medium for marine dinoflagellates
(Keller et al., 1987) at 10 °C under continuous light provided by
cool white fluorescent lights. After 5 weeks, the culture was har-
vested by centrifugation at 1500 rpm for 10 min.
The cells of A. carterae (195 mg) were extracted with 3 ꢁ 30 ml
of CHCl3–MeOH (2:1), yielding total lipids (6.8 mg), which were
dissolved in toluene (1 ml) and 1% sulfuric acid in methanol
(2 ml) was added. The mixture was left overnight in a stoppered
tube at 50 °C, then water (5 ml) containing sodium chloride (5%)
was added and the esters (2.17 mg) were extracted with hexane
(2 ꢁ 5 ml). The hexane layer was washed with water (4 ml) con-
taining potassium bicarbonate (2%), dried over anhydrous sodium
sulfate, filtered and the solvent was removed under reduced
pressure.
Ag+-TLC. The FAMEs were fractionated by Ag+-TLC using petro-
leum ether–diethyl ether–acetic acid (90:8:2, v/v) for the first sep-
aration and (70:28:2, v/v) for the second separation. Detection was
done with 0.2% 2,7-dichlorofluorescein (in ethanol) under UV lamp
at 366 nm. The zone of silica gel corresponding to PUFA methyl
100
N
520
262
75
50
25
0
COO
302
93
342
382
109
559
151
150
164
2
422
382
368
342
328
422
573
220
302
288
462
67
192
248
262
55
178 206 234
448
450
408
490
504
50
100
200
250
300
350
400
500
550
600
650
700
m/z
Fig. 4. Mass spectrum of picolinyl ester of 29:5n-6 acid. For explanation of values see the text.

ˇ
T. Rezanka et al. / Phytochemistry 69 (2008) 2849–2855
2854
Fig. 5. Hypothesis of odd VLCPUFAs biosynthesis. Black – n-6 isomers, red – n-3 isomers, bold – isomers found in the alga, underlined – isomers not found. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
esters was scraped off and FAMEs were extracted from the silica
gel using hexane–diethyl ether (9:1). The FAMEs were washed
with saturated NaCl and 2 M NH3 to remove Ag+ and dichlorofluo-
rescein, respectively. The structure of FAMEs (1.42 mg) was con-
firmed by LC–MS as described above.
Picolinyl esters of PUFA. A solution of potassium tert-butoxide
in tetrahydrofuran (0.5 ml, 1.0 M) was added to nicotinyl alcohol
(1 ml). After mixing, the appropriate fatty acid methyl esters
(ꢂ0.5 mg) in dry dichloromethane (1 ml) were added, and the mix-
ture was held at 40 °C for 30 min in a closed vial. After cooling to
room temperature, water and hexane were added, and the organic
phase was collected, dried over anhydrous sodium sulfate, and
evaporated.
was added followed by a derivative of pyran (99 mg, 0.20 mmol)
in absolute ethanol (0.5 ml). Hydrogen uptake was quantitative
in 4 h. The reaction mixture was filtered, diluted with water, and
extracted with diethylether. The combined ether extracts were
washed with water, cold 1 M HCl, brine, dried, and concentrated
to give of crude product as brown colored oil. Chromatography
on preparative TLC plates, using diethylether–hexane (15:85), gave
87 mg (88%) of polyene 20-((all-Z)-nonacosa-11,14,17,20,23,26-
hexaenyloxy)tetrahydro-2H-pyran as pale yellow colored oil. HRE-
IMS m/z calcd. for 496.4280 C34H56O2 [M]+, found 496.4276.
A solution of polyene THP ether (85 mg, 0.17 mmol) and PPTS
(pyridinium p-toluenesulfonate) (3 mg, 0.012 mmol) in ethanol
(2 ml) was stirred at 45 °C (bath temperature) for 10 h (Miyashita
et al., 1977). The solvent was evaporated in vacuo, and the residue
was chromatographed on preparative TLC, using hexane–diethyl-
ether (3:1) to afford pure alcohol ((all-Z)-nonacosa-11,14,
17,20,23,26-hexaen-1-ol) (69 mg, 98%). HREIMS m/z calcd. for
412.3705 C29H48O [M]+, found 412.3701.
Hydrogenation of PUFAs was carried out in 1 ml of methanol
with catalytic amounts of PtO2. FAMEs were extracted with n-hex-
ane and subjected to GC–MS.
3.3. (all-Z)-Nonacosa-11,14,17,20,23,26-hexaenoic acid (1)
To a solution of polyene alcohol (66 mg, 0.16 mmol) in 5 ml
acetone was added under stirring at ꢀ2 °C in 30 min a solution of
To an EtMgBr solution (4 ml of a 2 M solution in THF, 8.0 mmol)
at 0 °C was added dropwise a solution of 5 (149 mg, 0.56 mmol) in
dry THF (2.5 ml). The reaction mixture was warmed up to room
temperature over 10 min and then heated under reflux for
10 min (Heitz et al., 1989). After cooling at 0 °C, copper bromide
(114 mg, 0.8 mmol) was added, followed by dropwise addition of
bromide 6 (173 mg, 0.56 mmol) in dry THF (2.5 ml). The resulting
reaction mixture was heated under reflux for 12 h, then cooled to
room temperature, quenched by addition of water, and filtered.
The reaction mixture was extracted with ether, and the combined
organic extracts were washed with brine, dried, and concentrated.
Purification of the crude product 20-((all-Z)-nonacosa-14,17,
20,23,26-pentaen-11-ynyloxy)tetrahydro-2H-pyran on silica gel
(hexane-diethylether 9:1) gave 100 mg (36%) of a colorless oil,
HREIMS m/z calcd. for 494.4124 C34H54O2 [M]+, found 494.4121.
P-2 nickel was prepared via NaBH4 reduction of Ni(OAc)2 ꢃ 4H2O
(74 mg, 0.3 mmol) in absolute ethanol (5 ml). The flask was purged
Na2Cr2O7ꢃ2H2O (48 mg, 0.16 mmol) in 50
ll conc. sulfuric acid
and 0.5 ml water (Kunau, 1971). The mixture was stirred for an-
other 2 h at 0 °C and, subsequently, poured into 3 ml of ice water.
After the acetone had been completely evaporated under reduced
pressure, the aqueous phase was thoroughly extracted with ether.
The ether was washed neutral with water, dried, and evaporated,
leaving a crude product. After purification by TLC on silica gel (hex-
ane–diethylether, 2:1) the yield was 32.7 mg (48%) of acid 1 as col-
orless oil, purity 99% (GLC as methyl ester). IR (neat) 3550–2550
(br OH), 1710 (C@O) cmꢀl 1H NMR d 0.88 (t, 3 H, CH3), 1.22–1.34
;
(brm, 12 H, CH2), 1.70 (t, 2 H, CH2CH2COOH), 2.08 (m,
2H,@CHCH2CH2), 2.03 (m, 2 H, CH3CH2CH@), 2.37 (t, 2H,
CH2COOH), 2.82 (brm, 10H,@CHCH2CH@), 5.36 (m, 12 H, CH = ),
10.5 (brs, 1 H, COOH). 13C NMR d 14.3 (q, C-29); 20.7 (t, C-28),
24.5 (t, C-3), 25.0-32.0 (t, 7xC, C-4–C-10), 25.4–26.0 (t, 5x@CH–
CH2–CH@), 33.7 (t, C-2), 127.0–129.1 (d, 10xC), 129.8 (d, C-11),
132.3 (d, C-27), 179.4 (s, C-1, COOH); EIMS of methyl ester m/z
with hydrogen, and ethylenediamine (50 ll, 45 mg, 0.75 mmol)

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T. Rezanka et al. / Phytochemistry 69 (2008) 2849–2855
2855
440 (M+, 0.1), 408 (M-32, 0.9), 264
(7), 187 (15), 145 (19), 131 (22), 119 (36), 108
a
-ion (4), 241 (5), 215 (5), 201
Heitz, M.P., Wagner, A., Mioskowski, C., Noel, J.P., Beaucourt, J.P., 1989. Synthesis of
all-cis-1-bromo-4, 7, 10, 13-nonadecatetraene - a precursor to C-1 labeled
arachidonic-acid. J. Org. Chem. 54, 500–503.
Kakela, R., Ackman, R.G., Hyvarinen, H., 1995. Very long-chain polyunsaturated
fatty-acids in the blubber of ringed seals (Phoca hispida sp.) from Lake Saimaa,
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Keller, D.K., Selvin, R.C., Claus, W., Guillard, R.R.L., 1987. Media for the culture of
oceanic ultraphytoplankton. J. Phycol. 23, 633–638.
x
-ion (48), 105 (45),
91 (89), 79 (100), 67 (61), 55 (37); HREIMS m/z calcd. for 426.3498
C29H46O2 [M]+, found 426.3494.
3.4. (all-Z)-Nonacosa-11,14,17,20,23-pentaenoic acid (2)
Kroumova, A.B., Xie, Z., Wagner, G.J., 1994. A pathway for the biosynthesis of
straight and branched, odd- and even-length, medium-chain fatty acids in
plants. Proc. Natl. Acad. Sci. USA 91, 11437–11441.
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Mansour, M.P., Volkman, J.K., Holdsworth, D.G., Jackson, A.E., Blackburn, S.I., 1999a.
Very-long-chain (C28) highly unsaturated fatty acids in marine dinoflagellates.
Phytochemistry 50, 541–548.
Mansour, M.P., Volkman, J.K., Jackson, A.E., Blackburn, S.I., 1999b. The fatty acid and
sterol composition of five marine dinoflagellates. J. Phycol. 35, 710–720.
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lipid class, fatty acid and sterol composition in the marine dinoflagellate,
Gymnodinium sp. In batch culture. Phytochemistry 63, 145–153.
Mansour, M.P., Frampton, D.M.F., Nichols, P.D., Volkman, J.K., Blackburn, S.I., 2005.
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unusual C24–C28 polyunsaturated fatty acids. J. Appl. Phycol. 17, 287–300.
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mild and efficient catalyst for tetrahydropyranylation of alcohols. J. Org. Chem.
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Yoshizumi, H., 2000. C19 odd-chain polyunsaturated fatty acids (PUFAs) are
metabolized to C21-PUFAs in a rat liver cell line, and curcumin, gallic acid, and
their related compounds inhibit their desaturation. Biosci. Biotechnol. Biochem.
64, 1641–1650.
This compound, prepared from the THP ether (5) (149 mg,
0.56 mmol) and bromide (7) (174 mg, 0.56 mmol) following mul-
ti-step synthesis as described for the synthesis of the acid 1, was
obtained (after purification by TLC on silica gel–hexane-diethyl-
ether, 2:1) as colorless oil (28.7 mg, 12%); purity 98% (GLC as
methyl ester). IR (neat) 3550–550 (br OH), 1710 (C@O) cmꢀl 1H
;
NMR d 0.88 (t, 3H, CH3), 1.20–1.33 (brm, 18H, CH2), 1.71 (t, 2H,
CH2CH2COOH), 2.10 (m, 4H,@CHCH2CH2), 2.36 (t, 2H, CH2COOH),
2.83 (brm, 8H,@CHCH2CH@), 5.37 (m, 10H, CH@), 10.4 (brs, 1H,
COOH); 13C NMR d 14.2 (q, C-29); 22.6 (t, C-28), 24.6 (t, C-3),
25.2-31.8 (t, 10xC, C-4–C-10, C-25–C-27), 25.4–25.8 (t, 4x@CH–
CH2–CH@), 33.5 (t, C-2), 126.9–128.7 (d, 8xC), 129.1 (d, C-11),
131.9 (d, C-24), 179.6 (s, C-1, COOH); EIMS of methyl ester m/z
442 (M+, 0.1), 410 (M-32, 0.7), 264
159 (14), 150 -ion (25), 145 (23), 119 (34), 105 (51), 91 (88),
a-ion (5), 241 (6), 215 (7),
x
79 (100), 67 (62), 55 (29); HREIMS m/z calcd. for 428.3654
C29H48O2 [M]+, found 428.3651.
Poulos, P., Sharp, D., Johnson, I., White, A., Fellenberg, A.J., 1986. The occurrence of
polyenoic fatty-acids with greater than 22 carbon-atoms in mammalian
spermatozoa. Biochem. J. 240, 891–895.
Acknowledgement
Rezanka, T., 1989. Very-long-chain fatty-acids from the animal and plant kingdoms.
Progr. Lipid Res. 28, 147–187.
Rezanka, T., 1993. Polyunsaturated and unusual fatty-acids from slime-molds.
Phytochemistry 33, 1441–1444.
This work was supported by the Institutional Research Concepts
AV0Z50200510, AV0Z60050516 and MSMT 002162082.
Rezanka, T., 2002. Identification of very long chain fatty acids by atmospheric
pressure chemical ionization liquid chromatography-mass spectroscopy from
green alga Chlorella kessleri. J. Sep. Sci. 25, 1332–1336.
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