Synthesis of long chain n-3 and n-6 fatty acids having a photoactive conjugated tetraene group

Article abstract of DOI:10.1016/j.chemphyslip.2004.02.006

Fatty acids of the n-3 and n-6 families containing a photoactive conjugated tetraene group near the carboxylate were prepared from several naturally occurring fatty acids by sequential iodolactonization and treatment with excess 1,8-diazabicyclo[5.4.0]undec-7-ene. The new conjugated fatty acids include 5E,7E,9E,11Z,14Z- and 5E,7E,9E,11E,14Z-eicosapentaenoic acids derived from arachidonic acid; 5E,7E,9E,11Z,14Z,17Z- and 5E,7E,9E,11E,14Z,17Z-eicosahexaenoic acids from eicosapentaenoic acid; and 4E,6E,8E,10Z,13Z,16Z,19Z- and 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acids from docosahexaenoic acid. All of the newly synthesized fatty acids were characterized by UV, ¹H NMR and mass spectroscopy. These new products represent the first examples of directed conjugation of methylene interrupted double bond systems. The products can be synthesized in gram quantities and in high yields (>50%). Interestingly, it did not prove possible to synthesize fatty acids having a triene group near the carboxyl group even using mild conditions and different synthetic approaches. Once initiated, the isomerization always continued until a tetraene group was formed. Because of the sensitivity of the tetraene group to light, these fatty acids have the potential for being used in tracking fatty acid movements in cells employing fluorescence techniques and in UV light-induced cross linking to membrane proteins.

Full text of DOI:10.1016/j.chemphyslip.2004.02.006

Chemistry and Physics of Lipids 130 (2004) 145–158
Synthesis of long chain n-3 and n-6 fatty acids having a
photoactive conjugated tetraene group
Dmitry V. Kukleva,b, William L. Smitha,b,∗
a
Department of Biological Chemistry, University of Michigan Medical School, 5416 Medical Science I,
1301 E. Catherine Street, Ann Arbor, MI 48109, USA
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA
b
Received 11 December 2003; received in revised form 25 February 2004; accepted 25 February 2004
Available online 26 April 2004
Abstract
Fatty acids of the n-3 and n-6 families containing a photoactive conjugated tetraene group near the carboxylate were prepared
from several naturally occurring fatty acids by sequential iodolactonization and treatment with excess 1,8-diazabicyclo[5.4.0]
undec-7-ene. The new conjugated fatty acids include 5E,7E,9E,11Z,14Z- and 5E,7E,9E,11E,14Z-eicosapentaenoic acids derived
from arachidonic acid; 5E,7E,9E,11Z,14Z,17Z- and 5E,7E,9E,11E,14Z,17Z-eicosahexaenoic acids from eicosapentaenoic acid;
and 4E,6E,8E,10Z,13Z,16Z,19Z- and 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acids from docosahexaenoic acid. All of
1
the newly synthesized fatty acids were characterized by UV, H NMR and mass spectroscopy. These new products represent
the first examples of directed conjugation of methylene interrupted double bond systems. The products can be synthesized in
gram quantities and in high yields (>50%). Interestingly, it did not prove possible to synthesize fatty acids having a triene group
near the carboxyl group even using mild conditions and different synthetic approaches. Once initiated, the isomerization always
continued until a tetraene group was formed. Because of the sensitivity of the tetraene group to light, these fatty acids have the
potential for being used in tracking fatty acid movements in cells employing fluorescence techniques and in UV light-induced
cross linking to membrane proteins.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Subj. Classification: Parinaric acid; Arachidonic acid; Docosahexaenoic acid; Eicosapentaenoic acid; Conjugated fatty acids
Keywords: Photoactive; Tetraene group; Fatty acid
Abbreviations: AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; Qa, fluorescent yield; MTAD,
4-methyl-1,2,4-triazoline-3,5-dione; 4-HDHE, 4-hydroxy,5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid; TMS, trimethylsilyl; 5-HEPE,
5-hydroxy,6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid; 5-HETE, 5-hydroxy,6E,8Z,11Z,14Z-eicosatetraenoic acid; THR, tetrahydrofuran;
PUFA, polyunsaturated fatty acid; HUFA, highly unsaturated fatty acid; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; eq, molar equivalent;
BHT, butylated hydroxytoluene; MS, mass spectrum
∗
Corresponding author. Tel.: +1-734-647-6180; fax: +1-734-734-3509.
E-mail address: smithww@umich.edu (W.L. Smith).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.02.006

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D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
1. Introduction
of devising synthetic techniques for constructing other
novel fatty acids.
Fatty acids with conjugated double bonds have
been known for many years to be naturally occur-
ring compounds (Solodovnik, 1967), and more re-
cently there has been an interest in exploring the
biological activities of these fatty acids (Banni and
Martin, 1998; Pariza et al., 2000; Scimeca et al., 1994;
Solodovnik, 1967; Yurawecz et al., 1999). Conjugated
fatty acids are claimed to exhibit anti-atherosclerotic
effects, to potentiate immune responses and to mod-
ulate energy metabolism (e.g. by promoting protein
as opposed to fat deposition). Another aspect of
the interest in conjugated fatty acids has been in
their use as fluorescent probes for studying mem-
brane structure (Sklar et al., 1975). Sklar et al.
(1975, 1976, 1977) demonstrated that parinaric acid
(9Z,11E,13E,15Z-octadecatetraenoic acid) can be
used to detect phase transitions in bilayers as well as
interactions among lipids and proteins (Comfort and
Howell, 2002). cis-Parinaric acid can be incorporated
into phospholipids by lipid biosynthetic pathways
and spectroscopic investigations can be performed.
cis-Parinaric acid has become widely used as a mem-
brane probe and is now available commercially. Ad-
ditional information about the dynamics of membrane
behavior as well as interactions between proteins and
lipids can be obtained by using other conjugated dou-
ble bond systems (e.g. with the chromophore in the
middle of the fatty acid or near the carboxyl group
(Goerger and Hudson, 1988).
2. Materials and methods
2.1. Reagents
Arachidonic acid (AA; 90%) and 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU) were purchased from
Sigma Chemical Co. (St. Louis, MO). Fish oil
(28% eicosapentaenoic acid (EPA) and 23% do-
cosahexaenoic acid (DHA)) was purchased from
Wal-Mart. Trifluoroacetic anhydride, ethanolamine,
isobutylchloroformate, pyridine, pyrrolidine and 4-
methyl-1,2,4-triazoline-3,5-dione were products of
Aldrich Chemical Co. (Milwaukee, WI) with purities
≥ 96%. Benzene, hexane, ether and acetonitrile were
distilled over phosphorus pentoxide and triethylamine,
tetrahydrofuran (THF) and methanol were distilled
over metallic sodium before use. DBU was distilled
over CaH2 in vacuo. Silica gel "Selecto" 32–63 ␮m
was purchased from Selecto Scientific (Suwanee,
GA). Thin layer chromatography (TLC) plates were
purchased from Sigma. Compounds on TLC plates
were visualized by spraying the plates with a 5% so-
lution of phosphomolybdic acid in methanol and then
heating the plates for 2–3 min at about 100 ◦C.
2.2. Mass spectrometry and HPLC
During the past fifteen years several polyconjugated
and polyunsaturated fatty acids have been isolated
from marine plants mostly by Gerwick and co-workers
(Michailova et al., 1995; Wise et al., 1994). In devel-
oping biochemical tools that might be used to study
fatty acids containing n-3 and n-6 double bonds, we
synthesized fatty acids bearing a photoactive conju-
gated tetraene group near the carboxyl group and a
natural methylene interrupted n-3 or n-6 grouping. We
believe this to be the first report of a directed syn-
thetic method for creating this type of polyconjugated
system of double bonds close to carboxyl group from
a methylene interrupted system of double bonds in
natural polyunsaturated fatty acids. Another aspect of
our studies involved investigating the stability of the
methylene interrupted system of cis-double bonds in
naturally occurring essential fatty acids in the context
Mass spectra were obtained using a Hewlett-
Packard 5890 gas chromatograph coupled to a
Hewlett-Packard 5970 series mass selective detector
operated with a Hewlett-Packard 7946 computer. Gas
chromatography conditions were as follows: He was
used as the carrier gas at a flow rate of 35 cm/s; the
oven temperature was kept at 210 ◦C; the injector
temperature was 250 ◦C; the interface temperature
was 250 ◦C; separations were on a capillary column
DB-5ms (30 m ×0.32 mm, 1 ␮m; J&W, USA); the in-
jector split ratio was kept constant at 1:60. The mass
detector conditions were as follows: the electron en-
ergy was 70 eV; the emission current was 0.8 mA; the
accelerating voltage was 8 kV; the scale was from 50
to 1000. HPLC analysis and preparative separations
were performed on an Alliance HPLC system (Wa-
ters, USA) equipped with a Waters 2695 separation

D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
147
module and a Waters 2996 photodiode array detector.
Analytical RP-HPLC was performed on a Nucleosil-
C18 analytical column (4.6 mm × 250 mm, 5 ␮m;
Xpertek, USA). Solid phase extraction was performed
on Luna-2 C18 guard column (10 mm × 50 mm,
10 ␮m; Phenomenex, Torrance, CA). Preparative sep-
arations were performed on a Kromasil C18 column
(10 × 250, 5 ␮m; Xpertek, Cobert Associates, St.
Louis, MO). 1H NMR spectra were recorded on a Var-
ian INOVA-300 spectrometer operated at 300 MHz;
for samples dissolved in CDCl3, tetramethylsilane
was used as the internal standard. All of the signal
assignments were performed on the basis of selective
decoupling experiments. All of the UV-Vis spectra
were recorded on a Hewlett-Packard 8453 instrument;
the UV-Vis spectrophotometer was operated with
ChemStation data processing software.
In the case of arachidonic acid its ␦-iodolactone
(Fig. 1, Ia) was synthesized from a commercially avail-
able concentrate with an AA content of about 90%.
The yield of chromatographically pure product was
62%.
2.4. Reaction of iodolactones with DBU to form
polyconjugated fatty acids (Fig. 1, reaction (1))
The ␦-iodolactone of eicosapentaenoic acid (Fig. 1,
Ib) (1.30 g, 3.0 mmol) was dissolved in 15 ml of dry
benzene and a solution of 1.2 g (7.9 mmol) of dry DBU
in 15 ml of dry benzene was added. The reaction mix-
ture was left at room temperature under nitrogen for
72 h in the dark. The yield of the expected conjugated
fatty acids (Fig. 1, IIIb) determined spectrophotomet-
rically was 93% (ε (303 nm) 74,000 l mol−1 cm−1).
=
Spectral information is presented in Section 3.
Similarly from 1.4 g (3.1 mmol) of the ␥-iodolactone
of docosahexaenoic acid (Fig. 1, Ic) through the
action of 1.2 g (7.9 mmol) of dry DBU, the conju-
gated fatty acids (IIIc) were obtained with a yield of
95%. The composition of the mixture of tetraenoic
fatty acids was ∼90% 4E,6E,8E,10Z,13Z,16Z,19Z-
docosaheptaenoic acid and ∼10% 4E,6E,8E,10E,13Z,
16Z,19Z-docosaheptaenoic acid as determined by
HPLC.
2.3. Preparation of iodolactones of DHA, EPA and
AA
A mixture of fish oil fatty acids (43.5 g, 28% EPA
and 23% DHA) was dissolved in 150 ml of ethanol
and 230 ml of a 7.5% solution of KHCO3 (17.3 g of
KHCO3; 1.2 eq. per total fatty acid) was added to give
a clear solution of fatty acid salts. A solution of 27 g
of I2 in 300 ml of ethanol (1.5 eq. per EPA plus DHA)
and 500 ml of hexane were added to the clear solu-
tion with vigorous stirring. The reaction mixture was
kept at room temperature (24 ◦C) for 16 h, the hex-
ane layer was removed, and the reaction mixture was
extracted with hexane (3 × 500 ml). The combined
hexane layers were washed sequentially with aqueous
5% Na2S2O3 (250 ml), water (500 ml) and saturated
aqueous NaCl (200 ml) and then dried over anhydrous
Na2SO4 and evaporated under vacuum. The crude oily
product (24.9 g, indicating ≥75% yield) was dissolved
in dry benzene (30 ml) and after addition of 2 g of BHT
was stored at −80 ◦C. Aliquots of this crude mixture
of the ␥-iodolactone of DHA (Fig. 1, Ic) and the ␦-
iodolactone of EPA (Fig. 1, Ib) were purified by col-
umn chromatography on silica gel as required to iso-
late the individual iodolactones. The purification needs
to be performed quickly using a 25–33-fold weight
excess of silica gel as the adsorbent and elution with
a gradient of ether in benzene (0–5%). To prevent on
column degradation of the iodolactones, the column
is prewashed with 1% BHT in dry benzene.
4E,6E,8E,10Z,13Z,16Z,19Z-Docosaheptaenoic acid
exhibited the following spectral properties: UV (me-
=
thanol): λmax = 290, 303, 317 nm; ε (303 nm)
74,000 l mol−1 cm−1. Fluorescence (methanol): Em
λmax = 428 nm; Qa = 0.017. MS of oxazoline (m/z,
(I%)): [M]+ 351 (17), [M−1]+ 350 (10), [M–CH3]+
336 (5), [M–C2H5]+ 322 (7), 202 (12), 242 (12),
282 (18), 322 (5), 85 (100), 98 (55), 111(30) (ꢀ4
double bond) and 141 (33). RP-HPLC (C18 column
eluted with 85:15:0.1, methanol–H2O–acetic acid):
1
k = 8.8. H NMR: δ 0.95 (3H, t, J = 7.5 Hz, H-
22), 2.06 (2H, m, H2-21), 2.35 (2H, m, H2-3), 2.4
(2H, m, H2-2), 2.80 (4H, m, H2-15,18), 2.88 (2H, m,
H2-12), 5.36 (7H, m, H-11,13,14,16,17,19,20), 5.66
(1H, dt, J5,6 = 14.4 Hz, J54 = 6.9 Hz, H-4), 6.08
(1H, m, H-10), 6.18 (4H, m, H-5,6,7,8), 6.46 (1H, dd,
J9,8 = 13.5, J9,10 = 11.1, H-9).
4E,6E,8E,10E,13Z,16Z,19Z-Docosaheptaenoic acid
yielded the following spectral data: UV (methanol):
λmax = 289, 300, 315 nm; ε 300 nm) ∼ 80,000 l mol−1
cm−1. Fluorescence: Em λmax = 422 nm; Qa
=

148
D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
Fig. 1. Formation of polyconjugated fatty acids from naturally occurring highly unsaturated fatty acids. Reagents and conditions: (i) 2.2 mol
DBU, 72 h, 24 ◦C; (ii) 1.1 mol DBU, 12 h, 24 ◦C; (iii) 0.2 N KOH, 40% ethanol, 16 h, 24 ◦C; (iv) CH2N2/ether; chlorotrimethylsilane/pyridine,
1 h, 70 ◦C; (v) 0.5% H2SO4 in methanol in a sealed ampule, 80 ◦C, 1 h; (vi) 3 mol triethylamine in methanol, boiling under reflux, 5 h;
(vii) 1.15 mol acetylbromide in ether, 1 h, 24 ◦C.
0.013. RP-HPLC (C18 column eluted with 85:15:0.1,
methanol–H2O–acetic acid) k = 9.5. MS of ox-
azoline the same as for its isomer noted above;
MS of the MTAD adduct (m/z (I%): [M]+453 (3),
[M–C4H7O2]+ 366(11), [M–C4H7O2–C2H3NO]+
309 (6), [M–C11H17]+ 304(8). 1H NMR: δ 0.95
(3H, t, J = 7.5 Hz, H-22), 2.06 (2H, m, H2-21),
2.35 (2H, m, H2-3), 2.4 (2H, m, H2-2), 2.80 (4H,
m, H2-15,18), 2.88 (2H, m, H2-12), 5.36 (6H, m,
gated fatty acids (Fig. 1, IIIa) were obtained in a yield
of 95% (determined spectrophotometrically). The
composition of the mixture of tetraenoic fatty acids
determined by HPLC was 85% 5E,7E,9E,11Z,14Z-
eicosapentaenoic acid and 15% 5E,7E,9E,11E,14Z-
eicosapentaenoic acid.
5E,7E,9E,11Z,14Z-Eicosapentaenoic acid ex-
hibited the following spectral properties: UV
=
(methanol): λmax = 290, 303, 317 nm; ε (303 nm)
H-13,14,16,17,19,20), 5.66 (2H, m, J4,5 ≈ J10,11
=
74,000 l mol−1 cm−1. RP-HPLC (C18 column eluted
14.5, J4,3 ≈ J11,12 = 7, H-4,11), 6.13 (6H, m, H-
with 85:15:0.1, MeOH–H2O–acetic acid) k = 9.7.
5,6,7,8,9,10).
Fluorescence (methanol): Em λmax
=
428 nm;
Similarly from 1.58 g (3.7 mmol) of the ␦-
iodolactone of arachidonic acid (Fig. 1, Ia) and the
action of 1.4 g (9.2 mmol) of dry DBU, the conju-
Qa = 0.015. MS of oxazoline (m/z, (I%)): [M]+
327 (41), [M − 1] + 326 (35), [M–1Me]+ 312
(15), [M–C5H11]+ 256 (11), [M–C7H13]+ 230 (7),

D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
149
[M–C8H11]+ 216 (10), 98 (35), 85 (100). H NMR
(300 MHz, CDCl3, δ, ppm): 0.89 (3H, t, J = 7 Hz,
H3-20), 1.33 (6H, m, H2-17,18,19), 1.73 (2H, tt,
J32 = 7.5; J34 = 7.4, H-3), 2.03 (2H, m, H2-16),
2.23 (2H, bdt, J43 = 7.4, J45 = 6.9, H2-4), 2.33
(2H, t, J23 = 7.5, H2-2), 2.95 (2H, m, H-13), 5.36
(3H, m, H-12,14,15), 5.64 (1H, dt, J5,6 = 14.4 Hz,
J54 = 6.9 Hz, H-5), 6.09 (1H, m, H-6), 6.18 (4H, m,
H-7,8,9,11), 6.47 (1H, dd, J10,9 = 13.7, J10,11 = 11,
H-10).
0.031 in., 2 ␮m PEEK frits (Alltech)). After concen-
trating the fatty acid, the guard column was attached
to a preparative HPLC column (C18, 250 × 10 mm,
5 ␮m, equipped with PEEK frits), and the system was
washed with methanol–water–acetic acid (85:15:0.1).
The fractions containing the purified fatty acid product
were combined and placed −80 ◦C. After an overnight
crystallization, a white precipitate of the conjugated
fatty acid was collected.
1
5E,7E,9E,11E,14Z-Eicosapentaenoic acid ex-
hibited the following spectral properties: UV
(methanol): λmax = 289, 300, 315 nm; ε (300 nm)
∼80,000 l mol−1 cm−1. RP-HPLC (C18 column eluted
with 85:15:0.1, MeOH–H2O–acetic acid) k = 10.6.
Fluorescence (methanol): Em λmax = 428 nm; Qa =
0.013. MS of oxazoline is the same as that noted above
for its isomer. Mass spectrometry of methyl ester of
MTAD adduct (m/z (I%)): [M]+ 429 (8), [M–C8H15]+
2.5. Reaction of iodolactones with DBU to form
allylic lactones (Fig. 1, reaction (2))
The ␥-iodolactone of docosahexaenoic acid (Fig. 1,
Ic) (2.10 g; 4.63 mmol;) was dissolved in 15 ml of
dry benzene and a solution of dry DBU (770 ␮l;
5.1 mmol) in 15 ml of dry benzene was added. The
reaction mixture was stirred at room temperature
under nitrogen for 12 h in the dark, then transferred
to a centrifuge tube and centrifuged at 1000 × g for
5 min. The clear solution was separated from the pre-
cipitate which was then vigorously stirred with dry
benzene (25 ml) and centrifuged again, and the clear
benzene solutions were combined. The resulting so-
lution was evaporated under vacuum, the dry residue
dissolved in a small amount of benzene and after ad-
dition of 0.25 g of BHT, the sample was purified by
column chromatography on 60 g of silica gel using
a gradient of dry ether in dry benzene (0–7%). The
yield was 1.03 g (68%) of the ␥-lactone of 4-HDHE
(4-hydroxy,5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic
acid (IIc, Fig. 1)) as a pale yellow oil.
Similarly, from ␦-iodolactones of EPA (Fig. 1,
Ib) and AA (Fig. 1, Ia) the corresponding ␦-
lactones of 5-HEPE (5-hydroxy,6E,8Z,11Z,14Z,17Z-
eicosapentaenoic acid (Fig. 1, IIb) and 5-HETE (5-
hydroxy,6E,8Z,11Z,14Z-eicosatetraenoic acid (Fig.
1, IIa) were synthesized in yields of 72 and 71%,
respectively. The spectral properties of these newly
synthesized compounds are in agreement with those
reported previously (Corey et al., 1983, 1980; Kuklev
and Bezuglov, 1998; Wright et al., 1987).
1
318 (50). H NMR: 0.89 (3H, t, J = 7 Hz, H3-20),
1.33 (6H, m, H2-17,18,19), 1.73 (2H, tt, J32 = 7.5;
J34 = 7.4, H-3), 2.03 (2H, m, H2-16), 2.23 (2H, bdt,
J43 = 7.4, J45 = 6.9, H2-4), 2.33 (2H, t, J23 = 7.5,
H2-2), 2.95 (2H, m, H2-13), 5.39 (2H, m, H-14,15),
5.68 (2H, m, J5,6 ≈ J11,12 = 14.5, J5,4 ≈ J12,13 = 7,
H-5,12), 6.13 (6H, m, H-6,7,8,9,10,11).
Conjugated fatty acids were purified as follows. The
reaction mixture was transferred to a centrifuge tube
and centrifuged at 1000 × g for 5 min. The clear solu-
tion was separated from the precipitate which was then
stirred vigorously with dry benzene (25 ml) and cen-
trifuged again, and the clear benzene solutions were
combined. The solution was transferred to an evap-
orating flask, 5 ml of dry diglyme (2-methoxyethyl
ether) was added, and the mixture was evaporated un-
der vacuum keeping the bath temperature at less than
30 ◦C. To the resulting solution of the reaction mixture
in diglyme were added 5 ml of methanol, 5 ml of wa-
ter and 600 ␮l of acetic acid. The mixture was stirred
for 30 s and put on a solid phase extraction cartridge
(pre-washed with 10 ml of methanol and then 10 ml
of water). The cartridge was eluted with 15 ml of wa-
ter, 30 ml of a methanol–water mixture (50:50, v/v)
and 2 ml of methanol. All the fatty acid was found in
the methanol fraction. To the solution of the conju-
gated fatty acid was added 6 ml of water, and the re-
sulting solution was pumped through a guard column
(50 × 10 mm, C18, 10 ␮m, equipped with 0.5 in. ×
2.6. Reaction of allylic lactones with excess DBU to
form polyconjugated fatty acids (Fig. 1, reaction (10))
The conditions for the reactions of allylic lac-
tones with excess DBU to form polyconjugated fatty

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D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
acids are as described above for the reaction between
iodolactones and DBU. Thus, from the ␥-lactone of
4-HDHE (Fig. 1, IIc) the corresponding polyconju-
gated fatty acids were obtained in a yield of 93%
(determined spectrophotometrically). Similarly, from
␦-lactones of EPA (Fig. 1, IIb) and AA (Fig. 1, IIa)
the corresponding polyconjugated fatty acids were
obtained in yields of 86%.
2.9. Cleavage of TMS-ethers of allylic alcohols
under acidic conditions: formation of tetraenoic
polyconjugated and polyunsaturated fatty acid
methyl esters (Fig. 1, reaction (8))
The dry oil of the trimethylsilylether of the methyl
ester of 5-HEPE (∼50 mg) (Fig. 1, IVb) was dissolved
in 1 ml of a 0.5% solution of H2SO4 in methanol (1 ml
of diglyme and 100 ␮l of 1N HCl or 50 ␮l trifluo-
roacetic acid may be used as well), the reaction mix-
ture was put in a sealed ampule and kept at 80 ◦C for
2 h. After cooling, the ampule was opened, and the
contents were dissolved in 5 ml of methylene chloride
and washed in turn with water (3 × 5 ml) and satu-
rated aqueous NaCl (50 ml), and then dried over an-
hydrous Na2SO4, filtered and stored at −80 ◦C. The
trimethylsilylethers of methyl esters of 5-HETE and
5-HEPE were converted to their corresponding esters
using similar protocols.
The yields of the conjugated tetraenoic acid methyl
esters measured spectrophotometrically were 70–74%
based on the parent trimethylsilylether of the methyl
ester of the hydroxy acid. The isomer compositions
determined by HPLC analysis after a 2-h reaction time
were the same as for the synthesis involving the use
of excess DBU. There was a tendency for the amount
of the all trans-isomers to increase with the reaction
time (data not shown).
2.7. Base cleavage of allylic lactone ring—hydroxy
acid formation (Fig. 1, reaction (6))
To a solution of 236 mg (0.72 mmol) of the ␥-
lactone of 4-HDHE in 5 ml of ethanol was added 1 ml
of 1.5N KOH, 3 ml of ethanol and 3 ml of water, and
the reaction mixture was stirred for 16 h at room tem-
perature. Water (10 ml) was added, and the solution
was acidified to pH 4 with 1.5N HCl and extracted
with hexane–ether (1:1) (3 × 10 ml). The resulting
organic extract was washed with water (2 × 20 ml)
and then a saturated solution of NaCl and dried over
Na2SO4. The dry extract was filtered and evaporated
under vacuum to yield 214 mg (86%) of 4-HDHE.
Similarly, 5-HETE and 5-HEPE were synthesized
from their corresponding ␥-lactones in yields of more
than 85%. The spectral properties of these hydroxy
fatty acids were identical to those reported previously
(Corey et al., 1980; Kuklev and Bezuglov, 1998;
Solodovnik, 1967).
2.10. Acid catalyzed cleavage of the allylic lactone
ring (Fig. 1, reaction (5))
2.8. Synthesis of methyl esters of TMS-ethers of
hydroxy acids (Fig. 1, reaction (7))
To a solution of 350 mg of the ␥-lactone of 4-
HDHE in 3 ml of absolute methanol was added 2 ml of
0.5% concentrated sulfuric acid in absolute methanol.
The mixture was put in an ampule, sealed and kept
at 80 ◦C for 1 h. The reaction mixture changed from
colorless to yellow. After cooling, the ampule was
opened and the reaction contents diluted with 50 ml of
water and 50 ml of methylene chloride. The organic
layer was separated and the aqueous layer extracted
with methylene chloride (2 × 50 ml). The combined
organic layers were washed sequentially with wa-
ter (50 ml) and saturated aqueous NaCl (50 ml),
dried over anhydrous Na2SO4, filtered and stored at
−80 ◦C. The yield determined spectrophotometrically
was 85%. The resulting crude mixture of methyl es-
ters of 4E,6E,8E,10Z,13Z,16Z,19Z-docosaheptaenoic
4-HDHE (60 mg) was converted to its methyl es-
ter using freshly distilled diazomethane in ether. The
solvent was evaporated and the sample dissolved in
1 ml of dry pyridine (freshly distilled over CaH2).
Chlorotrimethylsilane (50 ␮l) was added, and the re-
action mixture kept at 70 ◦C for 2 h. After cooling, the
reaction mixture was evaporated under a stream of ni-
trogen and the dry residue was dissolved in 2–5 ml of
hexane and filtered through 500 mg of silica gel. The
trimethylsilylether of the methyl ester of 4-HDHE had
an Rf ∼ 0.9 (hexane–ether, 1:1) and was used without
further purification. Similar procedures were used to
obtain the methyl esters of 5-HETE and 5-HEPE and
their corresponding trimethylsilylethers.

D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
151
acid (∼80%) and 4E,6E,8E,10E,13Z,16Z,19Z-doco-
saheptaenoic acid (∼20%) was purified by HPLC
and analyzed further (Fig. 1, VIIc). Similarly,
from the ␦-lactone of 5-HEPE, a mixture of
5E,7E,9E,11Z,14Z,17Z-eicosahexaenoic (∼80%) and
over Na2SO4. The dry extract was filtered, evaporated
under vacuum and purified by column chromatogra-
phy on 10 g of silica gel using a gradient of ether in
hexane (0 → 30%). The yield was 355 mg (87%) as a
colorless oil with Rf ∼ 0.65–0.8 (hexane–ether, 1:1).
The properties of the compounds in good agreement
with those reported earlier (Kuklev et al., 1996, 1997).
5E,7E,9E,11E,14Z,17Z-eicosahexaenoic
(∼20%)
acids (Fig. 1, VIIb) were synthesized with a total yield
determined spectrophotometrically of 81%; from the
␦-lactone of 5-HETE a mixture of 5E,7E,9E,11Z,14Z-
eicosapentaenoic (∼80%) and 5E,7E,9E,11E,14Z-
eicosapentaenoic (∼20%) acids (Fig. 1, VIIa) were
synthesized with a total yield determined spectropho-
tometrically of 83%. Spectral data for these newly
synthesized compounds were the same as those for the
conjugated polyunsaturated fatty acids synthesized
with the use of excess of DBU (as described above).
2.12. Reaction of bromoacetates with DBU to form
tetraenoic conjugated fatty acids (Fig. 1, reaction (9))
To a solution of isomeric bromoacetates (Fig. 1, V)
(200 mg) in 5 ml of dry benzene was added 200 ␮l (ca.
1.5 eq.) of dry DBU. The reaction mixture was kept
at room temperature for 96 h. The yield of conjugated
tetraene determined spectrophotometrically was 55%.
The final reaction mixture was handled as for the syn-
thesis of conjugated fatty acids from iodolactones (as
described above) and stored in methanol at −60 ◦C.
2.11. Synthesis of epoxides from iodolactones and
epoxide ring opening to corresponding isomeric
bromoacetates (Fig. 1, reactions (3) and (4))
2.13. Mass spectrometry
The methyl ester of 4,5-epoxy,7Z,10Z,13Z,16Z,19Z-
docosapentaenoic acid was synthesized as follows. To
a solution of 9.7 g (21 mmol) of the ␥-iodolactone of
DHA (Fig. 1, Ic) in 50 ml of methanol was added 16 ml
of triethylamine (11.6 g, 5.5 eq.). The reaction mixture
refluxed for 3 h and then evaporated under vacuum.
The residue was stirred with hexane (3 × 70 ml),
filtered, evaporated under vacuum and purified by
column chromatography on 30 g of silica gel using a
gradient of ether in hexane (0 → 10%). The yield was
5.40 g (71%) of Vc (Fig. 1) as a colorless, mobile oil
with an Rf ∼ 0.58 (hexane–ether, 1:1). MS: m/z (I%):
[M]+ 358 (3), [M–MeO]+ 327 (8). 1H NMR (CDCl3,
δ, ppm): 0.99t (H-22; 3H), 1.86m—(H-3; 2H), 2.40m
(H-6; 2H), 2,50m (H-2; 2H), 2.84m (H-9,12,15,18;
8H), 2.98m—(H-4,5; 2H), 3.69s—(COOCH3, 3H),
4-Methyl-1,2,4-triazoline-3,5-dione
(MTAD)
adducts of conjugated fatty acids were prepared as
described by Dobson (Dobson, 1998). An aliquot of
the reaction mixture was analyzed by GC-MS imme-
diately after derivatization. Oxazolines of conjugated
fatty acids were prepared and analyzed by GC-MS as
reported recently (Kuklev and Smith, 2003).
3. Results and discussion
3.1. Overview
The overall goal of the work described in this re-
port was to prepare derviatives of n-3 and n-6 highly
unsaturated fatty acids (HUFAs) that could be em-
ployed as photoactive probes for defining further the
biological functions of essential fatty acids. Using nat-
urally occurring HUFAs as starting materials (Kuklev
and Bezuglov, 1998), we were able to develop photo-
sensitive conjugated tetraene systems neighboring the
carboxyl groups while leaving the ␻-termini of the
acids intact. This involved sequential iodolactoniza-
tion of the carboxyl group of the starting HUFAs and
then treatment with at 2–2.5 molar excess of 1,8-
diazobicyclo[5.4.0]undec-7-ene (DBU) (Fig. 1). The
H-7,8,10,11,13,14,16,17,19,20—5.37m).
Spectral
data for this compound were in full agreement with
those reported previously (Kuklev et al., 1991, 1992).
The mixture of isomeric bromoacetates (VI) was
synthesized as follows. To a solution of 305 mg of
the methyl ester of 4,5-epoxy,7Z,10Z,13Z,16Z,19Z-
docosapentaenoic acid in 10 ml of dry ether was added
a solution of 120 mg (1.15 eq.) of acetylbromide in
10 ml of dry ether. The reaction mixture was kept at
room temperature for one hour and then washed with
water (3×20 ml) and saturated NaCl (25 ml) and dried

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D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
iodolactonization protocol is mild and provides for
regioselective modification of the double bond clos-
est to the carboxyl group of HUFAs in high yield
while leaving other double bonds unaltered (Kuklev
and Bezuglov, 1994; Solodovnik, 1967). Treatment of
the iodolactone with an equimolar amount of DBU
yielded the corresponding lactone of an allylic alcohol
that, in turn, could be saponified to form the corre-
sponding hydroxyacids—widely distributed products
of the oxidative metabolism of HUFAs (Corey et al.,
1980; Wright et al., 1987).
Importantly, we discovered that using a 2–2.5-fold
molar excess of DBU to HUFA iodolactones led to the
formation of fatty acids having four conjugated dou-
ble bonds instead of the expected triene conjugation.
The success of this procedure required the use of dry
benzene (distilled over P2O5) as the solvent and dry
reagent—DBU (distilled over CaH2 at reduced pres-
sure); using benzene or DBU that had not been sub-
jected to these treatments led only to the formation of
various by-products (data not shown). We also found
that the formation of the conjugated tetraene cassette
does not depend on the size of the iodolactone ring.
This was seen in comparing of the reactions of the
␥-iodolactone of DHA having a ring containing five
atoms and the ␦-iodolactones of EPA and AA that have
lactone rings with six atoms (Fig. 1). In all the cases
the reactions involved an initial formation of the lac-
tone of an allylic alcohol followed by opening of the
lactone ring.
Interestingly, when alcoholysis of the lactone ring
of allylic lactones was performed under acidic con-
ditions (i.e. H+/methanol) methyl esters of the cor-
responding tetraene conjugated acids are the main
products. The double bond positions and structures
of the methyl esters correspond to the methyl es-
ters synthesized through the action of DBU on the
iodolactone and then esterified with diazomethane;
again the size of the lactone ring was unimportant.
An additional confirmation of the transformation of
the natural system of methylene interrupted double
bonds was obtained while investigating the reaction
of isomeric mixtures of vicinal bromoacetates with
DBU (Fig. 1). The bromoacetates were synthesized
from the corresponding epoxides that were obtained
from iodolactones as described in Section 2. The re-
action between these bromoacetates and DBU leads
to formation of methyl esters with the same conju-
gated tetraene group as those present in methyl esters
obtained by acidic alcoholysis of allylic lactones and
those obtained directly from iodolactones. We also
stress that treatment of trimethylsilyl ethers of allylic
alcohols (5-HETE, 5-HEPE, 4-HDHE) with aqueous
hydrochloric or trifluoroacetic acid leads to formation
of the same conjugated tetraene group as those ob-
tained directly from natural PUFAs as starting mate-
rials. Thus, an intermediate lactone is not required for
the formation of conjugated tetraenes.
Analysis of the reaction mixtures by HPLC demon-
strated: (a) the absence of any remarkable amounts
of conjugated triene-containing species (i.e. materials
with absorption maxima at 270–280 nm), (b) the iden-
tity of the products obtained from a given fatty acid
using different synthetic techniques as determined by
spectral and chromatographic properties, and (c) sys-
tems consisting only of carbon–carbon double bonds
possessing well-defined UV spectral features and in-
sensitivity to reduction by LiAlH4.
Purification of the conjugated polyunsaturated fatty
acids did present problems because these acids readily
undergo isomerization, polymerization and oxidation
(Frankel, 1979; Johnson, 1979; Johnson and Pryde,
1979; Lopez and Gerwick, 1987). It is essential to use
the procedures that are detailed in Section 2 paying
particular attention to the use of dry solvents, mild
temperatures and minimizing exposure to metals. Us-
ing the appropriate techniques it is possible to transfer
these fatty acids not only to methanol but to methylene
chloride or chloroform (or CDCl3) (e.g. for analysis
by 1H NMR).
Because the highest yields were observed in re-
actions of iodolactones with an excess of DBU, we
discuss further here formation of the conjugated
tetraenoic fatty acids involving these reaction condi-
tions. Because the procedures and outcomes were very
similar to one another for AA, EPA and DHA, we de-
scribe in the following sections only the compounds
derived from EPA. Experimental data on compounds
derived from AA and DHA are detailed in Section 2.
3.2. Preparation of iodolactone of EPA
A mixture of the ␥-iodolactone of DHA (Fig. 1, Ic)
and the ␦-iodolactone of EPA (Fig. 1, Ib) was pre-
pared in about 75% yield by treating a mixture of the
potassium salts of these two fatty acids with I2. The

D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
153
reaction was optimized to eliminate other I2 addition
products. The individual iodolactones were isolated by
column chromatography on silica gel which removed
any unreacted fatty acids (i.e. fatty acids lacking a ꢀ3,
ꢀ4 or ꢀ5 double bond). The spectral properties of
compounds Ia–c (Fig. 1) were in excellent agreement
with those reported earlier (Corey et al., 1983; Kuklev
et al., 1991; Wright et al., 1987).
UV spectra of the compounds separated by HPLC
showed characteristic UV absorption characteristics of
a trans, trans, trans, cis tetraene functionality (λ =
290.1, 303.2, 317.5 nm) for Compound 1 that was re-
placed in the absorption spectrum of Compound 2
with a pattern characteristic of an all trans tetraene
(λ = 289, 300.8, 315 nm) (Hamberg, 1995) (Fig. 3).
The main difference between the spectra is in the
hypochromic shift of the peak maxima for Compound
2 by about 3 nm. Preliminary observations of the spec-
troscopic properties of the two molecules, their mo-
bilities on HPLC and the spontaneous conversion of
Compound 1 to Compound 2 at room temperature (as
monitored by HPLC) suggested that these two com-
pounds were geometrical isomers.
The fluorescence emission spectrum of Compound
1 derived from EPA was determined in methanol at
23 ◦C. The emission origin is at about 350 nm and
there is a broad maximum at about 422 nm for the
all trans isomer and at about 428 for the trans, trans,
trans, cis isomer. The emission spectrum is essentially
independent of the solvent (data not shown). The flu-
3.3. Reaction of the iodolactone of EPA with DBU to
form polyconjugated fatty acids
Two major peaks were always present upon RP-
HPLC of reaction mixtures involving iodolactones and
excess DBU. Thus, HPLC analysis of the product mix-
ture from treatment of the ␦-iodolactone of EPA with
2.2 eq. of DBU for 72 h provided a Compound 1 with
k1 = 5.75 (10.87 min) and a Compound 2 with k2 =
6.16 (11.53 min) (Fig. 2). The difference in the reten-
tion times between the compounds was sufficient to
permit them to be separated on a preparative scale in
high purity (>95%).
Fig. 2. HPLC analysis of the reaction mixture (following solid phase extraction to remove DBU) of the conjugated fatty acids derived
from eicosapentaenoic acid. Column: Nucleosil-C18 (4.6 × 250, 5 ␮m); mobile phase: methanol–water–acetic acid (85:15:0.1); flow rate:
1.5 ml/min; detection: 300 nm.

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D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
Fig. 3. UV spectra (in methanol) of Compounds 1 and 2 derived from eicosapentaenoic acid.
orescence quantum yield was determined as described
earlier (Sklar et al., 1977; Solodovnik, 1967) and was
similar (i.e. Qa = 0.015) to that of cis parinaric acid
(Sklar et al., 1977). The fluorescence data on poly-
conjugated fatty acids derived from DHA and AA are
provided in Section 2.
the m/z below 216, represented a very complicated
fragmentation of the molecule at sites of conjugation.
Nonetheless, prominent peaks were clearly seen for
ions at m/z 85 (100%), 98 (65%), 138 (94%) (ꢀ5
double bond). Therefore, the normal fragmentation of
oxazolines peculiar to fatty acids having methylene
interrupted double bond systems terminates at C-12,
but starts, as anticipated, for non-conjugated fatty
acids with C-5. This indicates that the position of the
conjugated system of double bonds is between C-5
and C-12 in Compound 2.
To confirm the position of the conjugated system of
double bonds the 4-methyl-1,2,4-triazoline-3,5-dione
(MTAD) adduct of Compound 2 derived from EPA
was analysed by GC-MS (Dobson, 1998) (Fig. 4).
MTAD reacted with conjugated tetraene to form at
least three major 1:1 adducts together with some
amounts of products having the same spectra but
different mobilities on gas chromatography—a prob-
lem of the method originally described by Dobson
(Dobson, 1998). That is, the reaction between Com-
pound 2 and MTAD proceeded as if Compound 2
had three independent diene systems. We detected all
three regioisomers of the 1:1 adducts between MTAD
Compounds 1 and 2 derived from EPA were ana-
lyzed in as oxazolines (Kuklev and Smith, 2003). In
the mass spectrum of the oxazoline of Compound 2
(the mass spectrum of Compound 1 was almost the
same) a prominent molecular ion is present at m/z
325 (18%), and this is accompanied by a peak at m/z
324 (14%). This suggests that the molecular mass
of Compound 2 is two a.m.u. less than that of the
starting EPA and correlates with the structure of Com-
pound 2 being an eicosahexaenoic acid. The spectrum
can be separated into two parts (Kuklev and Smith,
2003). The first part, located in the higher mass re-
gion, contained prominent ions at m/z 216 (17%), m/z
256 (20%), m/z 296 (8%) and m/z 310 (45%) that
represent two methylene interrupted double bonds
in the ␻3 position (i.e. fragments [M–CH3]+ at m/z
310, and [M–CH2CH3]+ at m/z 296). The second
part of the spectrum, in the lower mass region with

D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
155
Fig. 4. Mass spectrum of C9–C12 isomer of the MTAD adduct of Compound 2 derived from eicosapentaenoic acid. Molecular m/z 427
and diagnostic m/z 318 ions are clearly visible.
and Compound 2 and all of them had a prominent
molecular ion [M]+: m/z 427, ≈15%; and diagnostic
ions of [M–C8H13]+: m/z 318, 22%—identifying the
location of the ring adduct between C-9 and C-12
(and therefore a 9,11-diene system in Compound 2)
(see Fig. 4); [M–C10H15]+: m/z 292, 17% as ex-
pected for an adduct ring located between C-7 and
C-10 (and therefore a 7,9-diene system in Compound
2; [M–C12H17]+: m/z 266, 20% and [M–C5H9O2]+:
m/z 326, 8%—identifying an adduct ring between
C-5 and C-8 (and therefore a 5,7-diene system in
Compound 2). Hence, in Compound 2 the conjugated
tetraene group is located between C-5 and C-12, and
thus, the double bonds are at positions C-5, C-7, C-9
and C-11. The data from mass spectroscopy of oxazo-
lines and MTAD adducts of the polyconjugated fatty
acids synthesized from DHA and AA are presented
in Section 2.
13.7, J10,11 = 11.1, H-10); and (b) for Compound 2
obtained from EPA: δ 0.95 (3H, t, J = 7.6 Hz, H-20),
1.73 (2H, m, H-3), 2.05 (2H, m, H2-19), 2.16 (2H,
m, H-4), 2.35 (2H, t, 7.2, H-2), 2.76 (2H, m, H-16),
2.86 (2H, m, H-13), 5.39 (4H, m, H-14,15,17,18), 5.68
(2H, m, J5,6 ≈ J11,12 = 14.5, J5,4 ≈ J12,13 = 7.1,
H-5,12), 6.13 (6H, m, H-6,7,8,9,10,11).
1
It is clear from these H NMR data (Fig. 5) that
there are 12 olefinic methines, 6 aliphatic methylenes,
1 methyl group and 1 carbonyl carbon in both com-
pounds. Two of the methylenes were bis allylic groups
(as indicated by their chemical shifts, one at δ 2.80
and another at δ 2.96). Selective decoupling experi-
ments demonstrated coupling from the C-2-methylene
at δ 2.35 through the aliphatic protons at δ 1.73 (C-3)
to the δ 2.14 (C-4) and further to the olefinic proton at
approximately δ 5.6 at C-5. Similarly, coupling from
the terminal methyl at δ 0.96 through the methylene
protons at δ 2.06 (C-19) to the cluster of olefinic pro-
tons at δ 5.3–5.4 established the structure at the ␻ ter-
minus of the molecule. The relative positions in the
conjugated tetraene functionality of the eight low-field
proton signals in Compound 1 at δ 5.65 (1H), δ 6.08
(2H), δ 6.18 (4H), and δ 6.47 (1H) were assigned on
the basis of selective decoupling experiments and sup-
ported by comparison with 1H NMR spectra of Com-
1
Complete data for the H NMR spectra shown in
Fig. 5 are: (a) for Compound 1 synthesized from EPA:
δ 0.96 (3H, t, J = 7.5 Hz, H-20), 1.73 (2H, m, H-
3), 2.06 (2H, m, H-19), 2.14 (2H, m, H-4), 2.35 (2H,
t, 7.4, H-2), 2.80 (2H, m, H-16), 2.96 (2H, m, H-
13), 5.37 (5H, m, H-12,14,15,17,18), 5.65 (1H, dt,
J5,6 = 14.4 Hz, J54 = 6.9 Hz, H-5), 6.08 (1H, m, H-
6), 6.18 (4H, m, H-7,8,9,11), 6.47 (1H, dd, J10,9
=

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D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
Fig. 5. Low field fragments of 1H NMR (δ, ppm, CDCl3, 300 MHz) spectra of Compounds 1 and 2 derived from eicosapentaenoic acid.
pound 2 where eight low-field proton signals formed
two clusters at δ 5.7 (2H, H-5,H-12) and at δ 6.13
(6H, H-6 to H-11). Additional support for the assign-
ments was obtained by comparison of our chemical
shifts and coupling constants with those from experi-
ments with naturally occurring conjugated fatty acids
(Lopez and Gerwick, 1987; Michailova et al., 1995;
Wise et al., 1994).
The configuration of the conjugated double bonds
in Compound 1 can be seen from the data presented
in Fig. 5. Thus, the doublet of doublets at δ 6.47 (H-
10) with coupling constants of 13.7 and 11.1 Hz is a
very common signal for a methyne proton at the third
carbon in from the end of a conjugated system of dou-
ble bonds if the terminal double bond (C-11 to C-12)
has a cis-configuration and the second double bond
has a trans configuration (C-9 to C-10) (Michailova
et al., 1995; Wise et al., 1994). One more diagnostic
signal is the nicely resolved doublet of triplets at δ
5.65 that is well known for a terminal methyne pro-
ton at a trans double bond in a conjugated system
(Wise et al., 1994). Isomerization of Compound 1 to
Compound 2 results in the disappearance of the well
shaped signals at δ 5.65 and δ 6.47 and formation of
only two signal clusters at 6.1 (6H) and 5.7 (2H). The
coupling constants for the external methyne protons
were almost the same with values at 14.5 Hz (trans
double bond) and 7.1 Hz (aliphatic methylene). These
data are in excellent agreement with the structures of a
5E,7E,9E,11Z-tetraene system for Compound 1 and a

D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
157
Fig. 6. Structures of newly synthesized polyconjugated and polyunsaturated fatty acids (A) 5E,7E,9E,11Z,14Z-eicosapentaenoic acid;
(B) 5E,7E,9E,11E,14Z-eicosapentaenoic acid; (C) 5E,7E,9E,11Z,14Z,17Z-eicosahexaenoic acid; (D) 5E,7E,9E,11E,14Z,17Z-eicosahexaenoic
acid; (E) 4E,6E,8E,10Z,13Z,16Z,19Z-docosaheptaenoic acid; (F) 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acid.
5E,7E,9E,11E-conjugated system in Compound 2 de-
rived from EPA.
instead the apparently more stable conjugated tetraene
system forms spontaneously. This occurs independent
of the position of the most carboxyl proximal dou-
ble bond or the length of the methylene interrupted
double bond system. Formation of the tetraene is also
independent of the nature of the reagents (e.g. acidic
versus basic). The location of the conjugated double
bond system remains the same as the position of the
first double bond in the parent fatty acid (e.g. the
5,8,11 methylene interrupted system of AA (C-5 to C-
12) converts to the conjugated 5,7,9,11 system (C-5
to C-12). In surveying the literature, we were unable
to find any naturally occurring fatty acids with a con-
jugated triene system linked to a system of methylene
interrupted double bonds. This along with the results
of our synthetic studies led us to conclude that this
type of structure is unstable and would not exist in na-
ture. We synthesized and characterized the conjugated
tetraene system, which in contrast to the conjugated
triene system, is quite stable in the form of E,E,E,Z-
system before undergoing a slow spontaneous isomer-
ization to a E,E,E,E-system. This rearrangement has
not been described previously. All of the fatty acids
Based on results from HPLC, UV spectroscopy,
1
fluorescence spectroscopy, H NMR and GC-MS of
derivatives we assigned structures for the compounds
derived from AA: Compound 1 as 5E,7E,9E,11Z,14Z-
eicosapentaenoic acid (Fig. 6, structure A) and Com-
pound 2 as 5E,7E,9E,11E,14Z-eicosapentaenoic acid
(Fig. 6, structure B); for compounds derived from
EPA: Compound 1: 5E,7E,9E,11Z,14Z,17Z-eicosahe-
xaenoic acid (Fig. 6, structure C) and Compound 2:
5E,7E,9E,11E,14Z,17Z-eicosahexaenoic acid. (Fig. 6,
structure D); and for compounds derived from DHA:
Compound 1: 4E,6E,8E,10Z,13Z,16Z,19Z-docosahe-
ptaenoic acid (Fig. 6, structure E) and Compound
2: 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acid
(Fig. 6, structure F).
3.4. Formation of tetraene versus triene group
The results of our synthetic studies suggest that it is
not possible to introduce a triene structure neighbor-
ing a methylene interrupted system of double bonds;

158
D.V. Kuklev, W.L. Smith / Chemistry and Physics of Lipids 130 (2004) 145–158
bond positions by mass spectrometry. J. Lipid. Res. 44, 1060–
1066.
that we have synthesized have spectral properties and
chemical properties that may prove useful in investi-
gating the biochemistry of essential fatty acids.
Kuklev, D.V., Shevchenko, V.P., Nagaev, I.Y., Latyshev,
N.A., Bezuglov, V.V., 1991. The synthesis of 4,5-
dehydrodocosahexaenoic and 5,6-dehydroeicosapentaenoic
acids and their selectively tritium labeled derivatives.
Bioorganicheskaya Khimiya 17, 1574–1581.
Kuklev, D.V., Aizdaicher, N.A., Imbs, A.B., Bezuglov, V.V.,
Latyshev, N.A., 1992. All-cis-3,6,9,12,15-octadecapentaenoic
acid from the unicellular alga Gymnodinium kowalevskii.
Phytochemistry 31, 2401–2403.
Acknowledgements
This work was supported in part by Grant No.
DK22042 from the National Institutes of Health.
Kuklev, D.V., Christie, W.W., Durand, T., Rossi, J.C., Girard, J.P.,
Kasyanov, S.P., Akulin, V.N., Bezuglov, V.V.V., 1996. Synthesis
of ketodienoic compounds from natural unsaturated fatty acids.
1. Linoleic Acid. Bioorganicheskaya Khimiya 22 (8), 622–627.
Kuklev, D.V., Christie, W.W., Durand, T., Rossi, J.C., Vidal, J.P.,
Kasyanov, S.P., Akulin, V.N., Bezuglov, V.V., 1997. Synthesis
of keto- and hydroxydienoic compounds from linoleic acid.
Chem. Phys. Lipids 85, 125–134.
References
Banni, S., Martin, J.C., 1998. Conjugated linoleic acid and
metabolites. In: Sebedio, J.L., Christie, W.W. (Eds.), Trans
Fatty Acids in Human Nutrition. Oily Press, Dundee, Scotland,
pp. 261–302.
Lopez, A., Gerwick, W.H., 1987. Two new icosapentaenoic acids
from the temperate red seaweed ptilota filicina. J. Agardh.
Lipids 22, 190–194.
Comfort, S., Howell, N.K., 2002. Gelation properties of soya and
whey protein isolate mixtures. Food Hydrocolloids 16, 661–
672.
Michailova, M.V., Bemis, D.L., Wise, M.L., W.H., G., Norris,
J.N., Jacobs, R.S., 1995. Structure and biosynthesis of novel
conjugated polyene fatty acids from marine green alga
Anadyomene stellata. Lipids 30, 583–589.
Pariza, M.W., Park, Y., Cook, M.E., 2000. Mechanisms of action
of conjugated linoleic acid: evidence and speculation. Proc.
Soc. Exp. Biol. Med. 223, 8–13.
Corey, E.J., Sih, C., Cashman, J.R., 1983. Docosahexaenoic acid
is a strong inhibitor of prostaglandin but not leukotriene
biosynthesis. Proc. Natl. Acad. Sci. USA 80, 3581–3584.
Corey, E.J., Albright, J.O., Barton, A.E., Hashimoto, S., 1980.
Chemical and enzymic syntheses of 5-HPETE, a key biological
precursor of slow-reacting substance of anaphylaxis (SRS) and
5-HETE. J. Am. Chem. Soc. 102, 1435–1436.
Scimeca, J.A., Thompson, H.J., Ip, C., 1994. Effect of conjugated
linoleic acid on carcinogenesis. Adv. Exp. Med. Biol. 364,
59–65.
Sklar, L.A., Hudson, B.S., Simoni, R.D., 1975. Conjugated polyene
fatty acids as membrane probes. Preliminary characterization.
Proc. Natl. Acad. Sci. USA 72, 1649–1653.
Sklar, L.A., Hudson, B.S., Simoni, R.D., 1976. Conjugated polyene
fatty acids as fluorescent membrane probes: model system
studies. J. Supramol. Struct. 4, 449–465.
Sklar, L.A., Hudson, B.S., Simoni, R.D., 1977. Conjugated polyene
fatty acids as fluorescent probes: synthetic phospholipid
membrane studies. Biochemistry 16, 819–828.
Sklar, L.A., Hudson, B.S., Petersen, M., Diamond, J., 1977.
Conjugated polyene fatty acids on fluorescent probes:
spectroscopic characterization. Biochemistry 16, 813–819.
Solodovnik, V.D., 1967. Chemistry of natural aliphatic acids
containing a conjugated system of double bonds. Russ. Chem.
Rev. 36, 272–283.
Dobson, G., 1998. Identification of conjugated fatty acids by
gas chromatography-mass spectrometry of 4-methyl-1,2,4-
triazoline-3,5-dione adducts. J. Am. Oil Chem. Soc. 75, 137–
142.
Frankel, E.N., 1979. Fatty acids. Homogeneously catalyzed
reactions. In: Pryde, E.H. (Ed.), Fatty Acids. American
Chemical Society, Champaign, pp. 426–456.
Goerger, M.M., Hudson, B.S., 1988. Synthesis of all-trans-
parinaric acid-d8 specifically deuterated at all vinyl positions.
J. Org. Chem. 53, 3148–3153.
Hamberg, M., 1995. Biosynthesis and degradation of a-parinaric
acid and related conjugated tetraene fatty acids. In: Samuelson,
B., Paoletti, R., Ramwell, P. (Eds.), Advances in Prostaglandin,
Thromboxane and Leukotriene Research. Raven Press, New
York, pp. 193–198.
Johnson, R.W., 1979. Fatty acids. Dimerization and polymerization.
In: Pryde, E.H. (Ed.), Fatty Acids. American Chemical Society,
Champaign, pp. 342–352.
Wise, M.L., Hamberg, M., Gerwick, W.H., 1994. Biosynthesis of
conjugated triene-containing fatty acids by novel isomerase
from the red marine alga Ptilota filicina. Biochemistry 33,
15223–15232.
Wright, S.W., Kuo, E.Y., Corey, E.J., 1987. An effective process
for the isolation of docosahexaenoic acid in quantity from cod
liver oil. J. Org. Chem. 52, 4399–4401.
Yurawecz, M.P., Mossoba, M.M., Kramer, J.K.G., Pariza, M.W.,
Nelson, G.J., 1999. Advances in Conjugated Linoleic Acid
Research. AOCS Press, Champaign.
Johnson, R.W., Pryde, E.H., 1979. Fatty acids. Isomerization,
conjugation, and cyclization. In: Pryde, E.H. (Ed.), Fatty Acids.
American Chemical Society, Champaign, pp. 319–342.
Kuklev, D.V., Bezuglov, V.V., 1994. Halolactonization and
halolactones. Bioorganicheskaya Khimiya 20, 341–366.
Kuklev, D.V., Bezuglov, V.V., 1998. Chemical modification of
natural polyenoic fatty acids. In: Kuklev, D.V. (Ed.), Modified
Fatty Acids. Dalpribor PbsH, Vladivostok, Russia, pp. 1–16.
Kuklev, D.V., Smith, W.L., 2003. A procedure for preparing
oxazolines of highly unsaturated fatty acids to determine double