I.R. Chechetkin et al.
Phytochemistry157(2019)92–102
whether linolipins are unique for flax or also biosynthesized in other
plant species, we carried out the screening of linolipins in the leaves of
several higher plants. These studies led to the detection and identifi-
cation of eight molecular species of linolipins in the leaves of meadow
buttercup (Ranunculus acris L., Ranunculaceae). The results of this work
are described here.
dinor-(ω5Z)-etherolenic and (ω5Z)-etherolenic acids (respectively) via
the cleavage of ester bridges. The second pair corresponds to anions of
(ω5Z)-etherolenic and dinor-(ω5Z)-etherolenic acids, respectively.
The positive ion mode mass spectrum of compound 1 (Table S1)
exhibited the adduct [M + NH4]+ at m/z 792.4865 (C43H70O12N). MS/
MS of the latter yielded several positive ions (Table S1). The daughter
ions at m/z 677.3910 and 579.3144 are consistent with the neutral
losses of hexenals via the ether bond cleavages within the esterified
residues of (ω5Z)-etherolenic and dinor-(ω5Z)-etherolenic acids. The
ions at m/z 595.4019 and 613.4111 were formed through the frag-
mentation of glycoside bonds that accompanied by the neutral losses of
galactose and dehydrated galactose, respectively. The most abundant
ion (m/z 529.3881), as well as the daughter ions at m/z 497.3239,
435.2765 and 417.2657, are consistent with the neutral losses of ga-
lactose and dehydrated galactose alongside with hexenyne or hexenal.
Both the MS and MS/MS data indicate that compound 1 has an MGDG
structure containing one residue of dinor-(ω5Z)-etherolenic acid and
one residue of (ω5Z)-etherolenic acid.
The deduced structure was confirmed by NMR spectral data. The
possessed the identical signals of glycerol and β-D-galactopyranose
moieties. The signals of glycerol protons H1a,b (4.32 and 4.18 ppm)
and H2 (5.19 ppm) were shifted downfield compared to signals of
H3a,b (3.89 and 3.68 ppm), thus indicating the presence of ester sub-
stituents at sn-1 and sn-2 and β-D-galactopyranose residue at the sn-3
position. The olefinic part of spectrum demonstrates the presence of
dinor-(ω5Z)-etherolenic acid residue (signals of eight double bond
protons H7″-H15″) and (ω5Z)-etherolenic acid residue (signals of eight
double protons H9‴-H17‴) between 5.25 and 6.75 ppm (Table 1). The
signals of olefinic protons of (ω5Z)-etherolenic acid moiety overlap
those of dinor-(ω5Z)-etherolenic acid moiety due to a small difference
in the chemical shifts but some of them can be distinguished from each
other (Fig. 5). The spectral parameters (Table 1), particularly the spin
2. Results
2.1. Detection of linolipins in the injured leaves of meadow buttercup
Leaves of basket willow (Salix viminalis L., Salicaceae,
Malpighiales), cabbage (Brassica oleracea L., Brassicaceae, Brassicales),
pea (Pisum sativum, Fabaceae, Fabales), roseroot (Rhodiola rosea L.,
Crassulaceae, Saxifragales), meadow buttercup (R. acris L.,
Ranunculales), garlic (Allium sativum L., Amaryllidaceae, Asparagales)
and Ipomoea tricolor Cav. (Convolvulaceae, Solanales) were examined
for the presence of linolipins. Galactolipids were isolated from the in-
tact leaves as well as from the freeze-thaw injured leaves and separated
by column chromatography and TLC. The ultraviolet (UV) spectrum of
monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol
(DGDG) fractions did not reveal the presence of linolipins (λmax at
267 nm) in the intact leaves. Only galactolipid fractions from the freeze-
thaw injured leaves of meadow buttercup, as well as from meadow
buttercup leaves infected with powdery mildew (Erysiphe aquilegiae var.
ranunculi (Grev.)) exhibited a strong UV absorption band at 267 nm,
suggesting the possible presence of linolipins there.
The molecular species of galactolipids isolated from meadow but-
tercup leaves were further separated by RP-HPLC. The analysis revealed
at least seven galactolipid molecular species exhibiting λmax at 267 nm
in the freeze-thaw injured leaves of meadow buttercup (Fig. 1b).
Compound 1 was prominent in the profile of galactolipid molecular
species isolated from meadow buttercup leaves infected with E. aqui-
legiae var. ranunculi, while compounds 2–7 could be detected only in
constant
values
J7″,8'' = 11.0 Hz,
J9″,10'' = 12.0 Hz,
J12″,13'' = 6.3 Hz, J14″,15'' = 10.9 Hz, as well as J9‴,10‴ = 11.0 Hz,
J11‴,12‴ = 12.0 Hz, J14‴,15‴ = 6.3 Hz, J16‴,17‴ = 10.9 Hz un-
ambiguously confirm (Z), (E), (Z), and (Z) configuration of the corre-
sponding double bonds in both acyl residues and overall the structures
of (7″Z,9″E,12″Z,14″Z)-10-oxa- 7,9,12,14-hexadecatetraenoyl [dinor-
(ω5Z)-etherolenoyl] and (9‴Z,11‴E,14‴Z,16‴Z)-12-oxa-9,11,14,16-
octadecatetraenoyl [(ω5Z)-etherolenoyl] residues, respectively.
To clarify the allocation of dinor-(ω5Z)-etherolenic acid and (ω5Z)-
etherolenic acid moieties between the glycerol sn-1 and sn-2 positions,
compound 1 was subjected to the hydrolysis by the sn-1-specific R.
arrhizus lipase. The liberated fatty acids (converted to Me esters) were
analyzed by GC-MS. Only (ω5Z)-etherolenic acid (as Me ester) but not
dinor-(ω5Z)-etherolenic acid (Me ester) was detected. These data de-
monstrate that the (ω5Z)-etherolenic acid and dinor-(ω5Z)-etherolenic
acid moieties are esterified at the sn-1 and sn-2 positions, respectively.
Based on the described data, compound 1 was assigned the structure of
1-O-(ω5Z)-etherolenoyl-2-O-dinor-(ω5Z)-etherolenoyl-3-O-β-D-galacto-
pyranosyl-sn-glycerol (Fig. 6). We suggest the trivial name “linolipin E”
for compound 1, the new complex oxylipin of linolipin family.
Compounds 1–7 were collected and finally purified by NP- HPLC for
structural elucidation. NP-HPLC enabled the separation of the ga-
lactolipid molecular species coeluted in the peak 3 to yield pure com-
pounds 3a and 3b (Fig. 2). Compounds 4, 6 and 7 were separated from
non-oxidized galactolipid species on
a cyanopropyl coated silica
column. Based on the high resolution electrospray ionization mass
spectral (ESI MS) data recorded in negative ion mode, the non-oxidized
galactolipid species coeluted in the peak 4 was identified as 18:3/16:3-
DGDG, that coeluted in the peak 6 as 18:3/16:3-MGDG, and that in
peak 7 as 18:3/18:3-DGDG. However, the sn-1/sn-2 distribution of the
acyl residues within the non-oxidized galactolipids was not studied in
this work.
2.2. Identification of compound 1 (linolipin E)
Pure compound 1 possessed a UV absorption spectrum identical to
those of divinyl ether (ω5Z)-etherolenic acid as well as linolipins A-D,
2009, 2008). Transesterification of compound 1 with sodium meth-
oxide afforded the methyl esters of dinor-(ω5Z)-etherolenic and (ω5Z)-
etherolenic acids, as judged by the data of GC-MS analyses (not shown).
The negative ion mode mass spectrum of compound 1 (Fig. 3 and
Table S1) exhibited a quasimolecular ion [M – H]- at m/z 773.4421
2.3. Identification of compounds 2, 3b and 4 (linolipins D, B and C)
The retention times of compounds 2, 3b and 4 on a C18 column, as
well as their UV absorption and ESI MS data (Tables S2, S3, S4), were
identical to those of authentic standards of linolipins D, B and C, re-
pounds 2 and 3b with sodium methoxide yielded only the methyl ester
of (ω5Z)-etherolenic acid, while transesterification of compound 4 af-
forded the methyl esters of α-linolenic and (ω5Z)-etherolenic acids, as
judged by the data of GC-MS analyses (not shown). R. arrhizus lipase
liberated (ω5Z)-etherolenic acid but not α-linolenic acid from
(C43H65O12
) and the adduct [M +
CH3COO]- at m/z 833.4650
(C45H69O14). The obtained high resolution ESI MS data are consistent
with the empirical formula C43H66O12 for compound 1. The MS/MS
spectrum of [M – H]- ion showed several diagnostic ions (Fig. 3 and
Table S1). The daughter ions at m/z 527.2842, 499.2514, 291.1948 and
263.1616 provide information about the fatty acid composition. The
first pair of ions is consistent with the neutral losses of dehydrated
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