1-Acetyl-3-[(3R)-hydroxyfatty acyl]glycerols: Lipid Compounds from Euphrasia rostkoviana Hayne and E. tetraquetra (Bréb.) Arrond

Article abstract of DOI:10.1002/cbdv.201500233

Five homologous acetylated acylglycerols of 3-hydroxyfatty acids (chain lengths C(14) - C(18)), named euphrasianins A - E, were characterized for the first time in Euphrasia rostkoviana Hayne (Orobanchaceae) by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MSⁿ). In addition to mass spectrometric data, structures of euphrasianins were verified via a three-step total synthesis of one representative homologue (euphrasianin A). The structure of the latter was confirmed by 1D- and 2D-NMR experiments as well as high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). The absolute configuration of the 3-hydroxyfatty acid moiety at C(3) was found to be R in the natural euphrasianins, which was determined by alkaline hydrolysis and methylation of a purified fraction, followed by chiral GC analysis. Furthermore, in extracts of Euphrasia tetraquetra (Bréb.) Arrond. euphrasianins C and E were detected exclusively, indicating that this subclass of lipid constituents is possibly valuable for fingerprinting methods.

Full text of DOI:10.1002/cbdv.201500233

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Chem. Biodiversity 2016, 13, 602 – 612
FULL PAPER
1-Acetyl-3-[(3R)-hydroxyfatty acyl]glycerols: Lipid Compounds from Euphrasia
ꢀ
rostkoviana HAYNE and E. tetraquetra (BREB.) ARROND.
a
a
b
c
c
by Peter Lorenz* ), Diana N. Knittel ), Jurgen Conrad ), Eva M. Lotter ), Jorg Heilmann ), Florian C. Stintzing ), and
a
€
€
Dietmar R. Kammerera)
a) Department of Analytical Development & Research, Section Phytochemical Research, WALA Heilmittel GmbH,
€
Dorfstr. 1, DE-73087 Bad Boll/Eckwalden (phone: +4971649307099; fax: +4971649307080;
e-mail: peter.lorenz@wala.de)
b) Institute of Chemistry, Bioorganic Chemistry, Hohenheim University, Garbenstraße 30, DE-70599 Stuttgart
€
) Pharmaceutical Biology Department, University of Regensburg, Universitatsstraße 31, DE-93053 Regensburg
c
Dedicated to Prof. Ulrike Lindequist on the occasion of her 65th birthday.
Five homologous acetylated acylglycerols of 3-hydroxyfatty acids (chain lengths C(14) – C(18)), named euphrasianins
A – E, were characterized for the first time in Euphrasia rostkoviana HAYNE (Orobanchaceae) by gas chromatography/mass
spectrometry (GC/MS) and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass
spectrometry (HPLC/APCI-MSn). In addition to mass spectrometric data, structures of euphrasianins were verified via a
three-step total synthesis of one representative homologue (euphrasianin A). The structure of the latter was confirmed by 1D-
and 2D-NMR experiments as well as high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). The absolute
configuration of the 3-hydroxyfatty acid moiety at C(3) was found to be R in the natural euphrasianins, which was determined
by alkaline hydrolysis and methylation of a purified fraction, followed by chiral GC analysis. Furthermore, in extracts of
ꢀ
Euphrasia tetraquetra (BREB.) ARROND. euphrasianins C and E were detected exclusively, indicating that this subclass of lipid
constituents is possibly valuable for fingerprinting methods.
Keywords: Eyebright, Orobanchaceae, Acetyl monoacylglycerols, Hydroxyfatty acids, Euphrasianins.
or perennial flowering plants [5], belonging to the
Orobanchaceae family (formerly included in the Scrophu-
Introduction
Lipids are one of the most abundant classes of natural
constituents found in higher plants. With regard to the
chemical structure and physiological function, plant lipids
may be divided into seven groups: fatty acids, acylglyc-
erols, waxes, phospho- and sphingolipids, lipopolysaccha-
rides, and isoprenoids (e.g., steroids and carotenoids).
lariaceae), with a cosmopolitan distribution. These hemi-
parasitic species [6][7] are mostly found in areas with
temperate climate, with an emphasis on the mountains of
the northern hemisphere [8]. Several Euphrasia species,
mainly E. stricta D. WOLFF ex. F. J. LEHM and E. rostko-
viana HAYNE, including their subspecies and hybrids, were
Among these, triglycerides of long-chain fatty acids, synopsized by the term ‘E. officinalis L.’ [9]. Hence,
besides mono- and diglycerides, are the most common unambiguous taxonomic identification of Euphrasia spe-
storage lipids in plants (e.g., in seed oils) [1]. Plant lipids cies is mostly vague and presumably only possible based
comprise a multitude of biological functions, e.g., they on DNA sequences [8].
serve for energy production, as structure components of
The terms ‘eyebright’ or ‘eyewort’ indicate the long
membranes, as signal molecules or pigments, etc. [1]. In
history of the plant for eye disease treatment. Eyebright
plant cuticles, lipids represent a protective barrier against has been stated to exert astringent, anticatarrhal, and
environmental impacts, such as desiccation, temperature
extremes, microbial infection and exposure to intense UV
radiation [2 – 4].
anti-inflammatory activities [10]. Since the Middle Ages,
herbal decoctions of Euphrasia have traditionally been
applied to treat several diseases, such as nasal catarrh,
Within the scope of exploring biologically active lipid hoarseness, cough, sinusitis, gastric disorders, skin prob-
constituents from various medicinal plants, we took a clo-
ser look on the phytochemistry of European eyebright
(Euphrasia rostkoviana HAYNE). Euphrasia is a genus
lems and conjunctivitis [9][10]. To characterize the mode
of action more precisely anti-oxidant, radical scavenging,
antibacterial, antihyperglycemic, and cytokine inhibitory
comprising about 350 species of green herbaceous, annual activities of E. rostkoviana extracts have been ascertained
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Chem. Biodiversity 2016, 13, 602 – 612
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in vitro and in vivo [10 – 12]. Beside its use in folk medi- MeOH (w/v). After pH adjustment (pH 3.0), extraction
cine, preparations of E. rostkoviana are nowadays applied with AcOEt, solvent removal and silylation with BSTFA/
in phytotherapy, homeopathy, and anthroposophic medi- DMF GC/MS analyses were performed. Based on the
cine, e.g., for the treatment of clotty and inflamed eyes, mass spectra of their TMSi derivatives and by comparison
against conjunctivitis, stye, and blepharitis [9][13].
Numerous secondary metabolites have been found in
with NIST spectral data and implementation of silylated
reference compounds, five homologous 3-hydroxyfatty
E. rostkoviana, such as iridoids [14][15], lignans [16], phe- acids with chain lengths of C(14) – C(18) could be
nylethanoids [17 – 19], phenolic acids [20], flavonoids,
and tannins [21]. Furthermore, traces of alkaloids and
essential oil compounds have been reported [9][22][23].
Since studies on E. rostkoviana lipid constituents are still
assigned as major constituents of the mixture (data not
shown). Among these homologues, the even-numbered
C(14) and C(16) representatives were predominant.
Although 3-hydroxyfatty acids are an important class of
rare [24], this study shall focus on their closer investiga- microbial lipid constituents frequently found in the
tion. In addition to E. rostkoviana, a phylogenetically lipid A segment of bacterial lipopolysaccharides [25],
ulterior Euphrasia species, E. tetraquetra (seacliff eye- reports on their occurrence in higher plants are still rare.
bright), was included in this study for a phytochemical
comparison.
3-Hydroxyfatty acids have been identified in cuticular
waxes of Aloe arborescens [26], in Hypericum lysima-
chioides [27] and H. perforatum [28]. Further, 3-acetoxy
derivatives of 3-hydroxyfatty acids were found in floral
nectars of the Krameriaceae and Scrophulariaceae fami-
lies [29][30]. Glycerol esters of 3-hydroxyfatty acids were
Results and Discussion
For lipid constituent analysis, the fresh total plant mate-
rial of E. rostkoviana was extracted with CH2Cl2. Based analyzed in glandular trichome exudates of Verbascum
on GC/MS investigations, several known compounds were blattaria f. erubescens (Scrophulariaceae) [31].
detected in the obtained extract. By comparing their mass
spectra with those of the NIST database and reference
To determine the stereochemistry of the 3-hydroxy-
fatty acids, the AcOEt extract of the saponified material
compounds, a homologous series of n-alkanes (H–C from an E. rostkoviana CH2Cl2 extract was methylated by
(21) – C(31), odd-numbered representatives were domi- treatment with BF3/MeOH. Me esters of the 3-hydroxy-
nating) was identified, with n-nonacosane (H–C(29)) fatty acids (C(14) – C(18)) thus obtained, were separated on
being the predominant homologue (see GC/MS chro-
matogram, Fig. 1a). Moreover, free fatty acids, such as
palmitic (C(16):0) and linolenic acid (C(18):3), as well as
a chiral GC phase. By comparison with (3R)- and (3R,S)-
hydroxymyristic acid methyl ester as references, the absolute
configuration was found to be R (data not shown). Even the
a-tocopherol (a-Toco) and b-sitosterol (b-Sito) were absolute configuration of the fatty acid moiety was
assigned. Also coumarin could be detected in the CH2Cl2 only determined for compound 1, we assume it to be the
extract (data not shown). However, two compounds at tR same for the other homologous, because of biogenetic
45.4 and 49.2 min (1 and 3, respectively) could not be
readily assigned. Exhibiting high conformity in their mass
spectra, both 1 and 3 revealed signals at m/z 117 and 187
considerations.
Furthermore, silylation of the compounds in the
unsaponified fraction yielded the corresponding TMSi
as most intense fragments (see Table 1), demonstrating derivatives of 1 – 5 (for GC/MS data see Fig. 1c and
that these constituents could be homologues. However, Table 1). The latter compounds were characterized by
because of low intensity, the molecular ion signals of the
compounds could not be allocated. Interestingly, three
additional homologues of 1 and 3, compounds 2, 4, and 5
were detected in the extracted ion mode (MS scan of m/z
117 or 187).
two intense fragments each, one at m/z 129 and another
at m/z 189, as well as further characteristic fragments at
higher m/z values which increased by each additional
CH2 unit (see Scheme 1b and Table 1). Furthermore,
[MꢀCH3]+ fragments were formed as a result of the loss
For further analysis, the crude CH2Cl2 extract was of a Me radical from the TMSi group, and these frag-
subjected to normal-phase vacuum liquid chromatography
ments were detected as highest m/z signals in the mass
(VLC) purification on silica gel and subsequent Chroma- spectra (Table 1). By alignment with mass spectral data
totronâ centrifugal radial preparative TLC (silica gel/gyp- of similar structures reported [30], it was concluded that
sum) separation to enrich the target compounds 1 – 5 in 1 – 5 may be assigned to 1-acetyl-3-[(3R)-hydroxyfatty
a fraction. Fig. 1b exhibits a total ion chromatogram
(TIC) indicating the presence of 1 – 5, besides small
amounts of monoacyl glycerols of unsaturated fatty acids
acyl]-glycerol esters (Fig. 2). Additionally, isomers of the
main constituents TMSi-1 and -3 (TMSi-1* and 3*,
respectively, see Fig. 1c) were detected by GC/MS, which
(see insert, Fig. 1b). A gradual increase of the higher is presumably due to acetyl migration from the sn-1 to
fragments (m/z > 187) in the mass spectra between 1 and the sn-2 position of the glycerol unit upon derivatization.
5 by ‘CH2’ units indicated that these compounds to be
members of a homologous series (Table 1).
To analyze the fatty acid composition, an aliquot of
the purified fraction was saponified with 5% KOH in
To obtain additional structural information, com-
pounds 1 – 5 were separately investigated by HPLC-
DAD-MSn (Fig. 3, Table 2). After HPLC separation, com-
pound analysis was performed in the positive and negative
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Fig. 1. Sections of GC/MS profiles (EI) showing metabolites of the total plant of E. rostkoviana. a) Crude CH2Cl2 extract. b) Fraction as
obtained by chromatographic purification; the insert shows an expanded chromatogram (tR 44 – 48.5 min). c) Fraction after TMSi derivatization.
Assignment: C(16):0, hexadecanoic acid; C(18):3, a-linoleic acid; H–C(21) – H–C(31), n-alkanes C(21) – C(31); a-Toco, a-tocopherol; b-Sito,
b-sitosterol; 1 – 5, euphrasianins A – E; Glyc, Glycerol; 1 – 5-TMSi, euphrasianin-TMSi derivatives; 1*, 3*-TMSi, isomers due to acyl migration.
APCI mode. In the APCI+ mode (MS1), base peak ions at According to EI-MS studies on acetylated glycerol esters
m/z 343, 357, 371, 385, and 399 ([M+HꢀH2O]+) were of 3-acetoxy fatty acids [30] and acetylated cyclopentane
detected from the five homologues 1 – 5, respectively, diols [32], three 1,3-dioxolenium cationic structures were
generated by H2O expulsion (see Fig. 3 and Table 2). For postulated for the latter (Fig. 3b).
a staggered illustration of extracted ion chromatograms,
Interestingly, the occurrence of intense fragments at
see Fig. 3a. Upon subsequent fragmentation of the m/z 117 and 189 (base peaks) in the EI-MS spectra (see
[M+HꢀH2O]+ ions, daughter ions at m/z 159, 117, and 99 Table 1, Scheme 1a,b) of underivatized and silylated 1 – 5,
were detected in the MS2 and MS3 experiments (Fig. 3b). respectively, gave further evidence for this ion species. A
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Table 1. GC/MS data of euphrasianins (1 – 5) and their TMSi derivatives (1 – 5-TMSi), detected in a fraction of a CH2Cl2 extract from
E. rostkoviana L
Constituent
Underivatized
Characteristic fragment ion peaks,
m/z [% BPI]
TMSi derivatives
Characteristic fragment ion
peaks, m/z [% BPI]
tR [min]
Mr [Da]
tR [min]
Mr [Da]
1
2
3
4
5
45.4
360.5
374.5
388.5
402.6
416.6
342 (0.1)a), 324 (1), 287 (2), 269 (3), 227 (6),
209 (10)b)
44.7
504.8
518.9
532.9
546.9
560.9
489 (2)c), 299 (7), 257 (18)d)
503 (2)c), 313 (7), 271 (16)d)
517 (2)c), 327 (6), 285 (14)d)
531 (1)c), 341 (5), 299 (12)d)
545 (1)c), 355 (5), 313 (11)d)
47.3
49.2
50.9
52.7
356 (0.1)a), 338 (0.4), 301 (1), 283 (2), 241 (5),
223 (8)b)
46.5
48.2
49.9
51.5
370 (0.3)a), 352 (0.6), 315 (1), 297 (2), 255 (4),
237 (7)b)
384 (0.1)a), 366 (0.3), 329 (0.5), 311 (1), 269
(3), 251 (6)b)
398 (0.2)a), 380 (0.3), 343 (0.4), 325 (1), 283
(3), 265 (6)b)
a) [MꢀH2O]+, molecular ion signal not observed; b) In addition to characteristic fragments common to the compounds of one homologous ser-
ies, fragments at m/z 187 (88), 176 (4), 158 (22), 145 (16), 135 (20), 117 (100), 103 (13), 97 (8), 83 (7), 71 (14), 57 (31), 55 (22) were also found
c
(BPI corresponds to 1); ) [MꢀCH3]+, molecular ion signal not observed; d) Additional fragments at m/z 349 (5), 189 (100), 145 (14), 143 (32),
129 (71), 117 (14), 101 (12), 73 (68) for the TMSi derivatives.
Scheme 1. Proposed mechanism for MS fragmentation (EI). a) Euphrasianin A (1). b) TMSi derivative of euphrasianin A (1-TMSi),
corresponding to the mass spectral data in Table 1.
abundance (base peaks) of the proposed 1,3-dioxolenium
cation fragments at m/z 117 and 189 (Table 1).
In addition, the presence of an acetyl group was indi-
cated in the APCI+ mode (MS1) by an AcOH dissociation
fragment ion [M+HꢀAcOH]+ (Fig. 3b). Moreover, in the
negative ionization mode
a
solvent cluster anion
Fig. 2. General structural formula of the euphrasianins A – E.
[M + MeOH]ꢀ was observed for compounds 1 – 5 as base
peak in the MS1 experiment (Fig. 3c). The dissociation of
the corresponding ions afforded two fragment types, i.e.,
supposable mechanism leading to the formation of
1,3-dioxolenium cations via a homolytic cleavage of the [fatty acidꢀH]ꢀ and [fatty acid–H–COꢀH2O]ꢀ in the
fatty acyl moiety, with either subsequent or simultaneous
MS2 and MS3 experiments, respectively. In accordance
cyclization, induced by EI, is illustrated in Scheme 1 [30] with GC/MS data, these results supported the aforemen-
[32]. This mechanism is also in line with the very high tioned structural proposal.
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Fig. 3. HPLC/MS(APCI) sections of purified fraction of a CH2Cl2 extract of E. rostkoviana. a) Staggered illustration of extracted ion traces,
recorded in the APCI+ with a mass scan on most intense ions [M+HꢀH2O]+ of the homologous euphrasianins A – E: m/z 343 (1), 357 (2), 371 (3),
385 (4), and 399 (5). b) MSn data of 1 (C19H36O6, Mr = 360.5) as obtained in the APCI+ mode. c) MSn data of 1 as obtained in the
APCIꢀ mode.
Since further separation of the homologues 1 – 5 product rac-8 as a diastereomeric mixture (Scheme 2).
appeared difficult and structure elucidation exclusively
based on MS data was ambiguous, a facile three-step total
synthesis of one representative homologue was accom-
plished as racemic mixture, using an established protec-
The protection group was removed by treatment with 1N
HCl/MeOH to yield product rac-9. Finally, rac-9 was
regioselectively acetylated at the primary OH group with
€
AcCl in the presence of Hunig’s base (DIPEA) at low
tion group concept [33]. Accordingly,
esterification [34] of (3R,S)-6 with rac-1,2-isopropyliden the obtained (3R,S)-1 was consistent in its chromato-
glycerol (7) yielded the intermediate condensation graphic and spectroscopic properties with the main
a
Steglich temperature (ꢀ78°). After chromatographic purification,
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Chem. Biodiversity 2016, 13, 602 – 612
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constituent 1 in the Euphrasia fractions (see Fig. 4a,c).
Thus, based on highly consistent mass spectrometric simi-
larities, the other constituents were assigned to homolo-
gous acetylated acylglycerols of 3-hydroxy fatty acids (C
(14) – C(18)), henceforth called euphrasianins A – E
(1 – 5, Fig. 2).
Moreover, the positive-ion high-resolution electrospray
ionization-mass spectral analysis of synthesized euphrasia-
nin A ((3R,S)-1) revealed a molecular formula C19H36O6,
as deduced from the [M+Na]+ ion at m/z 383.2387 (calcd.
for C19H36O6Na, 383.2404). Because of two stereogenic
centers and performing the synthesis of 1 with racemic
1
starting material, the H- and 13C-NMR spectra displayed
two sets of signals, due to a diastereomeric mixture (the
diastereomers could not even be separated by GC/MS).
Structure and signal assignment was verified by assessing
gHSQC and gHMBC spectra. The signals of CH3 and CH2
groups were allocated by gHSQC experiments (data not
shown). However, several signals overlapped in the NMR
spectra, and hence, interpretation of the data was not
1
straightforward (Table 3). The H-NMR spectrum of rac-1
1
revealed numerous different H-spin systems for the two
diastereomers: a CH group at d(H) 4.14 – 4.09 (2 9 1 H,
m), coupled with two CH2 groups at d(H) 4.26, 4.25
(1 9 2 H, dd, dd, each J = 11.5, 4.5 Hz) and 4.20 – 4.15
(3 9 2 H, m overlapped); a CH2 group at d(H) 2.57, 2.56
(1 9 2 H, dd, dd, each J = 15.9, 3.1 Hz), 2.45, 2.44 (1 9 2
H, dd, dd, each J = 15.9, 9.2 Hz), coupled with a CH
group at d(H) 4.06 – 4.01 (2 9 1 H, m); two CH3 groups
at d(H) 2.11 (2 9 3 H, s) and 0.88 (2 9 3 H, t,
J = 6.8 Hz), besides several overlapped signal groups for
the CH2 groups of the aliphatic side chain (Table 3). Nev-
ertheless, the 1D- and 2D-NMR data supported the struc-
tural assignment for the synthesized rac-1.
Finally, to demonstrate the occurrence of euphrasian-
ins in further Euphrasia species, a phylogenetically differ-
ent type, i.e., E. tetraquetra [8], was investigated.
Euphrasia tetraquetra, called sea cliff or maritime eye-
bright, is growing widespread on open, grassy, and turfy
areas, and chalk cliffs at the western coasts of Britain and
Ireland [35][36]. GC/MS screening of CH2Cl2 extracts of
total plants of E. tetraquetra in comparison with
E. rostkoviana revealed the appearance of euphrasianins
in both species, however, exhibiting different profiles.
While euphrasianins A and C (1, 3) were predominant in
E. rostkoviana, mainly euphrasianins C and E (3, 5) were
detected in E. tetraquetra (see extracted ion chro-
matograms, Fig. 4a,b). Thus, these compounds may serve
as chemotaxonomic markers, or these can be used in fin-
gerprinting methods for better identification on the spe-
cies level. A recent report on the incidence of structurally
similar compounds, i.e., partially acetylated acylglycerols
of (3R)-acetoxyfatty acids in floral oils of Diascia spp.,
belonging to the Scrophulariaceae [30], and our finding of
1 – 5 in the congeneric Orobanchaceae family (Euphra-
sia), might also suggest the significance of these com-
pounds in a coevolutionary context. Further investigations
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Scheme 2. Synthetic route to euphrasianin A (rac-1). Structural formula 1 together with arbitrary atom numbering.
Fig. 4. GC/MS extracted ion chromatograms (mass scan at m/z 187). a) CH2Cl2 extract of dried total plants of E. rostkoviana. Assignment:
1 – 5, euphrasianins A – E; 1*,3*, isomers due to acyl migration. b) CH2Cl2 extract of dried total plants of E. tetraquetra. c) synthetic (3R,S)-1 as
a reference.
on a higher number of other Euphrasia species are war- Because of the amphiphilic character of these molecules,
ranted to verify this first monitoring.
a membrane stabilizing function as structural lipids may
be possible [37][38]. However, the biological function of
euphrasianins still appears obscure. Since diacylglycerols
are important intermediates in the biosynthesis of triacyl-
Conclusions
The detection of euphrasianins A – E (1 – 5) as a novel glycerols and phospholipids [39], and play a role in cellu-
class of lipophilic constituents in Euphrasia sheds new
light on the lipid composition of this plant family.
lar signaling [40], it is assumed that the structurally
related euphrasianins could be also involved in such
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Table 3. 1H- and 13C-NMR data of rac-1 (diasteromeric mixturea), in CDCl3). d in ppm, J in Hz (ov, overlapping signals)
Positionb)c)
d(H)d)
d(C)e)
HMBC
1, 10
2, 20
3, 30
4.20 – 4.15 (m, ov)
4.14 – 4.09 (m)
4.26 (dd, J = 11.5, 4.5), 4.25 (dd, J = 11.5, 4.5),
4.20 – 4.15 (m, ov)
–
2.57 (dd, J = 15.9, 3.1), 2.56 (dd, J = 15.9, 3.1),
2.45 (dd, J = 15.9, 9.2), 2.44 (dd, J = 15.9, 9.2)
4.06 – 4.01 (m)
1.56 – 1.50 (m), 1.47 – 1.42 (m, ov)
1.47 – 1.42 (m, ov), 1.33 – 1.28 (m, ov)
1.26 (br. s, ov)
65.05 (CH2)
68.00, 67.97 (CH)
65.11, 65.07 (CH2)
C(2), C(3), C(18)
C(1), C(3)
C(1), C(2), C(4)
4, 40
5, 50
172.73, 172.67 (C=O)
41.56, 41.52 (CH2)
–
C(4), C(6), C(7)
6, 60
68.30, 68.28 (CH)
36.70 (CH2)
25.46 (CH2)
C(4), C(7), C(8)
C(5), C(6), C(8)f)
C(7)f)
C(15), C(16), C(17)f)
7, 70
8, 80
9, 90 – 14, 140
29.62, 29.60, 29.55, 29.54,
29.47, 29.32 (CH2’s)
31.89 (CH2)
15, 150
16, 160
17, 170
18, 180
19, 190
1.26 (br. s, ov)
1.33 – 1.28 (m, ov), 1.26 (br. s, ov)
0.88 (t, J = 6.8)
–
2.11 (s)
22.66 (CH2)
14.09 (Me)
C(15), C(17)f)
C(15), C(16)
–
171.08, 171.07 (C=O)
20.77 (Me)
C(1), C(18)
a) Obtained by synthesis; b) For atom numbering, see Scheme 2; c) Primed numbers refer to the other diastereoisomer; d) Recorded
f
at 600 MHz; e) Recorded at 150 MHz, 13 signals overlapped; ) Due to overlapping signals HMBC correlations were not completely assigned.
functions. However, further investigations are required to
investigate this hypothesis in more detail.
chromatograph with split injection (split ratio 30 : 1, injec-
tion volume 1.0 ll) coupled to a mass detector. The col-
umn used was
a
Zebron ZB-5ms cap. column
The authors are grateful to Mr. C. Metherell (Botanical
Society of Britain and Ireland, Northumberland, UK) and
(60 m 9 0.25 mm i. d. 9 0.25 lm film thickness, 5%
phenylpolysiloxane and 95% dimethylpolysiloxane coat-
Prof. Dr. O. Spring (Hohenheim University, DE-Stutt- ing; Phenomenex, Torrance, CA, USA). Carrier gas:
Helium at a flow rate of 1 ml/min. The injector used was
a PSS (programmed-temperature split/splitless injector;
gart) for the exact taxonomy of plant specimens. We wish
to thank Dr. I. Lederer (PhytoLab GmbH, Vestenbergs-
greuth) for HR mass spectra and H. Schneider (WALA temp.: 250°). The temperature program for the column
€
Heilmittel GmbH, DE-Bad Boll/Eckwalden) for providing
a photograph of E. rostkoviana.
oven was 100 – 320° with a linear gradient of 4°/min and
a final hold time of 30 min. The mass spectrometer was
run in electron ionization mode (70 eV).
NMR Spectroscopy
Experimental Part
NMR spectra were recorded in CDCl3 with a Varian
Unity Inova 500 MHz spectrometer and a Bruker Avance
III 600 MHz spectrometer. The measurement tempera-
ture was 298 K. Chemical shifts were expressed in
d (ppm) and referenced to residual (non-deuterated) sol-
vent signals of CDCl3 (1H: d(H) 7.27; 13C: d(C) 77.00).
13C-NMR signal assignment of the novel compounds 1, 8,
and 9 was based on two-dimensional heteronuclear NMR
experiments (gHMBC and gHSQC). The assignment of
the diastereotopic H-atom signals was supported by 1H-
decoupling experiments (data not shown). For evaluation
of NMR spectra the program, SpinWorks 3.1.7. (Copy-
rightâ 2010, K. Marat, University of Manitoba, USA) was
used.
General
Source of the standards for chiral GC analysis: (3R)- and
(3R,S)-hydroxymyristic acid ((3R)- and (3R,S)-6) were
purchased from Santa Cruz Biotechnology, DE-Heidel-
berg, Germany and ABCR GmbH, DE-Karlsruhe, Ger-
many, respectively. Liquid chromatographic analyses were
carried out on an Agilent 1200 HPLC system (Agilent
Technologies Inc., DE-Waldbronn, Germany) coupled to
an HCTultra Ion Trap mass spectrometer (Bruker Dal-
tonik GmbH, DE-Bremen, Germany) with an APCI
interface. HR-ESI-MS was performed on a Thermo Scien-
tific Exactive Orbitrap mass spectrometer (Thermo Fisher
Scientific, DE-Dreieich, Germany), by direct admittance
of the samples dissolved in MeOH.
Plant Material
GC/MS Analyses
E. rostkoviana HAYNE (aerial parts with roots) was col-
lected in August 2012 and 2014 near Markstein, in the
southern Vosges Mountains (France), mechanically
GC/MS of all analytes was performed with a Clarus 500
(PerkinElmer,
Inc.,
Shelton,
CT,
USA)
gas
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.cb.wiley.com

610
Chem. Biodiversity 2016, 13, 602 – 612
cleaned from impurities and stored at ꢀ80° until investi-
BSTFA (300 ll) at 105° for 15 min. From the obtained
gation. A separate sample of E. rostkoviana was air-dried. solution, 1 ll was injected into the GC/MS system.
ꢀ
E. tetraquetra (BREB.) ARROND. (seacliff eyebright) was
collected close to Dunlough Castle, at the northern tip of
RP-HPLC/APCI (pos/neg)-MSn Analyses
the Mizen Peninsula (Ireland), in August 2014, air-dried
and stored frozen at ꢀ80° until extraction. Voucher speci- For chromatographic separation a YMCTM carotenoid S-5
mens of E. rostkoviana and E. tetraquetra (voucher Nos.
HOH-010792 – 010795 and HOH-014239, respectively)
were deposited in the herbarium of the Institute of Bot-
any, Hohenheim University (Germany).
C30 reversed-phase column (5 lm particle size,
250 9 3.0 mm i.d., Waters Corporation, Milford, MA,
USA) was used at 25 °C and a flow rate of 0.42 ml/min.
The mobile phase consisted of MeOH/MTBE/H2O (90:5:5,
v/v/v; mobile phase A) and MeOH/MTBE (8:92, v/v;
mobile phase B). Elution started with 0% B for 5 min, fol-
lowed by a linear gradient to 30% B at 20 min, then
increasing to 100% B at 25 min, continuing for 5 min,
Extraction and Chromatographic Purification of
Euphrasianins A – E
Frozen plant material of E. rostkoviana was extracted in before re-equilibration to starting conditions. The injection
two batches (2 9 100 g) with CH2Cl2 (500 ml each) by
Ultra-turraxâ treatment (3 min at 12,400 rpm; IKA-Werke
GmbH & Co. KG, D-Staufen). After maceration for 24 h euphrasianins (Fig. 3a) was performed by mass spectrome-
volume of euphrasianin samples (1 mg of the fraction dis-
solved in 1 ml MeOH) was 10 ll. The detection of
at +4°, insoluble plant material was filtered by vacuum
€
suction over Celite through a Buchner funnel, followed by
a second extraction in the same manner. Subsequently,
try with an APCI interface operating in the positive and
negative ionization mode applying the following parame-
ters: HV capillary: ꢁ4000 V; dry gas N2: 5.00 l/min with a
the pooled extracts were dried (Na2SO4), and the solvent dry gas temperature of 350°; nebulizer: 50 psi; vaporizer
was removed by rotary evaporation in vacuo to yield a
dark green viscous extract (0.84 g). The extract was
loaded onto a silica gel VLC column (100 g TLC grade
silica gel 60 G, column dimension: 100 9 60 mm i.d., pre-
temperature: 400 °C. Full scan mass spectra (mass range
m/z from 50 to 1000) of the HPLC eluates were recorded
during chromatographic separation. To obtain further
structural information, collision-induced dissociation
conditioned with petroleum ether (PE), b.p. 40 – 60°) and (CID) experiments were performed. MSn data were
subsequently flushed with PE, CH2Cl2, and AcOEt acquired in the auto-MS/MS mode. The instruments were
(500 ml each). After TLC control and solvent evaporation controlled by Agilent ChemStation (Rev. B.01.03 SR1) and
a PE (0.070 g), a CH2Cl2 (0.188 g), and three AcOEt Bruker Daltonik EsquireControl Software (V6.1).
(0.249, 0.105, and 0.178 g) fractions were obtained. All
fractions were analyzed by GC/MS revealing the second
Configuration Analysis of the 3-Hydroxyfatty Acids
AcOEt fraction to contain euphrasianins 1 – 5. This frac-
tion (0.105 g) was further purified applying a Chroma-
Configuration analysis was performed according to a
totronâ centrifugal radial preparative TLC (2 mm layer, modified literature protocol [41]. In brief, a purified
silica gel/gypsum/fluorescence indicator 254 nm, 45:18:1.2,
w/w/w; preconditioned with CH2Cl2). Elution was per-
formed with a CH2Cl2/MeOH linear gradient (100:0 –
90:10, v/v). One fraction (CH2Cl2/MeOH, 95:5, v/v) was
euphrasianin fraction (9.3 mg) was treated with 1N
NaOH (10 ml) and stirred at room temperature (24 h).
Subsequently, the slurry was acidified with 1N HCl to pH
1.1 and extracted with AcOEt (2 9 20 ml). The com-
separated, and the solvent was removed in vacuo to yield bined AcOEt extracts were dried (Na2SO4), the solvent
27 mg of a beige residue, containing the highly enriched
target compounds 1 – 5.
For the screening of euphrasianins in air-dried sam-
removed by evaporation, and the residue obtained was
treated with 20% BF3 in MeOH (w/v; 1 ml) for 45 min
at 105°. Subsequently, the mixture was transferred into
ples, 10.0 g of total plants of E. rostkoviana and E. tetra- satd. aq. NaCl solution (5 ml) and extracted with hexane
quetra, respectively, were minced in CH2Cl2 (200 ml) by
(2 9 5 ml). The hexane extract was dried (Na2SO4) and
Ultra-turraxâ treatment (see above). Subsequently the evaporated to dryness by a vigorous stream of N2. From
mixture was macerated for 24 h (+4°) and then filtered the residue thus obtained, 1 ll of an hexane solution
over Celite by vacuum suction. The solvent was removed
in vacuo from the obtained extract to yield a dark green
(1 mg/ml) was injected into a GC-FID system (PerkinEl-
mer, Autosystem XL), equipped with a chiral capillary
residue (0.2 g). A solution of the residue (1 ll) in CHCl3 column (IVADEX-7, 25 m 9 0.25 mm i. d. 9 0.25 lm
(0.014 g/1 ml) was directly subjected to GC/MS analysis.
film thickness, diethyl tert-butylsilyl b-cyclodextrine coat-
ing; IVA Analysentechnik, DE-Meerbusch). Helium was
used as carrier gas at a flow rate of 1.3 ml/min. The tempera-
ture program for the column oven was 130 – 170 °C at
0.5 °C/min with a total run time of 80 min. For comparison
(3R)- and (3R,S)-hydroxymyristic acid methylesters,
Silylation of Samples for GC/MS Analyses
For GC/MS analysis, fractions or purified compounds
(4 mg) were dissolved in DMF (0.5 ml) and treated with
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
www.cb.wiley.com

Chem. Biodiversity 2016, 13, 602 – 612
611
obtained by methylation of (3R)- and (3R,S)-6, respec- derivatization.
tively, were used as reference compounds.
1,2-Isopropyliden-3-(3-trimethylsiloxy-
(=(2,2-dimethyl-1,3-dioxolan-4-yl)
myristoyl)glycerol
methyl 3-[(trimethylsilyl)oxy]tetradecanoate; TMSi-8).
GC/MS (70 eV) at tR 42.6 min: m/z 415 (10, [MꢀCH3]+)*,
Synthesis of the Reference Compound rac-1-Acetyl-3-
(3-hydroxymyristoyl)glycerol (= rac-Euphrasianin A; rac-
3-(Acetyloxy)-2-hydroxypropyl
anoate; rac-1). rac-1,2-Isopropyliden-3-(3-hydroxymyri-
3-hydroxytetradec- 357 (2, [MꢀTMSi]+), 275 (7), 257 (26), 217 (20), 143 (9),
115 (100), 101 (19), 73 (28); *molecular ion not observed.
stoyl)glycerol
(= rac-(2,2-Dimethyl-1,3-dioxolan-4-yl)
rac-1-(3-Hydroxymyristoyl)glycerol
(= rac-2,3-Dihy-
methyl 3-Hydroxytetradecanoate; rac-8). A mixture of (3R, droxypropyl 3-Hydroxytetradecanoate; rac-9). A quantity
S)-6 (1.00 g, 4.10 mmol), rac-1,2-isopropyliden glycerol (7,
0.65 g, 4.92 mmol) and 4-(dimethylamino)pyridine
of 1N HCl (4.35 ml) was added to a soln. of 8 (0.83 g,
2.32 mmol) in MeOH (22 ml), and the mixture was stirred
under N2 (5 h). Afterwards, the reaction was quenched by
adding satd. NaHCO3 solution (45 ml) and the aq. phase
was extracted with Et2O (3 9 80 ml). The combined
organic extracts were dried (Na2SO4), and the solvent was
removed in vacuo to yield the crude product (0.91 g).
Purification by VLC (conditions see above) gave pure 9.
(DMAP, 0.50 g, 4.10 mmol) in dried CHCl3 (20.5 ml) was
cooled in an ice/NaCl bath, while a solution of N,N0-dicy-
clohexylcarbodiimide (DCC, 0.84 g, 4.00 mmol) in 10.5 ml
CHCl3 was added under stirring. Then the mixture was stir-
red for 21 h at room temperature, and afterwards the sol-
vent was removed by vacuum rotoevaporation. The target
product 8 was isolated from the crude residue, thus Wax-like crystals; yield: 0.65 g (89% of the theory). GC/
obtained, by VLC (110.0 g TLC grade silica gel 60 G, pre-
conditioned with n-hexane). Elution of 8 was performed
with an n-hexane/AcOEt linear gradient (100:0 – 50:50, v/
v), and the corresponding compound was monitored by
MS purity (70 eV): 99% (tR 43.9 min, diastereomers were
not separated by GC) m/z 300 (0.1, [MꢀH2O]+)*, 287 (1,
[MꢀCH2OH]+), 269 (2, [MꢀCH2OHꢀH2O]+), 227 (6),
209 (7), 163 (7, [C6H11O5]+), 145 (100, [C6H9O4]+), 134
GC/MS. Two fractions, containing 8 were evaporated to (14, [C5H10O4]+), 116 (6), 97 (10), 83 (10), 75 (23), 71
dryness in vacuo; total yield: 1.24 g, 84.5% of the theory;
wax-like compound. GC/MS purity (70 eV): 98 and 93%,
(27), 57 (38); *molecular ion not observed. 1H-NMR
(CDCl3, 500 MHz): 4.23 (1 H, dd, J = 11.5, 4.3, Ha–C(3));
respectively (tR 42.8 min, diastereomers were not separated 4.22 (1 H, dd, J = 11.5, 6.5, Ha–C(30)); 4.17 (1 H, dd,
by GC) m/z 343 (24, [MꢀCH3]+)*, 325 (6,
J = 11.5, 4.2, Hb–C(30)); 4.15 (1 H, dd, J = 11.5, 6.4, Hb–C
[MꢀCH3ꢀH2O]+), 243 (4), 203 (28, [C9H15O5]+), 183 (3)); 4.06 – 4.00 (2 H, m, H–C(6, 60)); 3.95 – 3.91 (2 H, m,
(5), 159 (56, [C7H11O4]+), 145 (100, [C7H13O3]+), 116 (42), H–C(2, 20)); 3.68 (1 H, dd, J = 11.6, 3.9, Ha–C(1)); 3.67 (1
101 (51), 83 (7), 71 (21), 57 (42); *molecular ion not H, dd, J = 11.6, 3.9, Ha–C(10)); 3.59 (1 H, dd, J = 11.6,
1
observed. H-NMR (CDCl3, 500 MHz): 4.36 – 4.31 (2 H,
6.0, Hb–C(1)); 3.58 (1 H, dd, J = 11.6, 6.0, Hb–C(10)); 2.54
m, H–C(2, 20)); 4.22 (1 H, dd, J = 11.5, 4.5, Ha–C(3)); (2 H, dd, dd, J = 15.4, 3.0, 15.5, 3.0, Ha–C(5, 50)); 2.43 (2
4.18 – 4.17 (2 H, m, Ha,b–C(30)); 4.13 (1 H, dd, J = 11.5, H, dd, dd, J = 15.6, 9.6, 15.6, 9.6, Hb–C(5, 50)); 1.55 – 1.50
6.0, Hb–C(3)); 4.08 (2 H, dd, J = 8.5, 6.5, Ha–C(1, 10));
(2 H, m, H–C(7)); 1.48 – 1.39 (4 H, m overlapped, H–C
4.04 – 3.98 (2 H, m, H–C(6, 60)); 3.75 (2 H, dd, dd, J = 8.6, (70, 8)); 1.31 – 1.27 (4 H, m overlapped, H–C(80,16)); 1.26
5.8, 8.6, 5.8, Hb–C(1, 10)); 2.56 (1 H, dd, J = 16.3, 5.6, Ha–C (30 H, br. s, H–C(9,90 – 15, 150, 16)); 0.88 (6 H, t,
(5)), 2.55 (1 H, dd, J = 16.3, 5.6, Ha–(50)); 2.45 (1 H, dd, J = 7.0 Hz, H-–C(17, 170)). 13C-NMR (CDCl3, 125 MHz):
J = 16.3, 9.1, Hb–C(5)), 2.44 (1 H, dd, J = 16.3, 9.2, Hb–C 172.82 (C(4, 40)); 69.99, 69.97 (C(2, 20)); 68.47, 68.45 (C(6,
(50)); 1.55 – 1.50 (2 H, m, H–C(7)); 1.44 – 1.43 (6 H, m, H– 60)); 65.40, 65.39 (C(3, 30)); 63.26, 63.25 (C(1, 10)); 41.94,
C(20, 200)); 1.43 – 1.41 (4 H, m overlapped, H–C(70, 8)); 41.91 (C(5, 50)); 36.88, 36.87 (C(7, 70)); 31.88 (C(15, 150));
1.37 – 1.36 (6 H, m, H–C(19, 190)); 1.31 – 1.27 (4 H, m 29.65, 29.62, 29.60, 29.58, 29.52, 29.32 (C(9, 90-14, 140));
overlapped, H–C(80, 16)); 1.26 (30 H, br. s, H–C(9,90 – 15, 25.54 (C(8, 80)); 22.65 (C(16, 160)); 14.08 (C(17, 170)), 11 sig-
150, 160)); 0.88 (6 H, t, J = 7.0, H–C(17, 170)). 13C-NMR nals overlapped. Primed numbers refer to the other
(CDCl3, 125 MHz): 172.68, 172.63 (C(4, 40)); 109.92, 109.89 diastereoisomer. APCI+-MS m/z 336* [M+NH4]+, 319
(C(18, 180)); 73.47, 73.42 (C(2, 20)); 68.02, 67.92 (C(6, 60)); [M+H]+, 283 [M+HꢀH2O]+ (MS1); 301* [M+HꢀH2O]+
66.15, 66.12 (C(1, 10)); 64.86, 64.77 (C(3, 30)); 41.42, 41.37
(MS2); 117*[C5H9O3]+ (MS3); 57 (MS4). APCIꢀ-MS m/z
(C(5, 50)); 36.54, 36.51 (C(7, 70)); 31.88 (C(15, 150)); 29.61, 350* [M+MeOH]ꢀ (MS1); 317 [MꢀH]+, 299 [M–HꢀH2O]+,
29.59, 29.54, 29.53, 29.48, 29.30 (C(9, 90)–C(14, 140)); 26.63
243 [fatty acid ꢀ H]+, 225 [fatty acidꢀHꢀH2O]+, 197 [fatty
(C(20, 200)); 25.45, 25.44 (C(8, 80)); 25.25, 25.22 (C(19, 190)); acidꢀ2HꢀCOOH]+, 133 (MS2); *fragment ions. HR-MS-
22.65 (C(16, 160)); 14.07 (C(17, 170)), 10 signals overlapped. ESI+ m/z 341.2289 [M+Na]+ (calcd. for C17H34O5Na,
Primed numbers refer to the other diastereoisomer.
APCI+-MS m/z 376 [M+NH4]+, 359* [M+H]+ (MS1); 301*
[M+HꢀOC(CH3)2]+ (MS2); 283, 227, 209, 191, 117*
[C5H9O3]+ (MS3); 57 (MS4). APCIꢀ-MS m/z 390*
341.2298). Further characterization was performed by
TMSi-derivatisation. 1,2-Bis(trimethylsilyl)-3-(3-trimethyl-
siloxymyristoyl)glycerol (=2,3-bis[(trimethylsilyl)oxy]pro-
pyl 3-[(trimethylsilyl)oxy]tetradecanoate; TMSi-9). GC/MS
[M+MeOH]ꢀ (MS1); 339* [M–HꢀH2O]ꢀ (MS2); 281, 251, (70 eV) at tR 44.2 min: m/z 519 (1, [MꢀCH3]+)*, 431 (13,
207* (MS3); 189, 80 (MS4); *fragment ions. HR-MS-ESI+ [MꢀCH3ꢀTMSi]+), 379 (5), 373 (9), 309 (5), 257 (32), 219
m/z 381.2601 [M+Na]+ (calcd. for C20H38NaO5, 381.2617). (11), 147 (19), 143 (26), 129 (16), 116 (8), 103 (40), 73 (100);
Further characterization was performed by TMSi-
*molecular ion not observed.
€
© 2016 Verlag Helvetica Chimica Acta AG, Zurich
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612
Chem. Biodiversity 2016, 13, 602 – 612
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rac-Euphrasianin
ristoyl)glycerol;
A
(= rac-1-Acetyl-3-(3-hydroxymy-
rac-3-(Acetyloxy)-2-hydroxypropyl
3-
[17] O. Sticher, O. Salama, R. K. Chaudhuri, T. Winkler, Helv.
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hydroxytetradecanoate; (3R,S)-1). A solution of 9 (0.23 g,
0.72 mmol) and DMAP (0.15 ml, 0.19 g, 1.47 mmol) in
dry CH2Cl2 (4.6 ml) was externally cooled to ꢀ78° in a
Me2CO/dry ice mixture. Subsequently, AcCl (52 ll,
0.0572 g, 0.73 mmol) was added, the mixture was slowly
warmed up to room temperature, and stirred for 1 h. Sub-
sequently, after addition of Et2O (100 ml) the ppt. ammo-
nium salts were filtered off. Finally, the solvent was
removed by vacuum evaporation, and the crude product
(0.40 g) was purified by VLC (conditions see above) to
yield 1 (0.15 g, 60.0% of the theory) as a colorless syrup;
GC/MS purity (70 eV): > 99% (at tR 45.4 min). For MS
and NMR data, see Tables 1 and 3, respectively. HR-
ESI+-MS m/z 383.2387 [M+Na]+ (calcd. for C19H36O6Na,
383.2404). In solution (AcOEt) 1 slowly isomerised at
room temperature by acyl migration (data not shown).
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