N.S. Radulovi ´c and M.Z. Zivkovic Stosic
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Phytochemistry 186 (2021) 112732
reactions; Busta and Jetter, 2017; Deki ´c et al., 2019).
without prior derivatization, and subsequently extensively by NMR.
Along with the expected flavonoid derivatives, which were not the
subject of this work, a very minor fraction, ca. 12 mg of which were at
hand, proved to be a prolific source of new constituents that possessed
unusual chain functionalization, the number and identity of functional
groups, in addition to having iso- and anteiso-branches at chain termini,
Structural elucidation of secondary metabolites usually requires an
access to a sample of sufficient purity to permit NMR analyses. In the
case of long-chain compounds, encountered in wax constituents, both
requirements are very difficult or impossible to meet. Homologous
compounds differ only minutely in their polarity and cannot be sepa-
rated using conventional liquid chromatography but are easily discerned
by gas chromatography. The long chains result in shift isochronicity and
obstruct locating appropriate correlations in 2D NMR spectra that would
produce connectivity data needed. For example, a functional group at
one chain terminus cannot be brought into direct link via NMR with the
other terminus. However, when one measures an NMR spectrum of a
mixture of homologous compounds the difference in the chemical shifts
among the long-chained homologous is usually negligible. Logically, the
mass spectra are crucial in piecing the molecule together, but these
cannot be easily used to differentiate among isomers. Thus, a third
characteristic is necessary, which would allow specific combinations of
isomeric moieties to be interconnected. Gas chromatography offers this
unique tool, the retention indices, which can distinguish close positional
isomers based on their different volatility. However, these are not easily
attainable or are non-existent in the literature in the case of rare or new
metabolites, and this problem can be only solved by employing synthesis
of the very metabolites or their homologs.
or branches in
α- or β-position to the functional group. The identification
of these plant metabolites would not have been possible without the
synthetic endeavors to produce analogs used for both direct comparisons
of spectral data and predicting of retention indices of higher homologs.
In the current study, in total 16 unreported syn-1-phenyl-1,3-alkanediyl
diacetates, 7 unreported 3-oxo-1-phenylalkan-1-yl acetates, 8 unre-
ported 1-phenylalkane-1,3-diones, 10 unreported 1-hydroxy-1-pheny-
lalkan-3-ones, 66 unreported sec-alkanols, 11 unreported
( -1)-,
ω
(
ω
-2)-, 2- or 3-methylalkanoic acids were identified. Except for the acids,
all other identified classes were present as both n-, iso- and ante-
iso-isomers at chain ends with the iso-isomer being the predominant one.
This work gives an account of the structural elucidation of these com-
pounds that was specific due to the VLC-nature of the constituents, the
low available amount of the material and impossibility of further
separation.
2. Results and discussion
The genus Primula L. is the largest genus among the Primulaceae and
includes more than 400 species distributed throughout the cold and
temperate regions of the Northern Hemisphere (Colombo et al., 2017).
The production of oily or farinose exudates (farina) on aerial surfaces of
leaves, stems, calyces, and inflorescences is universal among Primula
taxa with glandular trichomes. Primula L. is one of the most widespread
of all plant genera, possibly because of its successful adaptation to
extreme environmental conditions (high altitudes, low temperatures,
high level of ionizing radiation, aridity, etc.; Richards, 2003; Zhang
et al., 2013). These exudates consist primarily of unsubstituted flavone
accompanied by a series of other highly unusual, methoxylated flavones
lacking the typical oxygenations, some of which are substituted by a
single hydroxyl or methoxyl group (Bhutia and Valant-Vetschera, 2012;
Berim and Gang, 2015).
2.1. 1-Phenylalkane-1,3-diyl diacetates
The most dominant peaks in the GC-MS chromatogram of the frac-
+
tion displayed mass spectra having prominent ions at m/z 43 [CH CO] ,
3
+
as the base peak, and at m/z 105 [PhCO] , suggesting the presence of
one or more acetyl groups in their structure, and of a monosubstituted
phenyl group possessing an oxygenation in the benzylic position. Sixteen
compounds showed this MS pattern, and these were distributed into
three series having an approximate increment of ca. 101 RI units per CH2
group, (SI, Table S3; Figure S1), implying three different chain termini.
Our NMR entrance points, bearing in mind that these are NMRs of the
entire fraction, for this group of compounds were the recognizable sin-
glets of methyl groups within acetates, at 2.06 and 1.98 ppm (Fig. 1),
attached to carbon atoms at 21.2 and 21.3 ppm (SI, Figure S3), which
also correlated with ester carbonyls at 170.5 and 170.1 ppm via two
bonds (gHMBC). The only other long-range C–H correlations of these
carbonyls were to a pseudo triplet at 5.79 ppm (dd, J = 7.15, 6.70 Hz,
Although several phytochemical studies devoted to the analyses of
the chemical composition of Primula glandular trichome exudates have
been carried out in the last decades (Huck et al., 2000; Bhutia et al.,
2
2
013; Berim and Gang, 2015; Shostak et al., 2016; Colombo et al.,
017), these offer limited data on the non-flavonoid constituents. So far,
H ) and a complex multiplet at 4.78 ppm (dddd, J = 8.36, 6.30, 6.20,
a
d
2
c
only one diterpene (ent-kaur-16-en-19-oic acid; Elser et al., 2016), two
dihydrochalcones (Bhutia et al., 2013), and three chalcones (Wollen-
weber et al., 1989; Budzianowski and Wollenweber, 2007; Val-
ant-Vetschera et al., 2009) have been reported thereof. One of the most
renowned plant species from this genus is Primula veris L. that was the
subject of phytochemical and pharmacological (Aslam et al., 2014;
Chinou et al., 2014) studies on numerous occasions and found its place
in several pharmacopoeias: Primulae flos and Primulae radix (British
Herbal Medicine Association, 1974; L’Adapharm, 1988; Council of
Europe, 2019). Most recently, for the first time discovered in a natural
source, several flavones, substituted only in the B ring, have been found
in the wax of P. veris (Budzianowski et al., 2005). The waxes/farinas of
this taxon have been investigated for the presence/structural elucidation
of flavonoids from several regions (Valant-Vetschera et al., 2009;
Shostak et al., 2016; Apel et al., 2017; Bᶏczek et al., 2017), but never
previously from Serbia.
4.50 Hz, H ), respectively (Table 1). The two esterified carbinol C–H
were separated by only a single CH group (2.03 ppm, ddd, J = ꢀ 14.52,
6.70, 4.50 Hz, Hb and 2.23 ppm, ddd, J = ꢀ 14.52, 8.36, 7.15 Hz, H )
1
1
judged from the observed H– H COSY, NOESY, and gHMBC in-
teractions (Fig. 1B and C). gHMBC and NOESY provided further links of
the H proton to a phenyl group geminal to one of the acetates, while the
a
d
H proton was adjacent to an alkyl chain. Thus, the molecules contained
at one terminus the 1-phenylalkane-1,3-diyl diacetate moiety while the
other terminus should be the branched or non-branched alkyl chains (SI,
Figure S2; Table S2). The observed fragmentation patterns in the MS of
the wax constituents could now be readily interpreted, as presented in
the SI material (SI, Table S4; Figures S4-1 – S4-8).
Since there are at least two chiral centers in the molecule (two for n-
and iso-branched chains, and three from the anteiso-branched chains),
two (i.e. four) diastereoisomers are possible, which are frequently, but
not unambiguously, designated as syn (1S*,3S*) and anti (1S*,3R*)
(Fig. 3, Moss, 1996). To resolve the splitting patterns of H , H , H , and
In this work, the focus was directed at non-flavonoid-type constitu-
ents of waxy exudates from the entire plants in bloom since these have
been neglected entirely. The interest in these minor wax constituents
came from one of our preliminary studies on alkanes of P. veris and
P. acaulis L. (L.), which were characterized by a very high contribution of
a
b
c
H , the spin system was simulated (Radulovi ´c et al., 2019) using Mes-
d
trelab Research S.L. (MestReNova) software package (Fig. 1), with the
aim of inferring the relative configuration from the values of the perti-
nent coupling constants; this would also allow to additionally separate
the signals of these diacetates from the overlapping signals coming from
the other constituents of the fraction. The chemical shifts and the
extracted values of the coupling constants were compared with the data
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branched alkanes compared to the linear isomers (Zivkovic et al., 2016).
For this reason, chloroform washings of the entire intact blooming plants
were chromatographically separated and analyzed by GC-MS, with or
2