Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

DOI: 10.1002/cbdv.202100285

FULL PAPER

Very-Long-Chain Wax Constituents from Primula veris and P. acaulis:

Does the Paradigm of Non-Branched vs. Branched Chain Dominance

Universally Hold in all Plant Taxa?

a

b

Milena Z. Živković Stošić, Niko S. Radulović,* Marija S. Genčić, and Vladimir N. Ranđelović

a

Department of Chemistry, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33,

1

8000, Niš, Serbia, e-mail: nikoradulovic@yahoo.com

Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33,

8000, Niš, Serbia

b

1

Herein n-, iso- and anteiso-series of very-long-chained (VLC) alkanes (C –C ), fatty acid benzyl esters (FABEs;

21

35

C –C ), and 2-alkanones (C –C ) were identified in the wax of Primula veris L. and P. acaulis (L.) L.

2

0

32

23

35

(Primulaceae). For the very first time in a sample of natural origin, the presence of iso- and anteiso-VLC FABEs

and 2-alkanones was unequivocally confirmed by synthetic work, derivatization, and NMR. It should be noted

that the studied species produced unusually high amounts of branched wax constituents (e.g., >50% of 2-

alkanones were branched isomers). The domination of iso-isomers, probably biosynthesized from leucine-derived

starters, is a unique feature in the Plant Kingdom. The plant organ distribution of these VLC compounds in P.

acaulis samples (different habitats and phenological phases) pointed to their possible ecological value. This was

supported by a eutectic behavior of binary blends of FABEs and alkanes, as well as by high UV-C absorption by

FABEs.

Keywords: Primula veris L., Primula acaulis (L.) L., branched very-long-chain wax constituents, unusual odd-even

ratio, thermal properties.

predominant ones.^[¹^]In the non-frequent cases when

significant amounts of the branched series were

Introduction

[

4]

[5]

The epicuticular waxes of the aerial organs of land reported (Antirrhinum majus L., Papaver rhoeas L.,

[

6]

[7]

plants are complex mixtures that mostly contain very- Solanum tuberosum L., and Brassica oleracea L. ) a

long-chain (VLC) compounds such as alkanes, primary specific plant organ was found to display this domi-

and secondary alcohols, aldehydes and ketones, esters, nance of branched over non-branched. Non-linear

etc. As we previously discussed, these compounds compounds were accumulated on the surfaces of

could be linear (major) or branched-chained that are reproductive organs, flowers in general, or more

[1–3]

[6]

only found in low amounts.

In our previous work, specifically petals (Solanum tuberosum L. and Tarax-

[

8]

we identified and spectrally characterized n-, iso- and acum officinale ). Despite more numerous analyses of

anteiso-isomers of new syn-1-phenyl-1,3-alkanediyl leaf compared to other organ waxes, these findings

diacetates, 3-oxo-1-phenyl-1-alkyl acetates, 1-phenyl- could be the consequence of differing function/

1

,3-alkanediones, 1-hydroxy-1-phenylalkan-3-ones, sec- development of plant organs.

alcohols (2- to 10-alkanols), and (ω-1)-, (ω-2)-, 2- or 3- The most used and important analytical techniques

methylalkanoic acids, and except for the acids, in all for detailed analysis of wax VLC constituents are gas

other identified classes the iso-isomers were the chromatography (GC) and gas chromatography

coupled with mass spectrometry (GC/MS), especially in

[

1]

combination with NMR, and IR. Besides the lack of

Supporting information for this article is available on the VLC commercial standards in general, especially the

WWW under https://doi.org/10.1002/cbdv.202100285

branched ones, another problem with the GC ap-

Chem. Biodiversity 2021, 18, e2100285

proach is the very low volatility of these VLC main classes (n-alkanes (�90%), branched alkanes,

compounds, which dictates long (complex) high- fatty acids, primary and secondary alcohols) of P.

[

15]

temperature programs (it is the opinion of the authors acaulis petal wax constituents.

that many wax constituents never leave the injection In this work we decided to analyze in more detail

port or the column and are hence not detected in the non-flavonoid wax constituents of two Primula

most standard GC programs). Consequently, the species (P. veris L. and P. acaulis (L.) L.), i.e., the flower

complex temperature programs make the retention and leaf surface wax washings. This study represents a

data less straightforwardly utilizable due to restrictions continuation of our previous investigation of P. veris,^[¹^]

in the application of equations (Kovatch, Van den and based on the results presented herein, we think

Dool, etc.) providing access to retention indices. This that we can provide answers/arguments to the

becomes even more complicated since most VLC following questions in the case of Primula wax

compounds do not exhibit an intense molecular ion in constituents: 1) are the branched VLC compounds

their MS.

always minor plant constituents compared to their

The relative distribution of carbon-chain length linear counterparts? 2) is the accumulation/occurrence

within homologous series of wax constituents reflects of branched VLC organ-specific? 3) does the distribu-

their biosynthesis: the dominance of odd-numbered n- tion of homologs within the branched series support

alkanes mirrors the acetate building block buildup of the currently accepted biosynthetic scheme? Addition-

the chain which ends in a single decarbonylation ally, we tried to ascertain the possible functional role

step.^[For the branched series, the odd-even ratio is of the detected/identified VLC constituents; more

additionally determined by the number of carbon specifically, we considered them as providing UV

atoms in the biosynthetic starter; so, we would expect protection, and/or affording specific thermal proper-

an iso-series to have the prevalence of odd-numbered ties to the waxy coating of Primula spp.

homologs since the up to now recognized starter is

9]

the isobutyryl-CoA derived from valine, and for the

reversed situation, for the anteiso- compounds (even- Results and Discussion

numbered) due to a 5-carbon starter (2-methylbutyryl-

Primula veris and P. acaulis Epicuticular Alkanes

CoA coming from isoleucine).

Profiles

With approximately 425 species known, and new

ones are still being discovered, the Genus Primula is A sample of the surface washings of P. veris leaves and

one of the largest genera. Most species (over 300) are flowers (P-1) were subjected to gradient silica gel

[

10]

[1]

found in the Himalayas and western China.

In column chromatography as described previously.

Europe only 33 grow spontaneously, while 20 in North The same approach was applied in the case of a

America. These plant species are known for their sample of the surface washings of P. acaulis leaves and

[

16]

production of farinose glandular secretions. It was flowers (P-2). As preliminarily reported,

the hexane

recently revealed that these waxy depositions, cover- fractions represented a mixture of solely very-long-

ing aerial surfaces of leaves, stems, calyces, and chain (VLC) n-, iso- and anteiso-alkanes (C –C , Fig-

21

35

inflorescences, have an important role in improving ure 1 and Table S1, Supporting Information (SI)). n-

their freezing tolerance and helping them to success- Alkanes were straightforwardly identified by co-injec-

[

11–13]

fully adapt to extreme environmental conditions.

tion of authentic standards. Branched (iso- and ante-

Although the scientific community has been aware of iso-) alkanes were mutually distinguished, as well as

Primula exudates for almost a century, up to now the from n-alkanes, by their differences in mass spectral

ecological role of these lipophilic, mostly flavonoid in fragmentation patterns and their gas retention behav-

nature exudates has not been completely clarified, but ior. Upon electron impact, both branched isomeric

the fact they are stored externally indicates that their series members are fragmented more easily at the C –

2

+

functions could include UV-protection, defense against C linkage, giving characteristic ions at [MÀ C H ] and

3

3 7

+

(

microbial) pathogens, or herbivore feeding deterrent [MÀ C H ] for the iso- and anteiso-isomers, respec-

2

5

[10,14]

effects.

Also, contemporary research neglected tively (Figure S1, SI). Together with the comparison of

the non-flavonoid analysis of the chemical composi- gas chromatographic data with those reported in the

tion of Primula glandular trichome.^[Surprisingly, the literature, this allowed the identification of the two

1]

[9]

alkanes from Primula species have been studied only classes of isomers. However, the definitive proof for

[15]

once thus far.

However, this work gave only a the position of the methyl groups in these branched

graphical representation of the relative ratio of the

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Figure 1. Structures of VLC alkanes, 2-alkanones, and FABEs from P. veris and P. acaulis washings.

alkanes was obtained from 1D and 2D NMR spectra in

an analogous manner reported previously.^[¹^]

Interestingly, VLC-branched alkanes were present in

a significant amount in an approximate ratio of n-: iso-:

Quantification of VLC alkanes was carried out by anteiso-alkanes 73: 21: 6, and 54: 34: 12, for P. veris and

peak-area integration method using n-dodecane as P. acaulis, respectively. A rare example of a plant taxon

the standard to generate the calibration curve. The containing significant amounts (25–50% of total

alkanes’ content determined per 100 g of fresh P. veris alkanes) of iso- and anteiso-alkanes is the tobacco

[

17]

and P. acaulis leaves and flowers (the parts were plant, Nicotiana tabacum.

washed together) was within the range of 0.017– species, anteiso-alkanes had predominant even-carbon

.30 mg, and 0.046–2.20 mg, respectively (Table S1, numbered distributions. However, the affinity of P.

In both studied Primula

5

SI). If one assumes in this case that the total surface of acaulis for the production of lower weight homologs

flowers is negligible compared to the total area of was even more obvious in the case of anteiso-alkanes

leaves, an estimate of the total amount of alkanes per with a maximum at C₂₆(ACL=26.91), whereas the

2

cm of the leaves is 10.6 and 7.1 μg for P. veris and P. most prominent anteiso-alkane in P. veris was C₃₀

acaulis, respectively. The composition of the alkanes (ACL=29.33). It seems also that P. acaulis had a

differed between P. veris and P. acaulis both qualita- significantly higher preference to produce even-car-

tively and quantitatively. Primula veris was found to bon numbered anteiso-alkanes (CPI=26.54) in com-

produce longer-chained alkanes (C –C ) when com- parison to P. veris (CPI=10.76).

23

35

pared to P. acaulis (C –C ), but all medium- and long-

Unexpectedly, we also found predominantly even-

9

33

chain alkanes (C –C ) were detected only in trace carbon numbered distributions for iso-alkanes as well,

9

21

amounts (Table S2, SI). Both VLC-n-alkane profiles of characterized by very similar CPI values (4.76 and 4.54,

the combined leaf and flower washings had the usual for P. veris and P. acaulis, respectively). However, the

higher plant odd-even ratio, and it showed a max- most prominent iso-homolog from P. veris was again

imum at C . These n-alkane distributions had an C₃₀(like in the case of anteiso-alkanes), while the

29

average chain length (ACL) of 28.93 and 26.77 for P. maximum of the distribution for P. acaulis (C ) was

24

veris and P. acaulis, respectively, accompanied by the shifted to lower-weight homologs by three acetate

carbon preference index (CPI) values of 9.93 and 11.30, units. Normally, plant waxes contain odd-numbered

respectively. CPI and ACL were calculated according to chain length iso-alkanes more abundantly compared

a modified formula previously published.^[The ten- to the even-numbered homologs.

dency of P. acaulis to produce higher amounts of

lower-chain alkanes in comparison to P. veris is clearly

seen in their ACL values.

9]

[18]

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Identification of VLC Benzyl Esters in P. veris and P. easily recognized by the characteristic ions at m/z 91

acaulis Wax Washings

(tropylium cation; base peak) and m/z 108 (protonated

benzyloxy cation; Figure S2 and Table S3, SI).^[

19]

Slightly more polar chromatographic fractions of P. Although no molecular peaks were observable for

veris and P. acaulis wax washings were found to some of the FABEs, molecular weights of these esters

contain twenty-eight fatty acid benzyl esters (FABEs; could be inferred from a characteristic fragment ion

+

[19–20]

Figure 1 and Table 1). Twenty-five of these VLC benzyl [MÀ 91] .

One could easily observe groups of

alkanoates (C –C ), undetected in the GC/MS runs of three closely eluting peaks with almost identical mass

2

0

32

+

the unfractionated wax washings of P. veris, were spectra (the same [M] and/or [MÀ 91] ion). The

Table 1. Content of VLC FABEs in leaves and flowers wax washings of P. veris and P. acaulis.

RI^[^a^]

Compound

P. veris^[^b^]

P. acaulis^[^b^]

Method of identification^[^c^]

2

3

4

984

Benzyl eicosanoate

Benzyl docosanoate

Benzyl tricosanoate

Benzyl 22-methyltricosanoate

tr^[^d^]

tr

n.d.^[^e^]

tr

n.d.

tr

n.d.

tr

n.d.

tr

n.d.

tr

n.d.

tr

n.d.

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

RI, MS, NMR, D

RI, MS, CoI, NMR, D

190

294

360

397

462

473

507

563

575

603

660

674

706

762

776

810

861

875

913

962

976

016

063

074

119

162

222

Benzyl tetracosanoate

7.0ꢀ0.4

1.0ꢀ0.05

0.98ꢀ0.05

3.2ꢀ0.2

1.0ꢀ0.05

0.98ꢀ0.07

7.2ꢀ0.5

1.1ꢀ0.06

1.0ꢀ0.05

0.95ꢀ0.04

tr

3.7ꢀ0.3

0.96ꢀ0.08

0.95ꢀ0.04

1.0ꢀ0.05

0.96ꢀ0.05

tr

Benzyl 23-methyltetracosanoate

Benzyl 22-methyltetracosanoate

Benzyl pentacosanoate

Benzyl 24-methylpentacosanoate

Benzyl 23-methylpentacosanoate

Benzyl hexacosanoate

Benzyl 25-methylhexacosanoate

Benzyl 24-methylhexacosanoate

Benzyl heptacosanoate

Benzyl 26-methylheptacosanoate

Benzyl 25-methylheptacosanoate

Benzyl octacosanoate

Benzyl 27-methyloctacosanoate

Benzyl 26-methyloctacosanoate

Benzyl nonacosanoate

Benzyl 28-methylnonacosanoate

Benzyl 27-methylnonacosanoate

Benzyl triacontanoate

Benzyl 29-methyltriacontanoate

Benzyl 28-methyltriacontanoate

Benzyl hentriacontanoate

Benzyl 30-methylhentriacontanoate

Benzyl dotriacontanoate

1.4ꢀ0.08

0.94ꢀ0.04

tr

0.97ꢀ0.06

Total

36.39 (28)

tr (6)

Normal chain (N)

Even-numbered

Odd-numbered

Iso (I)

Even-numbered

Odd-numbered

Anteiso (A)

25.47 (12)^[^f^]

20.27 (7)

5.20 (5)

6.91 (9)

2.91 (5)

4.00 (4)

4.01 (7)

0.98 (3)

3.03 (4)

Even-numbered

Odd-numbered

[

a]

[b]

RI – Experimental retention indices on a DB-5 column calculated against a series of co-injected n-alkanes (C –C ). Content of

29

44

VLC FABEs expressed in micrograms (μg) per 100 g of plant material. ^[Identification method: RI – Retention index matching; MS –

c]

mass spectra comparison; CoI – additional confirmation by co-injection of an authentic sample; NMR – NMR analysis; D – derivation

[

d]

[e]

reaction to the corresponding methyl esters. tr – trace (detected but <0.007 μg/100 g of plant material). not detected.

[

f]

Number in brackets represents the number of VLC FABEs belonging to that class.

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isomers could be further classified based on their GC- beeswax are esters of palmitic, oleic, and tetracosanoic

retention behavior, i.e., the constant retention index acids with 1-alkanols ranging in length from 12 to 36

[

27]

difference of ca. 103 units (Table 1). Within the groups carbons,

beeswax was used as a source of these

of three, the slowest eluting homolog was presumed very-long-chain alcohols. After transesterification of

to be the normal-chained isomer; this, in the case of beeswax with MeONa in MeOH, a mixture of C –C n-

6

32

+

the group with [M] at m/z 430, was later verified by alcohols was obtained, which was further oxidized to

co-injection of a synthetic sample of benzyl behenate, the corresponding fatty acids. The composition of this

1

that was also completely spectrally characterized ( H- fatty acid mixture was determined by GC/MS analysis

(

400 MHz) and ¹³C-NMR (100.6 MHz) in CDCl , MS, IR, as the corresponding methyl esters obtained after

3

and UV/VIS; Figures S3–S7, SI) and found to be present derivatization of a small sample (ca. 10 mg) with

in P. veris and P. acaulis wax washings. Although this CH N . This GC/MS analysis, along with NMR analysis,

2

kind of qualitative GC/MS analysis is generally accept- proved the lack (even in trace amounts) of any

[

9,28]

able, a certain degree of ambiguity in this approach branched-chain fatty acids in our sample.

Finally,

exists in the case of VLC FABEs: the low abundance or esterification (under Steglich conditions) with benzyl

non-existence of molecular ions or other ions allowing alcohol, yielded a model mixture of benzyl n-alka-

the determination of molecular weight.^[

19,21]

Also, for noates (C –C ; Scheme S1, SI). The composition of

6 32

similar reasons, the differentiation of normal and this FABEs model mixture was also determined by GC/

branched (e.g., iso- or anteiso-) chain FABEs cannot be MS (Table S4, SI), while the presence of any non-

based solely on MS data. The lack of RI data for these volatile contaminants was excluded by NMR analysis.

classes of branched esters in the literature (there are Gas co-chromatography of the obtained model mix-

only reported RI data for benzyl C –C

alkanoates;

n- ture with the fraction of P. veris and P. acaulis wax

restricts their applicability. Moreover, washings unequivocally confirmed the presence of

neither one of the so-far detected VLC FABEs has been C –C (except for C ) homolog series of VLC FABEs.

21

24

[22–23]

20

32

21

1

completely characterized by spectral means (e.g. H- All twelve n-FABEs were found herein for the first time

1

3

and C-NMR, IR, or UV/VIS).

in the genus Primula, while the esters of heptacosa-

Our usual GC/MS temperature-programmed runs noic, nonacosanoic, and hentriacontanoic acids repre-

À 1

(

1

70(0’)/5°C min /315(isothermal

30’),

He

flow sent new natural products.

Two more groups of saturated FABEs (with the

À 1

.0 mLmin ) for the analysis of wax constituents,

ends the linear programming at ca. 49 min. The same molecular weights as the corresponding normal-

majority of the detected VLC FABEs eluted during the chain isomer) eluted slightly faster from the GC

isothermal part of the program (hexacosanoates and column than the mentioned normal chain homologs,

higher-weight esters, R >49 min), where there is no implying that these were their branched-chain isomers

t

longer a linear relationship existing between the (Table 1). The first group of branched VLC FABEs had

retention times (indices) and the number of CH₂retention indices (ΔRIs) 36–60 units lower in value

groups in the molecules, so the usual calculation of RIs compared to the related normal chain isomer, while

[24]

is not applicable. Thus, an apparent straightforward the second series of VLC FABEs had the ΔRI value of

mutual correlation of their retention times was not 23–47 units. When compared to the herein identified

[

25–26]

possible, not even in the case of the n-isomers.

Primula iso-alkane series (C –C ), for which ΔRI is

24 35

To determine with certainty the RIs of all FABEs, the 37 units, and anteiso-alkane series (C –C ), where

24

35

samples were reanalyzed under different GC/MS ΔRI equals 27 units, the noted ΔRI for iso-, and

À 1

conditions – 200(0’) 1°C min /315(isothermal 30’), He anteiso-VLC FABEs fit the general trend. Recently, a

À 1

flow 1.0 mLmin , so that, now, all detected com- similar trend in the case of long-chain fatty acids

pounds eluted in the linear part of the program.

methyl esters (FAMEs) was noted with ΔRI values of 36

Since natural sources of longer (than behenic) n- and 28 units, for iso- (C –C ) and anteiso-isomers

9

20

[

28]

fatty acids are quite limited, most of them are not (C –C ), respectively.

Moreover, it is well-known

11

19

commercially available, and if milligram scale amounts that both alkanes and fatty acids are produced in

are accessible (like in the case of lignoceric and plants through the same biosynthetic pathway, by

hexacosanoic acids), they are usually very expensive. which an even-numbered fatty acid is shortened by

Therefore, to unambiguously identify other longer one carbon to give the corresponding odd-numbered

[

9]

normal chain homologs in the wax washings, a non- alkane, and vice versa. Thus, it seemed reasonable to

standard synthetic approach was followed. As it is assume that P. veris and P. acaulis produce VLC iso-

well-known that the main constituents (ca. 40%) of and anteiso-FABEs along with iso- and anteiso-alkanes.

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Isomeric methyl branched FAMEs are easily mutu- our identifications. Precisely, the gHSQC (¹H-

1

3

ally distinguished according to their mass spectral (400 MHz) and C-NMR (100.6 MHz) in CDCl Figure S8,

3

fragmentation patterns since scission commonly oc- SI) spectrum pointed to the existence of four different

curs at adjacent bonds to the tertiary carbon atoms, types of (terminal) methyl groups. Values of proton

yielding characteristic ions of variable intensities for and carbon chemical shifts of these methyl groups

+

different classes of isomers ([MÀ 43] for iso-, and were almost identical to the data previously reported

+

[28,29]

[

MÀ 29] and [MÀ 57] for anteiso-FAMEs).

This for the equivalent methyl groups in the long-chain n-,

[

1,28]

prompted us to transform our FABEs to the corre- iso-, and anteiso-fatty acids.

Neither of these

sponding FAMEs by transesterification of a portion of branched VLC FABEs have been reported so far as

the fraction enriched with FABEs using a solution of natural products.

MeONa in MeOH. Detailed GC/MS analyses of the thus

The GC/MS calibration curve for the synthetic

obtained FAMEs (by selected ion monitoring (SIM); benzyl behenate was built up to quantify FABEs in the

Figure 2) allowed us to establish ΔRI values of 37 and plant material. The content of VLC FABEs determined

2

7 for the two series of branched VLC FAMEs. Excellent per 100 g of fresh P. veris leaves and flowers was

matching of these values with the previously pub- within the range 0.94–7.2 μg (Table 1), and the total

2

lished ΔRI data for long-chain iso- and anteiso-FAMEs, amount of FABEs per cm of the leaves surface was

along with the presence of characteristic ions in their 25.1 ng. Although benzyl esters of normal-chain VLC

mass spectra, enabling the assessment of the branch- fatty acid were predominant (with the total amount of

ing in (ω-1)- and (ω-2)-position, supported our identi- 25.47 μg per 100 g), P. veris biosynthesized a signifi-

fication of VLC iso- and anteiso-FABEs in P. veris and P. cant amount of esters of branched-chain VLC fatty

acaulis wax washings. Interestingly, the presence of acids, as well (6.91 and 4.01 μg per 100 g for iso- and

the methoxycarbonyl group did not cause any signifi- anteiso-fatty acids, respectively). As can be seen from

cant change in the ΔRI values of iso- and anteiso- Table 1, this type of quantification was not possible in

isomers compared to the corresponding alkanes, while the case of P. acaulis, as the content of FABEs therein

the presence of the benzyloxycarbonyl group induced was much lower.

a decrease in these ΔRI values. This means that there

If one considers common biosynthetic pathways for

is a distinct influence of different functional groups on n-, iso-, and anteiso-fatty acids, one should expect an

ΔRI values of branched-chain isomers. Also, in the case even-numbered predominance for the herein identi-

of benzyloxycarbonyl group, additional π-π interac- fied n- and iso-FABEs and an odd-numbered predom-

tions with the GC-column stationary phase should not inance for the anteiso-FABEs. Like in the case of iso-

be neglected. Finally, a detailed NMR analysis of the alkanes, only iso-FABEs did not follow this expected

fraction containing VLC FABEs additionally confirmed

Figure 2. Comparison of selected ion monitoring (SIM) chromatograms of the fraction containing FABEs (A, m/z 91) and the FAMEs

À 1

mixture obtained after the transesterification reaction (B, m/z 74). GC temperature-programmed runs: 70(0’)/5°Cmin /315

À 1

(

isothermal 30’), He flow 1.0 mLmin

.

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trend, showing a slight odd-to-even prevalence (CPI= isomeric 2-alkanones. For example, the isomers of 2-

+

1

.37).

heptacosanone (the same [M] at m/z 394, Figure S10,

Up to now, VLC FABEs have not been reported as SI) had RI(DB-5MS) values of 2883, 2894, and

secondary metabolites of any Primula spp., and their 2923 units. As branching introduced to a molecule

occurrence is very restricted in samples of natural lowers its boiling point (and the RI value), it was

origin. A homologous series of nine benzyl alkanoates reasonable to assume that the isomer with the highest

with chain lengths from C₂₀to C₃₀(except for C₂₇and RI value could be the normal chain isomer, i.e., 2-

C ) have been identified in foliage epicuticular waxes heptacosanone. Unfortunately, the identification was

2

9

of two Eucalyptus species (E. globulus and E. again challenged by the lack of available retention

[30–34]

nitens).

Interestingly, these studies showed that indices in the literature. Precisely, only a single

reduced defoliation of some E. globulus genotypes by previous report of an RI value for 2-heptacosanone

[

38]

the autumn gum moth (Mnesampela privata Guenée) could be found. Mahmood et al. reported the value

was associated with an increased abundance of the of 2840 for RI on an SPB-5 column (equivalent to our

even carbon chain-length benzyl alkanoates (C –C ) GC column) that is significantly lower than the value

2

0

26

in their foliar waxes.^[

33–34]

Even-numbered VLC FABEs found for any of the isomeric 2-heptacosanones

have been detected in the epicuticular waxes of a detected in the washings. However, the RI value for 2-

small number of other plant species: Jojoba (leaf, C – nonacosanone, RI(DB-5MS)=3122, another detected

2

4

[

20]

C ),

bread wheat (flag leaf blade and peduncle, 2-alkanone, was in better agreement with the data

3

0

[35]

[23]

[39]

[40]

C –C ), common hogweed (inflorescences, C ),

from the literature (3131 (DB-5),

3116 (DB-5MS),

2

8

32

24

[36]

[23]

and olive (fruits, C –C ).

Except for the study of and 3121 (DB-5MS) ). Also, ΔRI values for the

24

28

jojoba leaf wax composition, where FABEs were possible iso- and anteiso-2-alkanone isomers (38 and

isolated and the identity of some of them was 28 units, respectively), corresponding to the two faster

confirmed by GC co-injection experiments with au- eluting peaks within the isomeric groups, are just

thentic standards (C₂₆and C₂₈),^[all other studies slightly higher than the values established for the

based the identification of FABEs on the comparison corresponding Primula alkanes series.

20]

of their mass spectra with the mass spectra available

As far as we know, VLC methyl ketones have

in commercial databases (e.g., NIST) or within an ‘in- previously been reported as components of plant

house’ MS database of wax compounds, usually in cuticular waxes from only several species: common

tandem with retention index (or times) values match- hogweed (Heracleum sphondylium, inflorescences, C

2

7

[

23]

[41]

ing but frequently the retention data was not reported and C ), Festuca arundinacea (leaves, C ), potato

2

9

29

37]

[

at all. It is expected that the spectral and retention (Solanum tuberosum; leaves, C –C ),

data reported herein will make the identification of globulus (foliage, C ),

Eucalyptus

and Avicennia marina (aerial

VLC methyl ketones were also

this way we identified 19 previously unreported VLC identified in the soils, peat, sediments, and clay

2

5

33

[

33]

2

7

[

42]

these and related FABEs a much easier task. Hence, in parts, C –C ).

2

6

35

[

37]

benzyl esters, having from 24 to 32 carbon atoms in mineral sealing of waste disposal sites,

the chains and distributed unevenly in n-, iso-, and components of surface lipids or gland secretions of

as well as

[

43]

anteiso-series (Table 1 and Figures S9-1–S9-9, SI).

snakes (C , C –C , C ), lizards (C , C , C , C –

27

29

33

35

27

29

31

33

[

44]

[45]

[39]

C ), spiders (C₂₉and C₃₁), butterflies (C ), and

35

29

[

46–47]

bumble bees (C , C , and C ).

On the other

hand, there were no previous reports on any branched

VLC 2-alkanones.

25

27

29

Identification of VLC 2-Alkanones in P. veris and P.

acaulis Wax Washings

Our TIC chromatograms of P. veris and P. acaulis

Both chromatographies of Primula wax washings

washings contained a dozen of VLC compounds (C – yielded fractions, that eluted with hexane – Et O (85:

24

2

C ) with their mass spectra consistent with those of 15, v/v), composed solely of 2-alkanones (Figure 1 and

3

0

saturated methyl ketones, showing a molecular ion, Table 2). Direct NMR measurements (without further

and the losses of 15, 18, and 60 amu, as well as, separation) confirmed that methyl ketones constituted

intense ions at m/z 58 (McLafferty rearrangement) and this fraction, and gave the information on the

+

m/z 43 ([R CꢁO] ), and a base peak at m/z 59 existence, type, and position of branching of these

1

[37]

(protonated acetone).

In some cases (C –C ), molecules; the ketone carbonyl group appeared at

24 27

analogously to FABEs, there were two or three δ 209.3; a singlet at δ 2.13 and a triplet at δ 2.41

C

H

1

13

compounds with the same mass spectrum eluting at (J=7.5, H- (400 MHz) and C-NMR (100.6 MHz) in

different retention times, indicating the presence of CDCl , Figure S11, SI) were readily assigned to the

3

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Table 2. Content of VLC 2-alkanones in the leaves and flowers wax washings of P. veris and P. acaulis.

RI^[^a^]

Compound

Class

P. veris^[^b^]

P. acaulis^[^b^]

Method of identification^[^c^]

2

3

476

21-Methyl-2-docosanone

2-Tricosanone

22-Methyl-2-tricosanone

21-Methyl-2-tricosanone

2-Tetracosanone

23-Methyl-2-tetracosanone

22-Methyl-2-tetracosanone

2-Pentacosanone

24-Methyl-2-pentacosanone

23-Methyl-2-pentacosanone

2-Hexacosanone

25-Methyl-2-hexacosanone

24-Methyl-2-hexacosanone

2-Heptacosanone

26-Methyl-2-heptacosanone

25-Methyl-2-heptacosanone

2-Octacosanone

27-Methyl-2-octacosanone

2-Nonacosanone

28-Methyl-2-nonacosanone

27-Methyl-2-nonacosanone

2-Triacontanone

I

N

I

A

N

I

A

N

I

A

N

I

A

N

I

A

N

I

N

I

A

N

I

N

I

N

n.d.^[^d^]

tr^[^e^]

RI, MS, NMR

514

578

588

615

680

690

717

781

792

820

883

894

923

985

996

023

086

125

189

199

227

329

391

429

493

531

632

733

0.13ꢀ0.009

0.23ꢀ0.02

0.087ꢀ0.005

0.089ꢀ0.006

0.17ꢀ0.01

tr

0.56ꢀ0.04

0.80ꢀ0.06

0.43ꢀ0.03

0.13ꢀ0.006

tr

0.55ꢀ0.03

0.28ꢀ0.02

0.20ꢀ0.01

0.046ꢀ0.003

tr

_t0_r .057ꢀ0.003

tr

0.048ꢀ0.03

0.088ꢀ0.006

tr

t⁰r^.⁰⁵²^ꢀ⁰^.⁰⁰³

0.50ꢀ0.03

0.70ꢀ0.04

0.32ꢀ0.02

0.30ꢀ0.02

0.60ꢀ0.04

0.21ꢀ0.01

1.9ꢀ0.1

1.4ꢀ0.08

0.22ꢀ0.01

0.29ꢀ0.02

tr

0.90ꢀ0.05

0.41ꢀ0.03

0.34ꢀ0.02

tr

_t0_r .23ꢀ0.02

tr

2-Hentriacontanone

30-Methyl-2-hentriacontanone

2-Dotriacontanone

31-Methyl-2-dotriacontanone

2-Tritriacontanone

2-Tetratriacontanone

2-Pentatriacontanone

_t0_r .44ꢀ0.03

tr

n.d.

_t0_r .13ꢀ0.008

n.d.

0.45ꢀ0.03

n.d.

tr

Total

4.207 (27)

11.110 (27)

Normal chain (N)

Even-numbered

Odd-numbered

Iso (I)

Even-numbered

Odd-numbered

Anteiso (A)

1.880 (12)^[^f^]

0.365 (6)

1.515 (6)

1.610 (9)

1.310 (5)

0.30 (4)

0.717 (6)

0.717 (4)

tr (2)

4.940 (12)

0.520 (5)

4.420 (7)

3.90 (9)

3.01 (5)

0.89 (4)

2.27 (6)

2.06 (4)

0.21 (2)

Even-numbered

Odd-numbered

[

a]

[b]

RI – Experimental retention indices on a DB-5 column calculated against a series of co-injected n-alkanes (C –C ). Content of

24

38

VLC 2-alkanones expressed in micrograms (μg) per 100–g of plant material. ^[Identification method: RI – Retention index matching;

c]

[d]

MS – mass spectra comparison (Figures S12-1–S12-6, SI); NMR – NMR analysis. tr – trace (detected but <0.007–μg/100–g of

plant material). ^[not detected. Number in brackets represents the number of VLC FABEs belonging to that class.

e]

[f]

methyl and methylene protons in the α-position to 31.9, implying the existence of CH À CH À CH À struc-

3

2

[48]

the ketone. Alongside the mentioned CH CO group, tural fragment from the non-branched isomers. The

3

DEPT-135 and gHSQC spectra pointed to the existence nonet at δ 1.54 ( J=6.6), partially overlapped with

H

of four additional different methyl groups at δ 11.4, the signal of the β-methylene group, that coupled

C

3

1

4.1, 19.3, and 22.7 (Table S6, SI). Although the signals with a doublet at δ 0.86 ( J=6.6), indicated the

of these methyl groups overlapped in the H-NMR presence of an isopropyl moiety corresponding to the

H

1

spectrum, CH group that resonated at δ 0.88 and iso-series of 2-alkanones. The assignment of these

3

H

δ_C14.1 correlated through two or three bonds signals was further confirmed by comparison of the

gHMBC) with two methylene carbons, at δ 22.7 and carbon C-13 and proton chemical shifts with those

(

C

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[

49]

reported for the synthetic 11-methyl-2-dodecanone.

in any sample of natural origin. There is a previous

1

3

The C-NMR chemical shifts of the two remaining report on medium-length branched long-chain methyl

methyl groups, at 11.4 and 19.3 ppm, were almost ketones, iso-(C –C ) and anteiso-2-alkanones (C –

13

16

14

identical with those reported for ω- and (ω-2)-methyl C ), alongside normal chain isomers (C –C ), that

16

12

16

groups (11.4 and 19.2 ppm, respectively) from 11- are emitted by different marine arctic strains of the

methyl-2-tridecanone,^[

existence of anteiso-2-alkanones in the fractions.

To quantify these methyl ketones in the plant essential oil constituent of Ruta graveolens L. (C ).

49]

providing evidence for the Cytophaga-Flavobacterium-Bacteroides group.

even shorter chain iso-2-alkanone were identified as

[49]

Also,

[50]

11

material, a sample of 2-pentacosanone was synthe- Normal chain methyl ketones (C –C ) are also major

9

13

sized to be further used as a standard for the components of Ruta chalepensis L. essential oil and

construction of the corresponding calibration curve. some of them (C and C ) display biting deterrence,

9

11

These measurements revealed that P. acaulis produced repellence,

and

larvicidal

activities

against

[

51]

significantly higher quantities of VLC (C –C ) methyl mosquitos.

23

35

ketones compared to P. veris (11.1 vs. 4.2 mg per 100 g

of fresh plant material, Table 2; or 6.1 vs. 2.9 μg per

cm of the flower and leaf surfaces). Strikingly, both

Distribution of VLC Alkanes, FABEs, and 2-Alkanones in

Different P. acaulis Samples

2

species produced dominantly branched-chained 2-

alkanones with more than 50% of the total 2- After the initial study of non-flavonoid constituents of

2

alkanones (1.6 and 3.3 μg per cm of the flower and P. veris and P. acaulis wax washings, three additional

leaf surfaces, for P. veris and P. acaulis, respectively), samples (P-4 to P-6, Table 3) of P. acaulis, and a single

and mutually comparative shares of n-: iso-: anteiso-2- sample of a high mountain Primula spp. from

alkanones (45:38:17, and 44:34:20, for P. veris and P. Azerbaijan (P-3, Table 3) were collected. Three of them

acaulis, respectively). The chain branching distribution represented material gathered in the blooming phase

of VLC methyl ketones in P. veris and P. acaulis was from different locations in southeastern and eastern

characterized by two major, almost equally abundant Serbia, while a sample of Primula spp. (most likely a

2

1

-alkanones: 24-methyl-2-pentacosanone (0.80 and subspecies of P. acaulis) from northern Azerbaijan

.9 mg per 100 g, respectively) and 2-pentacosanone contained only leaves since it was collected in July.

(0.56 and 1.9 mg per 100 g, respectively). While n- and Moreover, the quantity of two samples allowed us to

anteiso-2-alkanones distributions reflected the conven- study the composition of wax washings of flowers and

tional higher plant pattern with an odd- (ACL=26.82 leaves separately (samples P-4 and P-6). GC/MS

and 26.60, and CPI=4.15 and 8.50, for P. veris and P. analyses (Tables S7–S9 in SI) of the obtained crude

acaulis, respectively) and even-carbon predominance chloroform washings revealed interesting features

(

ACL=26.32 and 25.93, CPI=23.90 and 9.80, respec- regarding the distribution of VLC alkanes, FABEs, and

tively), in the case of iso-isomers we have noted again methyl ketones in different plant organs.

an unusual even to odd prevalence (ACL=26.03 and Except for Primula spp. from Azerbaijan (P-3) and P-

5.77, CPI=4.37 and 3.38, respectively). 5, all other studied P. acaulis samples produced high

To the best of our knowledge, neither of the herein amounts of branched VLC alkanes (accounting for ca.

found VLC iso- (C –C , C , C ) and anteiso-2- 23–56% of the total alkanes) that were deposited in a

2

23

30

32

33

alkanones (C –C , C ) have not been found thus far larger amount (2–3 times) on leaves than on flowers

24

28

30

Table 3. Data of the collected and herein studied Primula species.

Sample designation Name^[^a^]

Habitat

mountain Suva planina, SE Serbia May 2015

Date of collection

Plant organ

[

b]

P-1

P-2

P-3

Primula veris L.

flowers and leaves

leaves

P. acaulis (L.) L.

village Gornja Slatina, SE Serbia

Tufandag, a mountain of the

Greater Caucasus, N Azerbaijan

April 2015

July 2015

Primula spp. (most likely

a subspecies of P. acaulis)

P. acaulis (L.) L.

[

c]

b]

c]

P-4

P-5

P-6

mountain Suva planina, SE Serbia March 2016

flowers and leaves

P. acaulis (L.) L.

village Gornja Slatina, SE Serbia

village Veliki Izvor, E Serbia

March 2016

[

a]

[b]

The Latin binominals are given according to The Plant List: http://www.theplantlist.org/. Plant organs of these species were

analyzed together. ^[Plant organs of these species were analyzed separately.

c]

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(Table S7, SI). The domination branched vs. normal unambiguously confirmed that the unusual predom-

isomers was most pronounced for leaves compared to inance of even-numbered VLC alkanes and 2-alka-

flowers. Additionally, not only that the mentioned nones is a unique feature of this plant species.

species from Azerbaijan biosynthesized lower amounts Unfortunately, it seems that the content of branched

of VLC iso- and anteiso-alkanes but also produced VLC FABEs in studied Primula waxes was too low so to

lower overall amounts of alkanes than the other three be directly detected in the crude wax washings.

P. acaulis species from Serbia. We noted an analogous

trend in the case of VLC 2-alkanones as well (Table S9,

Unusual Even-to-Odd Chain Preference of iso-Alkanes

SI). Interestingly, two lower iso-homologs, 14-methyl-

and 2-Alkanones and Odd-to-Even Chain Preference of

2

-pentadecanone, and 15-methyl-2-hexadecanone,

iso-FABEs in Primula Species

were detected in the flower wax of samples P-4 and P-

6

, which were not previously been identified in any of In higher plants, wax components with straight-chain

the analyzed Primula species. Moreover, separate hydrocarbon backbones are all formed from VLC n-

analyses of flowers and leaves wax washings revealed fatty acids that are biosynthesized by sequential

that VLC FABEs were primarily accumulated on flowers elongation of the precursor acetyl-CoA with C units

2

as these wax constituents either were not present or derived from malonyl-CoA. In this condensation-

were found only in trace amounts on leaves. However, elongation process, acyl chains of up to C₁₆and C₁₈

the subspecies from Azerbaijan again fell out of this formed by de novo synthesis are further extended to

rule, as significant amounts of VLC FABEs were found C₃₀or higher by a second elongation system. Finally,

in its leaves wax washings (Table S8, SI).

modification of the acyl chain gives products including

The fact that the wax washings were isolated from alkanes, aldehydes, primary alcohols, alkyl esters,

plant material collected in different phenological secondary alcohols, ketones, and various polyoxygen-

[

53]

phases (in the blooming and after anthesis for Serbian ated compounds. On the other hand, branched VLC

and Azerbaijan species, respectively) could be the fatty acids (and other derivatives resulting from them)

reason for the alteration in the biosynthesis of non- are synthesized by successive elongation of CoA

flavonoid wax constituents and their accumulation on thioesters of short branched-chain acids derived from

specific plant organs. Another factor that could cause branched-chain amino acids such as valine, leucine,

the differences in wax composition among these P. and isoleucine. Thus, isobutyryl-CoA (derived from

acaulis species is the adaptation to environmental valine), isovaleryl-CoA (derived from leucine), and α-

conditions, as Primula spp. was collected on Tufan- methylbutyryl-CoA (derived from isoleucine) are the

dang Mountain (north Azerbaijan) at an attitude great- termini precursors for iso-fatty acids having an even

er than 3300 m, whereas all other P. acaulis samples number of carbon atoms, iso-fatty acids having an odd

from Serbia grew at altitudes less than 870 m. It has number of carbon atoms, and anteiso-fatty acids

been previously reported for some Primula species having an odd number of carbon atoms,

[

18,54]

that they exhibit an essentially conservative, slow- respectively.

growing survival strategy throughout its climate Previous studies indicated that plant species that

envelope, from the highest to the lowest elevations produce higher amounts of iso-alkanes (and other wax

that is (among other things) reflected in increasing constituents) incorporate valine-derived starters (i) ex-

leaf size towards lower elevations associated with xclusively (like Nicotiana benthamiana), so only even-

[

52]

decreasing leaf nitrogen contents per unit mass.

numbered iso-alkanes are present in their waxes, or

Therefore, the declining production of branched VLC (ii) selectively (like Arabidopsis thaliana) leading to the

alkanes, as well as of total alkanes, and increasing predominance of even-numbered homologs com-

accumulation of VLC FABEs on the leaves of Primula pared to the odd-numbered ones (that are leucine-

[

18]

spp. from Azerbaijan could be a cold-adaptive survival derived products).

The herein studied Primula

strategy of this species, as a change in wax composi- species produce predominantly even-numbered iso-

tion could cause the change in the melting point and alkanes and 2-alkanones, as well as odd-numbered

fluidity of the leaf wax layer. Moreover, the accumu- fatty acid benzyl esters. Thus, it seems that these

lation of VLC FABEs could also be triggered by an species prefer more to incorporate the leucine-derived

increased flux of UV radiation at high attitudes (see starter, rather than valine-derived one, during the

Section Potential UV protective role of Primula FABEs). biosynthesis of the wax constituents with the iso-

VLC alkanes, FABEs, and 2-alkanones in waxes of P. backbone, which is a unique feature in the Plant

acaulis species originating from different habitats Kingdom. Considering the characteristic branching

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patterns of these wax constituents and the isomer isoleucine. However, significant amounts of some odd-

patterns predicted for the possible methyl branch numbered anteiso-alkanes (e.g., C , C , and C ) and

25

29

31

introduction mechanisms, we propose the most likely 2-alkanones (C₂₅and C ), as well as even-numbered

27

biosynthetic pathways by which these new (or rare) anteiso-FABEs (C ) were found in the herein analyzed

26

secondary metabolites could be formed in the plant Primula wax washings (Tables S7–S9, SI). Thus far, it

tissues. Very similar chain length profiles of iso-alkanes, has not been clarified by which biosynthetic route

iso-FABEs, and iso-2-alkanones suggest that these these anteiso-homologs are formed, and usually, plants

compounds share the same biosynthetic pathway. either produce them in trace amounts or do not

[

18,49]

Thus, even-numbered iso-2-alkanones could be formed produce them at all.

Although the logical amino-

by, initially, hydrolysis of odd-numbered 3-keto acyl acid derived starter for these types of anteiso-com-

CoA intermediates of elongation, followed by a pounds would be the one that arises from homoiso-

subsequent decarboxylation.^[⁵⁵^]Major iso-alkanes and leucine (2-amino-4-methylhexanoic acid, Scheme 1),

iso-FABEs are both, most probably, formed from the this is a quite rare amino acid the origin of which is

corresponding odd-numbered VLC acyl-CoA that is the attributed to the non-specificity of the enzymes

substrate for two parallel pathways. Specifically, iso- involved in leucine biosynthesis, and, so far, homoiso-

alkanes with the odd total carbon number could be leucine was described just once as a plant metabolite.

generated in the decarboxylation step,^[while the Precisely, it was found in the seeds of Aesculus

last step in the biosynthesis of iso-FABEs could be californica where it serves as a nitrogen-storage

catalyzed by some alcohol acyltransferases, which link material that is degraded during the subsequent

alcohols to acyl moieties (Scheme 1). Similar alcohol germination of the seeds, making the pool of nitrogen

18]

[

57]

acyltransferases have been previously found in melon, readily available.

strawberry, and banana.^[

56]

Among the anteiso-series, we noted the normal

predominance of homologs that are derived from

Scheme 1. Proposed biosynthetic pathways to even-numbered iso-alkanes and 2-alkanones, and to odd-numbered iso-fatty acid

benzyl esters (ACP – acyl carrier protein; CoA – coenzyme A; ox-TA – oxidative transamination; DE – (oxidative or reductive)

decarboxylation; LCFA biosynth – long-chain fatty acid de novo biosynthesis; FAE – fatty acid elongase; KCS – ketoacyl-CoA

synthase; KCR – ketoacyl-CoA reductase; HCD – hydroxyacyl-CoA dehydratase; ECR – enoyl-CoA reductase; AAT – alcohol acyl-

transferase).

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Structure-Retention Index Relationship

acyl chain) have a significant influence on both the

methylene value and the functional group increment.

It was previously reported that as the alkyl chain of

The presence of n-, iso-, and anteiso-FABEs and methyl

ketones homologous series in P. veris and P. acaulis the alcohol moiety of the esters lengthens from

wax washings enabled us to study structure-retention methyl to n-hexyl, the methylene value of the homolo-

index relationships for these two classes of natural gous series (C –C ) of esters decreases from 99.25 to

1

6

[

62]

products to provide more general conclusions helpful 85.97 accordingly (on SE-30 column).

Similarly, the

for future investigations. Due to the lack of GC- herein calculated M value for the methyl ester series

retention data for benzyl esters and methyl ketones in (C –C ) on our DB-5 column is 100.45ꢀ0.45. The

6

32

the literature, their identification by MS alone is only presence of the benzyl moiety in our esters caused an

tentative. Thus, a structure–retention index relation- increase in their methylene value. A similar effect was

ship, enabling retention data prediction, could facili- noted previously for the alkyl benzene series that is

tate and authenticate the identification of these characterized by an M value of 102.48 and 103.78 on

secondary metabolites.^[

58–59]

The correlation of the DB-1 and DB-35 columns, respectively.

experimentally derived retention indices (RI) for n-, iso- known that aromatic compounds are more retained

and anteiso-FABEs and methyl ketones with the on GC columns relative to aliphatic compounds with

number of carbon atoms in the aliphatic linear part of an increasing phenyl content of the column stationary

[59]

It is well-

,

[

26]

the molecule (without the atoms in the functional phase. Therefore, the effect of the column stationary

group) could be expressed by a general linear phase on M values of FABEs and alkyl benzenes, due

regression equation:

to π-π interactions between the phenyl groups in the

stationary phase (like in the case of DB-5MS and DB-

35) and aromatic rings in these analytes, should not be

neglected.

RI¼M ꢂ n ðCÞ þ ðFG þ S Me_iÞ

where M is the linear regression coefficient which is

In an analogous manner, linear regression analyses

usually defined as the methylene value as it represents of retention data of n-, iso- and anteiso-2-alkanones

the increment in the value of the retention index for (Figures S16–S18, SI) gave normal chain: RI =101.64×

n

2

each additional -CH - group in the homologous series, n(C)+380.25 (R =0.99999); branched chain: RI =

n is the number of carbon atoms in the alkyl chains of 101.68×n (C)+239.23 (R =0.99999), and RI

2

iso

2

=

anteiso

2

the fatty acids (without carboxylic one) and 2- 101.86×n (C)+347.36 (R =0.99997), where n (C) is

alkanones (without the acyl group carbon atoms), FG the number of carbon atoms minus 2 (from the acyl

is the functional group increment and Me is the group). All three homologous series have almost the

methyl group increment, while its value depends on same M value of ca. 102 units.

the position of the methyl group.^[

60]

Linear regression analysis of the plot of RI vs. n (C)

Thermal Properties of Primula FABEs and Alkanes

for the n-series of our FABEs (Figure S13, SI), yielded M

for n-series and FG for all FABEs regioisomers; there is The hydrophobic nature of the outer layers (cuticle) of

no branching in the molecules of the n-series, so the the plant helps to prevent uncontrollable water loss

[

63]

value of Me is zero. FG represents a value unique for and infiltration of exogenous factors. Although, the

the series of compounds and varies with chemical transport across the cuticle is essentially determined

structure, intermolecular electronic and steric interac- by wax composition, the physical properties of

[

58–59]

tions, and polarity of column liquid phases.

The cuticular lipids, such as the melting point, affect

[

63–65]

methylene value of ca. 103 units was found for the n- cuticular permeability as well.

series: RI =103.23×n(C)+1023 (R =0.99999). It is increases (sometimes within the physiological range),

As temperature

2

n

worth noticing that the intercept value, corresponding the lipids melt and become more permeable, and

to the function group contribution to the RI value, is water loss increases rapidly. The point at which water

comparable to the value of the retention index of loss begins to increase is termed the critical temper-

benzyl formate (1071).^[The analogous equations for ature (Tc) that is highly correlated with the melting

61]

the iso- and anteiso-FABEs series are: RI =100.17× point (Tm) of cuticular lipids (Tm values are usually just

iso

2

[66,67]

n(C)+1057.2 (R =0.99999); RI

1

=100.29×n(C)+ slightly higher than Tc).

067.1 (R =0.99998, Figure S14 and S15, SI). M

anteiso

2

As cuticular wax composition can be quite complex

amounted to ca. 100 units for both iso- and anteiso- (usually contains more than 100 different compounds),

series. It seems that the steric effects (branching in the it is also necessary to understand how these com-

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pounds interact with each other to affect the bulk lipid of the component lipids, suggesting that some

properties.^[

64,68]

While alkanes appear to be omnipre- packing disruption occurs in these mixtures.

[68–69]

sent in the cuticular waxes of higher plants, very-long- Contrary, the results of the very recent study of the

chain FABEs are restricted to just several plant species. phase behavior of binary blends of paraffin wax (PW;

Thus, we decided to study the thermal properties of formed of high molecular weight n-alkanes C –C ),

20

40

FABEs and alkane mixtures isolated from P. veris wax and rice bran wax (RBW; that consists of long-chain

washings. We prepared binary blends containing saturated aliphatic esters C –C ) imply that the n-

44

64

various ratios of benzyl behenate (BB), or model alkanes present in PW do not interact completely with

mixture of benzyl esters (FABEs), and alkanes (ALK) the esters in RBW, leading to a binary wax mixture

and measured their melting points. The obtained with partial co-crystallization forming a monotectic

[

70]

results are presented as a pseudo phase diagram of system. On the other hand, our blend containing (n-

the melting points of our mixtures in the function of , iso- and anteiso-) alkanes and normal chain FABEs

the percentage composition of the mixture (Figure 3). showed a eutectic behavior, with the eutectic point at

The minimum melting point (eutectic point; T ) for 53°C at 70 wt% FABEs, suggesting the upgraded

e

both blends (BB/ALK and FABEs/ALK) occurred at interaction of benzyl esters compared to fatty alcohol

3

0 mass percent of the higher melting component – esters with alkanes.

alkanes; n (ALK): n (BB or FABEs)=1:2.3. Pure benzyl

It has been reported previously that binary mix-

behenate caused a slightly higher melting point tures of n-alkanes and 2-alkanones show eutectic

depression of ALK (Te=51.9°C; ΔT=Tm

– Te= behavior as well. For example, at 30 wt% of 2-

alkanes)

(

1

1.4°C) than the model mixture of FABEs (Te=53.0°C; heptadecanone (T =48.5–49°C), a eutectic mixture,

m

ΔT=10.3°C). It seems that there was, also in both consisting of nearly pure ketone microcrystals mixed

cases, a peritectic point at 30 wt% of benzyl behenate with n-heneicosane (T =41.5–42°C) microcrystals, is

m

or FABEs mixture (n (ALK): n (BB or FABEs)=2.4: 1).

formed at 35.5–36°C (ΔT=6°C). For higher weight

Previously, the thermal properties of simple mix- homologs, it was noted that 10 wt% of 2-heneicosa-

tures between pairs of aliphatic wax esters found in none (T =62–62.5°C) in n-hexacosane (T =55.9°C)

m

insect cuticles (like stearyl arachidate, tetradecyl caused a depression in the melting point of the alkane

[

71]

stearate, and dodecyl myristate) and n-alkanes, con- for 8.3°C.

Unfortunately, we have not isolated

taining from 26 to 48 carbons, were studied. Those sufficient quantities of the mixture of 2-alkanones

pairs were chosen in such a way that wax ester and from P. acaulis and P. veris wax washings to study the

alkanes had similar Tm values so that the predicted Tm thermal behavior of its binary blend with the corre-

would be the same as that of the two components. sponding alkane mixture. Also, our synthetic standard

However, these mixtures melted 3–5°C below the Tm of 2-pentacosanone has a low melting (T =30.3°C)

m

point which makes it unsuitable for the study of

thermal properties.

Specifically, it was reported that the ice nucleation

temperature of fairy primrose (P. malacoides) leaves

covered with natural flavone was approximately 6°C

lower compared to those that had their flavone

artificially removed. Additionally, it was found that

these leaf farinose flavonoids reduced subsequent

electrolyte leakage from the cells exposed to freezing

temperatures. It is well-known that branching in the

carbon backbone also causes depression in the

melting point. For example, 2-methylpentacosane

[

72]

melts ~12°C lower than n-hexacosane.

Hence, the

preference of Primula species for high amounts of

various types of branched-chain wax constituents

certainly influences overall wax thermal properties.

Although most of the plant species biosynthesize

Figure 3. Melting temperatures of binary blends containing P. lipids that remain in the solid state at environmental

veris alkanes and benzyl behenate or the benzyl esters model temperatures, partial melting often occurs due to the

[

68]

mixture.

deposition of lipids with low melting points, or due

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to the presence of a certain combination of lipid radiation, especially in species that grow at higher

classes in waxes (like alkanes and esters, primary altitudes (like the herein studied Primula spp. from

alcohols or methyl ketones) whose mutual interactions Tufandang Mountain).

cause significant depression of the wax melting

[69,71,73]

point.

Although, this will tend to increase water

loss, there as some benefits as well, which may include

a better dispersal of lipids or other compounds across

Conclusions

the cuticle (like those that improve resistance to As we already published, the analyzes of the surface

microbial attack or host-plant resistance, or ‘fix’ the wax of P. veris revealed that this species produced

strain in the cutin matrix of leaf and fruit cuticles that high amounts of iso- and anteiso- compounds, along-

results from the expansion of the underlying surface side normal chain homologs^.^[¹^]This work provides

during growth) or improved release of cuticular results, in addition to previous research, continuous

pheromones that can act as visual and olfactory study of non-flavonoid constituents of the surface

markers for insects or may act as triggers to other waxes of P. veris and P. vulgaris (Primulaceae): VLC

[

63,69,74]

plants.

normal and branched (iso- and anteiso-) alkanes, FABEs

and 2-alkanones. To the best of our knowledge,

normal VLC FABEs and 2-alkanones have restricted

occurrence in the Plant Kingdom, while the branched-

Potential UV Protective Role of Primula FABEs

The amount of light falling on the plant and reaching chain ones have not been previously found in any

the leaf cells is critical for the wellbeing of the plant, sample of natural origin.

as plants, like all other living organisms, sense and

Although the presence of VLC FABEs and 2-

respond to UV radiation, both the wavelengths alkanones in any analyzed sample is readily recogniz-

present in sunlight (UV-A and UV-B) and the wave- able based on characteristic intense ions in their mass

lengths below 280 nm (UV-C).^[

63,75]

All types of UV spectra, the unavailability of reliable gas chromato-

radiation are known to affect various plant processes graphic data in the literature for these compounds

causing both DNA damage (which can cause heritable (especially for the branched-chain ones) brings un-

mutations)

and/or

damage

to

physiological certainty to their identification based solely on GC/MS

processes.^[Over the last three decades, as ozone analysis. Moreover, in contrast to VLC alkanes, the

depletion has become a major environmental prob- presence and position of methyl branching are not

lem, the particular role of flavonoids in screening clearly visible in mass spectra of branched VLC FABEs

short-wavelength solar UV-B radiation (over the 280– and 2-alkanones. Therefore, we were faced with a

75]

[

76]

3

15 nm waveband) has been strongly emphasized.

challenging situation in our efforts to undoubtedly

For example, simple flavone (2-phenyl-4H-1-benzopyr- confirm the identity (whether some homolog is normal

an-4-one) exhibits two major absorption peaks (in or branched-chain isomer) of the mentioned wax

methanol) at 307 nm (Band I associated with absorp- constituents.

tion due to the B-ring cinnamoyl system) and 250 nm

Luckily, studied Primula species produced signifi-

(

Band II with absorption involving the A-ring benzoyl cant amounts of these metabolites (including the

[76]

system),

so it could be expected that it acts as a branched-chain ones), which allowed isolation of every

good UV-B protectant in Primula species exudates.

class of compounds (alkanes, FABEs, and methyl

Shorter wavelength UV light (UV-C), with higher ketones) in separate chromatographic fractions. Sub-

associated energy, is the most hazardous range of UV sequent 1D and 2D NMR analyses of obtained mixtures

lights emitted by the sun. Fortunately, UV-C is strongly (containing three homologs series of isomers) enabled

affected by the ozone layer in the stratosphere, so that not only the unequivocal identification of certain

the amount of this radiation reaching the Earth’s compound classes but also the determination of the

[

77]

surface, except for high mountains, is still low.

branching type and its position. Alongside the men-

However, whether there will be an increase in UV-C tioned lack of RI data in the literature, GC/MS

flux on Earth, as a result of stratospheric ozone identification of the members of the mentioned

depletion due to atmospheric pollution, or not, is homologous series of branched and normal chain

unpredictable. Benzyl behenate absorbs almost over FABEs and 2-alkanones was also impeded by the low

the whole UV-C range having the maximum at 210 nm volatility of these wax constituents, so some of them

with high molar absorptivity. Thus, FABEs could help eluted from the GC column in the part of the

to minimize damage to the plant caused by UV-C chromatogram where the existence of linearity be-

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tween the retention times (indices) and the number of that is essential for control of water loss, or minimizing

CH groups in the molecule is somewhat uncertain. the damage to the plant caused by UV-C radiation)

2

Therefore, to unambiguous confirm the identity of throughout the lifecycle of the herein studied Primula

these compounds we had to convert them to more species. This is further sustained with the unique

volatile derivatives whose MS and GC data are well feature of these Primula species to predominantly

described in the literature, and/or to synthetized biosynthesize even-numbered iso-alkanes and 2-alka-

authentic standards, and/or to compare GC behavior nones, as well as odd-numbered iso-FABEs.

of all three series of FABEs and 2-alkanones with

corresponding alkane’s series under different GC

conditions (as GC elution pattern of n, iso, and anteiso- Experimental Section

alkanes is well-known).

General Experimental Procedures

Although neither of the branched-chain FABEs and

2

-alkanones have been previously described as natural All commercially available chemicals, purchased from

products, there is still a possibility that these VLC wax Sigma Aldrich (USA), Merck (Germany), and Acros

constituents were simply neglected due to their low Organics (Belgium), were used as received. Preparative

content in the plant waxes or misidentified (for chromatographic separations were carried out using

example as n-homolog) because of the lack of reliable silica gel 60 (particle size distribution 35–70 μm; Acros

retention data in the literature. Thus, we have studied Organics, Geel, Belgium). Alumina-backed silica gel 40

the structure-retention index relationship for homo- F₂₅₄plates (MACHEREY-NAGEL GmbH & Co. KG, Düren,

logs series of VLC n-, iso-, and anteiso-FABEs and Germany) were used for analytical TLC. The spots were

methyl ketones and established corresponding equa- visualized by UV light (254 nm), or by spraying with

tions for RI prediction that should make GC/MS 50% (v/v) aqueous H SO or phosphomolybdic acid

2

4

identification of these secondary metabolites more (12 g) in EtOH (250 mL) followed by a short period of

straightforward in the future.

heating.

Moreover, we have revealed some interesting facts

about the distribution of these wax constituents in

1

13

Proton ( H) and carbon ( C) NMR spectra were

different P. acaulis plant organs. Firstly, in the bloom- recorded on a Bruker Avance III 400 spectrometer

ing phase this Primula species accumulated two- to (Bruker Corporation, Fällanden, Switzerland) operating

three-fold more VLC iso-alkanes and 2-alkanones on at 400 and 100.6 MHz, respectively. 2D experiments

1

the leaves than on the flowers. However, VLC alkanes ( H, H-COSY, NOESY, gHSQC, and gHMBC) and DEPT-

and 2-alkanones represented the main constituents of 90/135 were run on the same instrument with the

flower wax washings, whereas Primula-type flavonoids built-in Bruker pulse sequences. Solutions were pre-

dominated the composition of leaves wax washings. pared in CDCl with chemical shifts (in ppm) refer-

3

1

13

Besides, VLC FABEs were almost exclusively deposited enced to tetramethylsilane in H- and C-NMR spectra

on the surface of flowers. The only exception was the and/or CDCl (δ 77.2) and residual CHCl (δ 7.3) in

1

3

C

3

H

surface wax isolated from the leaves of Primula spp. 2D NMR; abbreviations used: s – singlet, dd – doublet

collected on Tufandang Mountain (north Azerbaijan) of doublets, t – triplet, tt – triplet of triplets, q –

that contained considerably lower amounts of normal quartet, m – multiplet (denotes complex pattern), br.

and branched-chain alkanes and 2-alkanones, and – broad signal.

higher amounts VLC FABEs. The mentioned changes in

the leaves wax composition could be due to the

GC/MS analyses were performed on a Hewlett-

different phenological phases of this Primula spp. Packard 6890N gas chromatograph equipped with a

sample or could represent an adaptation survival fused silica capillary column DB-5MS (poly(5% diphen-

strategy to different climate conditions at such high yl/95% dimethylsiloxane), 30 m×0.25 mm, film thick-

attitudes. These findings, along with our discovery that ness 0.25 μm; Agilent Technologies, Santa Clara, CA,

binary bland containing Primula (n-, iso-, and anteiso-) USA) and coupled with a 5975B mass selective

alkanes and normal chain FABEs shows eutectic detector from the same company. The injector and

behavior and that FABEs could help plant in screening interface were operated at 250 and 320°C, respec-

solar UV-C radiation (whose flux is increased on higher tively. Two temperature programs were used; pro-

attitudes), indicates that normal and breached VLC gram 1: oven temperature was raised from 70 to

alkanes, FABEs, and 2-alkanones have important 315°C at a heating rate of 5°C/min and then,

adaptive roles (e.g., changing of the wax melting point isothermally held for 30 min; program 2: oven temper-

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ature was raised from 200 to 315°C at a heating rate 5 mg of P. veris (P-1) and P. acaulis (P-2 and P-5)

of 1°C/min and then, isothermally held for 30 min. As flowers and leaves surface wax bulk extracts

a carrier gas helium was used with a flow of 1.0 mL/ (5 batches), respectively. Along with a portion of wax

min for both programs. The samples, 1 μL of the washings, a sample of concentrated used solvent was

sample solutions in CHCl (10 mg dissolved in 1 mL), subjected to GC/MS analysis to identify any contami-

3

were injected in a split mode (split ratio 40:1). The nation possibly arising from the use of the described

mass selective detector was operated at the ionization procedures and/or solvents.

energy of 70 eV, in the 35—850 amu range and

scanning speed of 0.34 s. The relative abundance of

In the case of two additional P. acaulis samples (P-4

the detected constituents in the washings and and P-6; Table 3) from Serbia, the flowers (320 g and

fractions was calculated from peak-areas without the 140 g of fresh plant material, respectively) and leaves

use of correction factors, as described below. The IR (200 g and 240 g of fresh plant material, respectively)

measurements (ATR – attenuated total reflectance) were separated, and the surface of flowers and leaves

were carried out using a Thermo Nicolet model 6700 individually washed to yield 112ꢀ3, 43ꢀ8, 624ꢀ12

FT-IR instrument (Waltham, MA, USA). UV spectra (in and 435ꢀ10 mg of surface wax, respectively. As only

hexane) were measured using a UV-1800 UV-vis leaves were available to us for the taxon from

spectrophotometer (Shimadzu, Kyoto, Japan). Melting Azerbaijan (sample P-3; Table 3), only leaves wax

points were determined on MPM-HV2 melting point washings were studied (150 g of fresh leaves yielded

meter (Paul Marienfeld GmbH & Co. KG, Lauda- 96 mg of surface wax).

Königshofen, Germany). Elemental microanalyses for

carbon and hydrogen of the synthesized compounds

Chromatographic Fractionation of the Bulk Flower and

were carried out with a Carlo Erba 1106 microanalyzer

Leaf P. veris and P. acaulis Wax Washings

(Carlo Erba, Milan, Italy).

Primula veris (P-1) leaves and flowers wax washings

were subjected to dry-flash column chromatography

Plant Material

using a gradient of diethyl ether (Et O) and hexane

2

The data for the herein studied Primula species are (from pure hexane to pure Et O, with an increment

2

presented in Table 3. Their Latin binominals are given step of 5%, v/v; fraction volume: 100 mL). The

according to The Plant List: http://www.theplantlis- obtained fractions were pooled according to TLC and/

th

t.org/ (accessed on April 10 , 2021). Voucher speci- or GC/MS analyses. The alkanes (77.2 mg) were

mens were deposited in the Herbarium of the Faculty comprised the hexane fraction. The fraction that

of Sciences and Mathematics, University of Niš, eluted with hexane-Et O (90:10, v/v) mixture (17.3 mg)

2

Herbarium Moesiacum Niš (HMN), under the accession proved (after GC/MS analysis) to be a complex mixture

numbers 13906, 14437, 14438, 14439, 14440, 14441, containing, along with several other constituents, a

and their identity was confirmed by one of the co- series of benzyl esters with very-long-chain fatty acids,

authors (V.R.).

while the following fraction by polarity (8.3 mg

hexane-Et O (85:15, v/v)) contained 2-alkanones. An

2

analogous fractionation procedure was applied to P.

acaulis (P-2) wax washings. Alongside alkanes

Flower and Leaf Wax Washings

At first, fresh flowers and leaves of P. veris and P. (34.8 mg), two fractions representing complex mix-

acaulis were cleaned from dust by mild agitation. tures were isolated. One of them, among a number of

Pooled waxes from the surface of flowers and leaves, other constituents, contained a series of benzyl esters

samples P-1 (775 g of fresh plant material), P-2 (400 g), with very-long-chain fatty acids (90:10, v/v, 7 mg),

and P-5 (200 g; Table 3), were washed with CHCl in an while the other consisted of nearly 80% (based on GC/

3

ultrasound bath, at room temperature. Although each MS) of very-long-chain 2-alkanones (12.7 mg; hexane–

flower (leaf) was dipped in chloroform no more than Et O (85: 15, v/v)).

2

5

s, the two organs were not analyzed separately. The

chloroform wax washings were then filtered through a

Syringe Econofilter (25/0.45 lm RC, Agilent Technolo-

gies, Santa Clara, CA, USA) to remove any insoluble

Very-Long-Chain Alkanes, Benzyl Esters and

-Alkanones Identification and Quantification

2

material, dried with anhydrous MgSO and concen- Qualitative analysis of VLC alkanes, FABEs, and methyl

4

trated in vacuo to yield 926ꢀ25, 735ꢀ10 and 360ꢀ ketones from the chloroform wax washings of the

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herein studied Primula species and the corresponding C6’-H); ¹³C-NMR spectral data (100.6 MHz, CDCl ): δ

3

1

chromatographic fractions was based on at least three 14.1 (br. q, J(C,H)=124.3, C-22), 22.7 (br. t, J(C,H)=

of the following five criteria:^[¹^]positive matches of 125.2, C-21), 24.9 (br. t, J(C,H)=127.1, C-3), 29.1(C-4),

1

linear retention index (RI) values (or ΔRI values for the 29.2 (C-19), 29.4, 29.5 and 29.6–29.7 (br. t J(C,H)=

corresponding branched-chain isomers) and the mass 124.7, overlapped, from C-5 to C-18), 31.9 (C-20), 34.3

1

spectra with those from the literature, GC co-injection (br. t, J(C,H)=128.4, C-2), 66.0 (tt, J(C,H)=147.1,

2

experiments with an authentic sample, derivatization

J(C,H)=3.8 C-1’), 128.1 (overlapped m, C-4’), 128.2

reactions and NMR measurements (Table S6, SI). MS (overlapped m, C-3’ and C-5’), 128.5 (br. dd, J(C,H)=

data for all identified very-long-chain FABEs and 2- 160.4, J(C,H)=5.9, C-2’ and C-6’), 136.1 (m, C-1’), 173.7

alkanones are given in Tables S3 and S5 in the (m, C-1); EIMS (70 eV) m/z (rel. int.): 430 [M] (1), 339

1

2

+

Supporting Information (SI) document, respectively.

[MÀ PhCH ] (6), 321 [MÀ PhCH À H O] (11), 303

2

+

[

MÀ C H ] (2), 108 [PhCH OH] (86), 91 [PhCH ]

9

19

2

+ +

Quantification of VLC alkanes, benzyl esters, and 2- (100), 71 [C H ] (13), 57 [C H ] (21), 43 [C H ]

5

11

4

9

3 7

alkanones in the plant material, i.e., per plant leaf (20); Found: C, 80.8; H, 11.7, O, 7.5. Calc. for C H O :

29 50 2

surface, was carried out by the peak-area integration C, 80.87; H, 11.70; O, 7.43%; R =0.37 (hexane/Et O,

f

2

method. Authentic standards of n-dodecane, benzyl 24:1, v/v).

behenate, and 2-pentacosanone were injected at

seven different concentrations to build up seven-point

Preparation of a Model Mixture of Very-Long-Chain

Benzyl Esters

GC/MS calibration curves by plotting compound

concentration versus peak area (C=f(A)). Each sample

was analyzed in three consecutive runs. The parame- Beeswax (2 g) was added in small portions with stirring

ters of the calibration curves obtained are given in into a refluxing MeONa solution, prepared by dissolv-

Table S10 in SI. The leaf surface of each species was ing metallic sodium (2 g) in dry MeOH (100 mL). After

calculated from the corresponding digital images the entire amount of the added beeswax dissolved,

using AutoCAD software, while millimeter paper was the reaction mixture was heated for an additional 2 h

utilized for scaling.

and then, quenched with excess ice-water, followed by

extraction with dichloromethane (3×100 mL). The

organic layers were combined, dried with anhydrous

MgSO and the solvent removed in vacuo.

Synthesis of Benzyl Docosanoate (Benzyl Behenate) by

Steglich Esterification

4,

Behenic acid (0.510 g, 1.5 mmol), N,N’-dicyclohexylcar-

The sample of the transesterified beeswax was

bodiimide (0.330 g, 1.6 mmol), benzyl alcohol subjected to silylation and GC/MS analysis to verify the

(

0.468 mL, 4.5 mmol), and 4-(dimethylamino)pyridine presence and identity of long-chain alcohols. Ten

0.018 g, 0.15 mmol) were dissolved in dry tetrahydro- milligrams of the crude sample were placed in a GC/

furan (20 mL). The reaction mixture was stirred for MS vial, dissolved in dry pyridine (0.1 mL) and then,

4 h under anhydrous conditions. The precipitated N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA,

N,N’-dicyclohexylurea was filtered off and the solvent 0.1 mL), and (CH ) SiCl (0.01 mL) were added. The vial

2

3

was removed in vacuo. The pure ester (according to was capped and heated to 60°C for 20 min and after

GC/MS and NMR) was obtained after isocratic dry-flash that, the sample was directly analyzed by GC/MS. The

chromatography on silica gel using hexane-Et O (97: 3, crude mixture of very-long-chain alcohols was then

2

v/v) mixture as the eluent (0.505 g, yield: 78.3%).

purified by isocratic dry-flash chromatography on silica

gel (hexane-Et O, 88:12, v/v).

2

Benzyl behenate; white crystalline solid; M.p.

5

6.4°C; λ

nm (log ɛ): 210 (4.0), hexane; FT-IR-ATR

The isolated mixture of alcohols was further

max

[

78]

(neat): 3020–3060 (ν(C -H)), 2955 (ν (CH )), 2916 oxidized by Jones reagent. The oxidizing agent (8 N)

Ar as 3

(ν (CH )), 2848 (ν (CH )), 1734 (ν(C=O), 1472 (ν(C = was slowly added to a stirred solution of the alcohol

as

2

s

2

Ar

C )), 1462 (δ (CH )), 1389 (δ (CH )), 1166 (ν(C-O)), 746, mixture (0.500 g) in acetone (~0.1 M) at 0°C until the

Ar

as

2

s

2

1

7

30 (γ(C -H)), 719, 696 (Φ(C -C )); H-NMR spectral solution remained orange-colored. The reaction was

data (400 MHz, CDCl ): δ 0.88 (3H, t, J(H,H)=6.9, H- then stirred for 10 min before water (40 mL) was

Ar Ar Ar

3

2

2), 1.19–1.33 (36 H, m, from H-4 to C21-H), 1.63 (2H, added, and the aqueous layer was extracted with

3

tt, J(H,H)=7.6, 6.7, C3-H), 2.35 (2H, t, J(H,H)=7.6, C2- hexane (3×100 mL). The combined organic layers

H), 5.11 (2H, s, C1’-H), 7.29–7.41 (5H, m, from C2’-H to were washed with brine, dried with anhydrous MgSO₄,

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and the solvent was removed in vacuo. The complete

conversion of the very-long-chain alcohols to the

Acknowledgements

corresponding acids was confirmed by GC/MS analysis This work is a part of the Ph.D. thesis of Milena

of a small portion of the crude sample (10 mg) Živković Stošić supervised by Niko Radulović. This

[

28]

pretreated with CH N (~3% Et O solution).

The work was funded by the Ministry of Education, Science

2

obtained mixture of very-long-chain acids (without and Technological Development of Serbia [Project No.

further purification) was used for the Steglich-type 172061, No. contract 451-03-9/2021-14/200124].

esterification of benzyl alcohol as described for the

synthesis of benzyl behenate. After purification using

isocratic dry-flash chromatography (hexane-Et O, 97:3,

v/v), the percentage composition of the model mixture

2

Author Contribution Statement

of very-long-chain benzyl ester was determined by N.R., M.G., and M.Ž.S. performed the experiments for

GC/MS analysis (Table S4 in SI). The mixture of the thus the isolation, structure elucidation, analyzed the data,

obtained FABEs consisted of n-C -C , in the side and prepared the manuscript; V.R. confirmed the

6

32

chains.

identity of the plant species and read the final version

of the manuscript.

Synthesis of 2-Pentacosanone

2

-Pentacosanone was synthesized starting from meth-

yl acetoacetate in a classic three reaction sequence References

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Very-Long-Chain Wax Constituents from Primula veris and P. acaulis: Does the Paradigm of Non-Branched vs. Branched Chain Dominance Universally Hold in all Plant Taxa?

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