Synthesis of alkyl-glycerolipids standards for gas chromatography analysis: Application for chimera and shark liver oils

Article abstract of DOI:10.3390/md16040101

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20–24%), 16:0 (42–54%) and 18:1 (6–16%) and, to a lesser extent, (0.2–2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

Full text of DOI:10.3390/md16040101

marine drugs
Article
Synthesis of Alkyl-Glycerolipids Standards for Gas
Chromatography Analysis: Application for Chimera
and Shark Liver Oils
Michelle Pinault 1, Cyrille Guimaraes 1, Hélène Couthon 2, Jérôme Thibonnet 3,4
Delphine Fontaine 1, Aurélie Chantôme 1,5, Stephan Chevalier 1,5, Pierre Besson 1,5
,
ID
,
Paul-Alain Jaffrès 2,† and Christophe Vandier 1,4, ,†
*
1
Inserm, UMR1069, Université de Tours, 37000 Tours, France; Michelle.pinault@univ-tours.fr (M.P.);
cyrille.guimaraes@gmail.com (C.G.); delphine.fontaine@etu.univ-tours.fr (D.F.);
aurelie.chantome@univ-tours.fr (A.C.); stephan.chevalier@univ-tours.fr (S.C.);
pierre.besson@univ-tours.fr (P.B.)
CEMCA, CNRS UMR6521, Université de Brest, IBSAM, 6 Av V. Le Gorgeu, 29238 Brest, France;
helene.Couthon@univ-brest.fr (H.C.); Paul-Alain.Jaffres@univ-brest.fr (P.-A.J.)
Equipe SIMBA, Synthèse et Isolement de Molécules BioActives, EA 7502, Université de Tours, 37000 Tours,
France; jerome.thibonnet@univ-tours.fr
2
3
4
5
Faculté de Sciences et Techniques, Université de Tours, 37000 Tours, France
Faculté de Pharmacie, Université de Tours, 37000 Tours, France
*
†
Correspondence: christophe.vandier@univ-tours.fr; Tel.: +(33)-247366024; Fax: +(33)-247366226
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 January 2018; Accepted: 20 March 2018; Published: 23 March 2018
Abstract: Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty
alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues
but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only
a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl
chains of variable length are not available. These compounds are, however, necessary as standards
for analytical methods. Here, we investigated different reported procedures and we adapted some
of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various
lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these
standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify
the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0
(20–24%), 16:0 (42–54%) and 18:1 (6–16%) and, to a lesser extent, (0.2–2%) for each of the following:
16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours
and compare their composition to those of non-tumour tissues.
Keywords: ether-lipids; alkyl-glycerolipids; standards for gas chromatography; fish oil
1. Introduction
Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), have a long fatty alkyl chain
linked to the glycerol unit by an ether bond [1]. The most prevalent glycerol-backboned AEL found
in nature consist of an O-alkyl or O-alk-10-enyl group attached to glycerol at position sn-1. In cells,
AEL can be triglycerides (diacyl-glyceryl-ether lipids, DAGE) which possess two fatty acyl moieties
at positions sn-2 and sn-3, or phospholipids (1-O-Alkyl-2-Acyl-sn-glycerylphospholipids, AAGPL)
with one fatty acyl chain at the sn-2 position and one phosphodiester group (e.g., phosphocholine,
phosphoethanolamine). The common feature of these two series of compounds is the 1-O-alkyl-glyceryl
ether lipid (AGEL), as shown in Figure 1.
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Figure 1. Chemical structure of AEL (alkyl-ether-lipid) subtypes: DAGE, AAGPL and the structure of
the common fragment 1-O-alkyl-glyceryl ether lipid (AGEL). For simplification purposes, chirality is
not depicted.
Natural AEL were found to stimulate the immune system [
have anti-tumor properties [ ], reduce the side effects induced by radiotherapy [
reduction in the growth of tumor cells and metastases [ ]. Interestingly, ether-lipids have been also
2] and hematopoiesis [
3]. They also
4
5
], and lead to a
6
considered for potential use in the diagnosis of prostate cancer [7].
Some biological effects of these AEL can be explained by an interaction with their receptors, such as
the platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) [
integration in the phospholipids of cell membranes [10], and by a modification of the activity of Protein
Kinase C (PKC) [11 13] which has a high activity in breast tumors [14]. The integration of AEL into the
membrane results in the formation of DAG ether-lipid analogs, alkyl-acyl-glycerols, which inhibit PKC
activation unlike the “classical” ester-linked, PKC-activating diacyl-glycerol DAG [12 15]. In addition
8,9], or following their
–
,
to a decrease in PKC activity, it has been shown that these lipids inhibit the activity of other kinases
such as phosphatidylinositol-3-kinase [16] and MAP kinases [17], but also Na+/K+ ATPase [18]. AEL
derivative edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine) was found to inhibit
the activities of Na+/H+ exchanger [19] and K+ channel [20], as well as the transport of nutrients [21].
Thus, all of these effects can participate in the anti-proliferative effects of AEL. The replacement of the
phosphocholine moiety by an aminoglycoside group led to a large series of compounds exhibiting
anticancer properties [22
edelfosine by a saccharide unit to design glyco-AEL, we identified less toxic compounds whereas
the modulation of ion channels was preserved [24 27]. Among disaccharide-AEL, [28] we identified
,23]. Interestingly, with the replacement of the phosphocholine moiety of
–
ohmline (1-O-hexadecyl-2-O-methyl-rac-glycero-3-lactose) as a lead synthetic glycol-AEL that can
prevent metastasis development by modifying the activity of ion channel complexes (KCa-Ca2+)
located in cholesterol-rich nano-domain and regulating calcium signaling and migration of cancer
cells [29–31]. Some aspects of the mechanism of action of ohmline were recently reported, showing
that ohmline fluidified the plasma membrane, especially if this plasma membrane had a high level
of cholesterol [32]. Altogether, these results pointed out the great therapeutic potential of synthetic
ether lipids and contribute to questions about the role of natural ether lipids. AEL are abundant
in many tissues, and especially in shark liver oil, breast milk, red blood cells, blood plasma, and
bone marrow [33–35]. Among human tissues, bone marrow contained the highest percentage of
alkyl-glycerols in both triglycerides and phospholipids fractions [35]. In mammals, there is a higher
prevalence of alkyl-glycerols in phospholipid fractions compared to neutral lipid (triglycerides)
fractions [36]. Interestingly, tumors have been found to contain larger amounts of AEL than normal
tissues [36], but the physiological roles of these natural AEL have not been elucidated so far. The clinical
significance of their accumulation in tumors remains to be determined. Furthermore, no quantitative
analysis has been carried out in tissues to precisely determine the proportion of AEL molecular species
based on their alkyl chain length and unsaturation level. If some AELs are commercially available
(e.g., 1-O-hexadecyl-sn-glycerol, also known as chimyl alcohol, and 1-O-octadecyl-rac-glycerol also
known as batyl alcohol), others with saturated alkyl chain of variable length or monounsaturated alkyl

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chains are, unfortunately, not commercially available. These compounds are necessary as standards
for analytical methods. This led us to investigate reported synthesis procedures and to adapt some of
them to prepare a series of 1-O-alkyl-glycerols with saturated chain length (14:0, 16:0, 17:0, 19:0, 20:0,
22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by
NMR and GC-MS. Finally, we used these standards to precisely characterize alkyl-glycerols subtypes
in shark and chimera liver oils.
2. Results and Discussion
2.1. Alkyl-Glycerolipids Chemical Synthesis
Since our goal was to have alkyl-glycerol standards for analytical purpose, the chirality at the
sn-2 position was not an issue since the targeted compounds (4a–i; Figure 2) possess only one
asymmetric carbon and, therefore, on non-chiral support, both enantiomers will possess similar
physico-chemical properties including retention time. As a consequence, the syntheses of the standards
were achieved in racemic series. Solketal
1 is the common commercial precursor to prepare O-alkyl
glycerol since the free alcohol function can be deprotonated and then engaged in an alkylation
4
reaction with a lipid alcohol activated with a mesyl functional group or directly with a halogenoalkyl.
Prinz et al. [37] used KOH to deprotonate solketal in benzene and then myristoylmethanesulfonate
(C14:0) or palmitoylmethanesulfonate (C16:0) was added. After alcohol deprotection with HCl in
methanol, the alkylglycerol 4a (C14:0) and 4b (C16:0) were isolated in 77–85% yield (two steps).
Aranda et al. [38] only changed the solvent (DMSO) to prepare compound 4a (C14:0) in 80–85% yield.
Huang et al. [39] used NaH in toluene for deprotonation of solketal and a subsequent alkylation was
achieved with bromoalkane (1-Bromotetradecane, 1-Bromohexadecane or 1-Bromooctadecane), thus
producing compounds 4a
consisted of using lipid alcohol and racemic solketal
–
b
(yields were not reported). Another approach that retained our attention
activated with a tosyl group as the leaving
2
group. The use of this activated solketal was previously reported by Bhatia et al. [40] to produce
1-O-hexadecyl-sn-glycerol in 65% yield (including the acetal deprotection). We prepared the tosylate
2
from solketal 1 following a reported procedure [41]; it is worth noting that compound 2 is also
commercially available (analytical data are available in the Supporting Material, SI1.7, 1.8). Then, we
used the method of Bhatia et al. to introduce the alkylether moiety (Figure 2). First, the alkyl alcoholate
was prepared by deprotonation of the alcohol with NaH and then this alcoholate reacted with the
tosylated solketal
Then the deprotection of the acetal function was achieved in acidic conditions (HCl 2N) in methanol
at reflux and the final compounds 4a were isolated in 92–98% yield. If most of these compounds
2 [42]. After work-up, the expected compounds 3a–i were isolated in 60–96% yield.
–
i
were reported and prepared by other methods, it must be noted that to the best of our knowledge,
compounds 3/4c, 3/4d, 3/4g were not reported before.
For the analysis of the 1-O-alkyl glycerol by GC, protection of the alcohol function must be
achieved. For this derivation step, we have protected the alcohol function with acetate groups. For
this purpose, two methods were employed. Method A consisted to use acetic anhydride in pyridine
at 60 ◦C [43] and method B used acetyl chloride without solvent at 140 ◦C (Figure 2) [44]. Method A
used mild conditions that should reduce degradation but the work-up (treatment with acid and basic
solutions) induced sample losses. Method B is much faster and was efficient with a simplified work-up.
However, this method requires heating at 140 ◦C that can induce, depending on the structure of the
lipid chain, some degradation. In this series of compounds, no degradation was observed including
for the mono-unsaturated compound 5c and 5f. The final compounds 5a–i were fully characterized
and isolated in good yield (92–98%).

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Figure 2. Synthesis of alkyl-glycerolipids 4a
–
i
and derivatization as diacetyl esters 5a
Pyridine, 1 h, 0 C then 24 h, rt; (ii) preparation of the alcoholate: NaH, ROH, dimethoxyethane, 1 h,
then addition of
, 100 ◦C then 48 h, reflux; (iii) HCl 2N, Ethanol, 4 h, reflux. Method A (iv): acetic
–i. (i) TsCl,
◦
2
◦
◦
anhydride, pyridine, 60 C, 30 min. and then 1 night at 20 C; Method B (
v
): acetyl chloride, 5 min. 140
◦
C.
2.2. Liver Oils Alkyl-Glycerolipids Analysis
In order to precisely identify the alkyl-glycerol subtypes in shark and chimera liver oils we first
extracted the total lipids of chimera and shark liver oil and derived alkyl-glycerols to alkyl-glycerol
diacetates to be analyzed using gas chromatography as described in Sections 3.2 and 3.4.
Figure 3 shows an example of chromatogram of alkyl-glycerols diacetate obtained from chimera
(Chimaera monstrosa) liver oil.
Figure 3. Chromatogram of alkyl-glycerols diacetate from chimera (Chimaera monstrosa) liver oil.
Using the alkyl-glycerols standards, we analysed the alkyl-glycerol subtypes in shark and chimera
liver oils (Figure 4). We found that both oils contain mainly alkyl-glycerols with C14:0, C16:0, and C18:1
alkyl chains and low amounts of C16:1 and C18:0 alkyl chains. Interestingly, C17:0 and C20:0 were
found in Chimaera monstrosa and shark liver oils. Note, that we did not find C19:0 or C22:0 alkyl chains.

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After derivatization as acetate, we optimized the temperature program for the separation and
identification of alkyl-glycerolipids by GC-MS (see Supplementary Materials SI2.1, SI2.2). This method
confirmed the results obtained using GC-FID.
60
Centrophorus squamosus / Somniosus microcephalus
Chimaera monstrosa
50
20
10
0
19:0 20:0 22:0
18:0 18:1
Alkyl chain lenght
14:0
17:0
16:0 16:1
Figure 4. Comparative analysis of alkyl-glycerols of chimera (Chimaera monstrosa) and shark liver (a
mixture of two species: Centrophorus squamosus and Somniosus microcephalus). Bars represent mean
value and error bars represent SD, n = 6. Neither C19:0 nor C22:0 alkyl chains were detected.
We found the presence of 14:0 (20–24%), 16:0 (42–54%), and 18:1 (6–16%) alkyl-glycerols and, to a
lesser extent (0.2–2%), 16:1, 17:0, 18:0 and 20:0 alkyl-glycerols. It was generally reported that liver oil of
certain shark species contain high levels of alkyl-glycerolipids as a mixture of subtype that varied by
length and unsaturation of the alkyl chain with almost exclusively saturated and monounsaturated,
the main alkyl moieties being the C16 and C18 chains in both lipid classes [45,46]. Interestingly,
a high level of 18:1 was also found in the composition of alkyl chains in alkyl-glycerolipids from
Greenland shark (Centrophorus squamosus) liver oil [47]. Thus, our standards confirm the presence of
these alkyl-glycerolipids in shark and chimera liver oils.
3. Materials and Methods
3.1. Chemical Synthesis
Chemicals and solvents ACS grade or higher, commercially available, were used without further
purification (Carlo Erba, Val de Reuil, France; and Sigma-Aldrich, Saint-Quentin Fallavier, France).
NMR spectra (Bruker, Wissembourg, France) were recorded on a Bruker DPX Avance 300 spectrometer
(300 MHz for 1H, 75 MHz for 13C), Bruker DRX-500 (500 MHz for 1H, 125 MHz for 13C), and Bruker
Ascend-600 AVANCE III HD equipped with TCI cryoprobe (600 MHz for 1H, 150 MHz for 13C). CDCl3
was used as the solvent; chemical shifts are expressed in ppm relative to TMS as an internal standard.
Purification of crude products was carried out using Puriflash® 430 (Interchim, Montluçon, France)
equipped with an evaporative light-scattering detector (ELSD). Preparative thin-layer chromatography
(TLC, LK5) separation was carried out using 20
×
20 cm, glass-backed, 1 mm or higher thickness silica
gel Merck F254 plates (VWR, Fontenay-sous-Bois, France). Control TLC was performed using Merck
F254 aluminium-backed silica gel sheets (VWR). Lipid compounds on TLC plates/sheets were revealed
by 20,70-dichlorofluorescein.

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HMRS analysis was performed on the HRMS CNRS ICOA platform at the University of Orleans.
3.2. Analytical Chemistry
Chemicals and solvents, ACS grade or higher, commercially available, were used without further
purification (Carlo Erba).
After the extraction of total lipids by the Folch method [48] ester bonds were reduced using
the strong reducing agent Vitride® (sodium hydride and aluminum) (0.5 mL Red-Al® for 10 mg
total lipids) according to a described method [49], in a 80/20 v/v mixture of diethyl ether/hexane,
giving ether-lipids (1-O-alkylglycerols and 1-O-alk-1-enyl-glycerols) and fatty alcohols. The reaction
medium was neutralized at melting-ice temperature using ethanol/water (20/80, v/v). The reaction
products were recovered from the diethyl ether phase and evaporated to dryness, then were diluted
in chloroform/methanol (2/1, v/v ), and finally separated by TLC on 20
×
20 silica gel plate (LK5)
with chloroform/acetone (90/10, v/v ) as the eluent. The plate was allowed to dry and was then
sprayed with 2,70dichlorofluorescein (0.2% in ethanol) to reveal the compounds under UV light. In
parallel, batyl alcohol (C18:0 AGEL) was deposited on the same plate as a reference standard for
collecting ether lipid samples at the same Rf = 0.18. The silica gel containing the ether lipids was
scraped and transferred into a tube to be derivatized as 1-O-alkylglycerol-diacetate. Acetylation was
performed with a large excess (600 µL) of ethanoyl chloride directly on the silica contained in the
tube, by heating at 140 ◦C for 5 min. Only 1-O-alkylglycerol-diacetate were obtained since alkenyl
lipids are destroyed by the presence of acid in the reaction. Reaction medium was neutralized with
a saturated aqueous solution of sodium hydrogen carbonate. Diacetate of 1-O-alkylglycerols were
extracted with diethyl ether, evaporated to dryness and dissolved in hexane before analysis by gas
chomatography. Gas chromatographic separation of acetate-derivatized 1-O-alkylglycerolipids was
performed on a Shimadzu GC-2010 Plus system (automatic liquid sample injection unit, on-column
injector, flame ionization detector set at 325 ◦C) equipped with a Zebron 5-MS column (Phenomenex,
Le Pecq, France), (30 m, 0.25 mm ID, 0.25
µm film thickness). The temperature program was set as
follows: 60 ◦C (2 min. hold), to 180 ◦C at 14 ◦C/min (4 min. hold), to 185 ◦C at 0.1 ◦C/min (4 min.
◦
◦
hold), then to 250 C at 2 C/min (25 min. hold). Hydrogen was used as a gas vector at constant
pressure of 180 kPa.
3.3. Mass Spectrometry
Optimization of the Separation Method
GC-Ultra-ISQ (Thermo Fisher, Villebon sur Yvette, France) was used with a Zebron 5-MS column
(Phenomenex, Le Pecq, France) having a 30 m length, 0.25 mm of diameter, and 0.25
µ
m film thickness.
L, and the vector gas
min−1. The temperature program was as follows:
◦
The injector temperature was 250 C, the splitless injection volume was 1
µ
was hydrogen with a constant flow rate of 1.5 mL
·
140 ◦C for 1 min, followed by a ramp at 7
·
◦C min−1 up to 310 ◦C, then a 3 min hold. The transfer line
·
temperature was 310 ◦C. The source temperature was 300 ◦C. The solvent delay was 3 min and the
mass range 35–500 m/z.
3.4. Experimentation Section
3.4.1. Preparation of the (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (2)
◦
To a cold (0 C) solution of (
±
)-2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane(4.0 g, 30 mmol),
in anhydrous pyridine (6 mL), p-TsCl (7.0 g, 37 mmol) was added. After stirring for 24 h, diethyl
ether (200 mL) was added and the solution was washed with aqueous HCl (1 M, 2 200 mL), H2O
5 × 100 mL), dried over Na2SO4, and filtered. Diethyl ether was evaporated to give the desired
×
(
1
product as a clear oil. Yield = 81%. H NMR-CDCl3,
δ (ppm) 7.80 (d, J = 8.3 Hz, 2H, CHAr), 7.36 (d,

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J = 8.0 Hz, 2H, CHAr), 4.34–4.22 (m, 1H, CH), 4.10–3.92 (m, 3H, CH), 3.77 (dd, J = 8.8, 5.1 Hz, 1H, CH),
2.46 (s, 3H, ArCH3), 1.34 (s, 3H, CH3 acetate), 1.31 (s, 3H, CH3 acetate).
3.4.2. Preparation of 4-Alkoxymethyl-2,2-dimethyl-1,3-dioxolanes (3a–i)
Alcohol (1.0 eq.) and sodium hydride (60% in mineral oil, 2.8 eq.) were introduced in
dimethoxyethane (10 mL). The solution was refluxed up to the end of H2 emission. Then, a solution
of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (1.4 eq.) in ethane-1,2-diol was
added and the mixture was refluxed for 48 h. After cooling to room temperature, the solution was
poured into water. The organic layer was extracted with diethyl ether and dried over Na2SO4. The
solvents were removed under vacuum and the desired product was obtained after a purification by
flash chromatography in chloroform. The advancement of the purification was followed by TLC in
chloroform/acetone (90:10).
4-(n-Tetradecyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3a)
.
This product was obtained from
1-tetradecanol (0.5 g; 2.4 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68. Yield = 68%
1
(0.52 g). H NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m,
CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol),
1.48 (3H, s, CH3 acetal), 1.41 (3H, s, CH3 acetal), 1.29 (22H, m, CH2), 0.91 (3H, t, J = 6.8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Hexadecyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3b). This product was obtained from
1-hexadecanol (0.5 g; 2.1 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68. Yield = 96%
1
(0.71 g). H NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m,
CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol),
1.48 (3H, s, CH3 acetal), 1.41 (3H, s, CH3 acetal), 1.29 (26H, m, CH2), 0.91 (3H, t, J = 6.8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Hexadec-7-enyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3c). This product was obtained from
1-hexadec-7-enol (0.5 g, 2.1 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68.
1
Yield = 76% (0.56 g). H NMR-CDCl3,
δ (ppm) 5.35 (2H, m, CH=CH), 4.26 (1H, m, CH-glycerol), 4.01
(1H, m, CH2O-alkyl), 3.76 (1H, m, CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol),
2.03 (4H, m, CH2-CH=CH-CH2), 1.75–1.56 (2H, m, CH2CH2O-glycerol), 1.47 (3H, s, CH3 acetal), 1.40 (3H,
s, CH3 acetal), 1.29 (18H, m, CH2), 0.91 (3H, t, J = 7.0 Hz, CH3). 13C NMR-CDCl3, δ (ppm) 130.5, 109.6,
74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.3, 29.2, 29.0, 28.8, 28.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Heptadecyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3d). This product was obtained from
1-heptadecanol (0.5 g, 1.9 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68. Yield = 60%
1
(0.43 g). H NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m,
CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol),
1.48 (3H, s, CH3 acetal), 1.41 (3H, s, CH3 acetal), 1.29 (28H, m, CH2), 0.91 (3H, t, J = 6.8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Octadecyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3e). This product was obtained from
1-octadecanol (0.5 g, 1.8 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68. Yield = 65%
1
(0.46 g). H NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m,
CH2O-alkyl glycerol), 3.40-3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol),
1.48 (3H, s, CH3 acetal), 1.41 (3H, s, CH3 acetal), 1.29 (30H, m, CH2), 0.91 (3H, t, J = 6,8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Octadec-9-enyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3f). This product was obtained from
1-octadec-7-enol (0.5 g, 1.9 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68.
1
Yield = 78% (0.56 g). H NMR-CDCl3,
δ (ppm) 5.35 (2H, m, CH=CH), 4.26 (1H, m, CH-glycerol), 4.01
(1H, m, CH2O-alkyl), 3.76 (1H, m, CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol),

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2.03 (4H, m, CH2-CH=CH-CH2), 1.75–1.56 (2H, m, CH2CH2O-glycerol), 1.47 (3H, s, CH3 acetal), 1.40 (3H,
s, CH3 acetal), 1.29 (22H, m, CH2), 0.91 (3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
δ (ppm) 130.5, 109.6,
74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.3, 29.2, 29.0, 28.8, 28.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Nonadecyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3g). This product was obtained from
1-nonadecanol (0.5 g, 1.75 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.69. Yield = 70%
1
(0.49 g). H NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m,
CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol),
1.48 (3H, s, CH3 acetal), 1.41 (3H, s, CH3 acetal); 1.29 (32H, m, CH2), 0.91 (3H, t, J = 6.8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Eicosyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3h). This product was obtained from 1-eicosanol
1
(0.5 g, 1.7 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68. Yield = 68% (0.47 g). H
NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m, CH2O-alkyl
glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol), 1.48 (3H,
s, CH3 acetal), 1.41 (3H, s, CH3 acetal), 1.29 (34H, m, CH2), 0.91 (3H, t, J = 6,8 Hz, CH3). 13C NMR-CDCl3,
δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
4-(n-Docosyloxymethyl)-2,2-dimethyl-1,3-dioxolane (3i).
This product was obtained from
1-docosanol (0.5 g, 1.5 mmol). TLC performed in chloroform/acetone (90:10). Rf = 0.68. Yield = 79%
1
(0.53 g). H NMR-CDCl3,
δ (ppm) 4.26 (1H, m, CH-glycerol), 4.01 (1H, m, CH2O-alkyl), 3.76 (1H, m,
CH2O-alkyl glycerol), 3.40–3.30 (4H, m, CH2OC and CH2O-glycerol), 1.70–1.50 (2H, m, CH2CH2O-glycerol),
1.48 (3H, s, CH3 acetal), 1.41 (3H, s, CH3 acetal), 1.29 (38H, m, CH2), 0.91 (3H, t, J = 6.8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 109.6, 74.2, 71.8, 71.3, 67.3, 31.7, 29.7, 29.2, 29.0, 26.6, 25.7, 23.2, 14.0.
3.4.3. Preparation of the Ether-Glycerol 4a-i by Hydrolysis of Acetal 3a–i
To a solution of 4-alkoxymethyl-2,2-dimethyl-1, 3-dioxolane (1.0 eq.) in methanol (approx. 5 mL),
HCl (2 M, 2.0 eq.) was added and the solution was heated to reflux for 4 h. After cooling to room
temperature, the mixture was poured into water, the organic layers were extracted with ether, dried
over Na2SO4 and the solvents were removed under vacuum. The residue was purified by flash
chromatography with hexane/ethyl acetate 3/1 v/v.
3-(n-Tetradecyloxy)propane-1,2-diol (4a). This product was obtained from compound 3a (0.52 g,
1.58 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 98% (0.45 g). 1H
NMR-CDCl3,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol),1.29 (22H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
(ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 14.
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
δ
3-(n-Hexadecyloxy)propane-1,2-diol (4b). This product was obtained from compound 3b (0.71 g,
1.99 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 92% (0.58 g). 1H
NMR-CDCl3,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol), 1.29 (26H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
(ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 14.
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
δ
(7E)-3-(n-Hexadec-7-enyloxy)propane-1,2-diol (4c). This product was obtained from compound
3c (0.56 g, 1.58 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 98%
(0.49 g). 1H NMR-CDCl3,
δ (ppm) 5.35–5.30 (2H, m, CH=CH), 3.90 (1H, m, CH-glycerol), 3.82–3.75
(2H, m, CH2O-alkyl), 3.69–3.47 (4H, m, CH2OH and CH2O-glycerol), 2.60 (2H, bs, 2xOH), 2.03 (4H, m,
CH2-CH=CH-CH2), 1.63 (2H, m, CH2O-glycerol), 1.28 (18H, m, CH2), 0.91 (3H, t, J = 6.8 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 130.5, 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.3, 29.2, 29.0, 28.8, 28.0, 26.6, 23.2, 14.0.;
HRMS ([M+H]+) calcd for [C19H38O3H]+ 315.2894, found 315.2894.

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3-(n-Heptadecyloxy)propane-1,2-diol (4d). This product was obtained from compound 3d (0.43 g,
1.16 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 95% (0.36 g). 1H
NMR-CDCl3,
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol), 1.29 (28H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
δ (ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2,
14.; HRMS ([M+H]+) calcd for [C20H42O3H]+ 331.3207, found 331.3210.
3-(n-Octadecyloxy)propane-1,2-diol (4e). This product was obtained from compound 3e (0.46 g,
1.20 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 96% (0.40 g). 1H
NMR-CDCl3,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol), 1.29 (30H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
(ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 14.
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
δ
(9E)-3-(n-Octadec-9-enyloxy)propane-1,2-diol (4f). This product was obtained from compound 3f
1
(0.56 g, 1.46 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 96% (0.48 g). H
NMR-CDCl3,
δ (ppm) 5.37–5.30 (m, 2H, CH=CH), 3.88–3.83 (m, 1H, CH), 3.72 (ddd, J = 11.1, 7.0, 3.9 Hz,
1H, CH), 3.68–3.62 (m, 1H, CH), 3.54 (dd, J = 9.7, 3.8 Hz, 1H, CH), 3.50 (dd, J = 9.8, 6.0 Hz, 1H, CH), 3.48
(dd, J = 6.4, 9.2 Hz, 1H, CH), 3.45 (dd, J = 9.2, 6.5 Hz, 1H, CH), 2.58 (d, J = 5.1 Hz, 1H, CH), 2.15 (m, 1H,
CH), 2.01 (dd, J = 12.8, 6.6 Hz, 4H, CH2-CH=CH-CH2), 1.39–1.15 (m, 24H, CH2), 0.88 (t, J = 6.9 Hz, 3H,
CH3). 13C NMR-CDCl3,
δ (ppm), 130.5, 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.3, 29.2, 29.0, 28.8, 28.0, 26.6,
23.2, 14.0.
3-(n-Nonadecyloxy)propane-1,2-diol (4g). This product was obtained from compound 3g (0.49 g,
1.23 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 95% (0.42 g). 1H
NMR-CDCl3,
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol), 1.29 (32H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
δ (ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2,
14.; HRMS ([M+H]+) calcd for [C22H46O3H]+ 359.3520, found 359.3523.
3-(n-Eicosyloxy)propane-1,2-diol (4h). This product was obtained from compound 3h (0.47 g,
1.14 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 95% (0.40 g). 1H
NMR-CDCl3,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol), 1.29 (34H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
(ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 14.
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
δ
3-(n-Docosyloxy)propane-1,2-diol (4i). This product was obtained from compound 3i (0.53 g,
1.20 mmol). TLC performed in hexane/ethyl acetate (75:25). Rf = 0.10. Yield = 95% (0.46 g). 1H
NMR-CDCl3,
CH2OH and CH2O-glycerol), 2.62 (2H, bs, 2xOH), 1.62 (2H, m, CH2O-glycerol), 1.29 (38H, m, CH2), 0.91
(3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
(ppm) 72.1, 71.8, 71.0, 63.5, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 14.
δ (ppm) 3.90 (1H, m, CH-glycerol), 3.80–3.71 (2H, m, CH2O-alkyl), 3.70–3.45 (4H, m,
δ
3.4.4. Derivation Procedure: Preparation of 3-(Alkoxy)propane-1,2-diacetate
Method A [43]: To a solution of 3-(alkoxy)propane-1,2-diol (1.0 eq.) in pyridine (4.0 eq.), acetic
anhydride (5.5 eq.) was added and the solution was heated to 60 ◦C for 30 min and stirred at room
temperature overnight. The organic layer was extracted with diethyl ether, washed with HCl (1 M), an
aqueous solution of K2CO3 (10%), and a saturated solution of NaCl. The resulting organic layer was
dried over Na2SO4 and the solvents were removed under vacuum. The residue was purified by flash
chromatography using chloroform/acetone (90:10).
Method B [44]: In a screw cap tube, to the 3-(alkoxy) propane-1,2-diol (1.0 eq.), acetyl chloride (2.1
eq.) was added. The tube was closed under inert gas and heated at 140 ◦C for 5 min. After cooling at
room temperature, diethyl ether was added. The excess of acetyl chloride was neutralized by dropwise

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addition of a saturated aqueous solution of sodium hydrogen carbonate until no more bubbles were
observed. The organic layer was filtered over Na2SO4 and evaporated to dryness to give the pure
compound as a white powder.
3-(Tetradecyloxy)propane-1,2-diacetate (5a). This product was obtained from compound 4a by using
method A (0.45 g, 1.56 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 95%
1
(0.55 g). H NMR-CDCl3,
δ (ppm) 5.20 (1H, m, CH-glycerol), 4.50 & 4.07 (2H, m, CH2O-acetate), 3.67
& 3.46 (2H, m, CH2O-alkyl), 3.33–3.30 (2H, m, CH2O-glycerol), 2.07 (3H, s, CH3 acetate), 2.03 (3H, s,
CH3 acetate), 1.55–1.47 (4H, m, CH2-alkyl), 1.30–1.24 (20H, m, CH2), 0.89 (3H, t, J = 7 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 171.8, 170.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3, 20.7, 14.
3-(Hexadecyloxy)propane-1,2-diacetate (5b). This product was obtained from compound 4b by using
method A (0.58 g, 1.83 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 95%
1
(0.70 g). H NMR-CDCl3,
δ (ppm) 5.20 (1H, m, CH-glycerol), 4.49 & 4.09 (2H, m, CH2O-acetate), 3.67
& 3.46 (2H, m, CH2O-alkyl), 3.34–3.30 (2H, m, CH2O-glycerol), 2.07 (3H, s, CH3-acetate), 2.03 (3H, s,
CH3-acetate), 1.55–1.47 (4H, m, CH2-alkyl), 1.30–1.24 (24H, m, CH2), 0.89 (3H, t, J = 7 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 171.8, 170.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3, 20.7, 14.
(E)-3-(Hexadec-7-enyloxy)propane-1,2-diacetate (5c). This product was obtained from compound
4c by using method B (0.49 g, 1.56 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77.
Yield = 98% (0.61 g). 1H NMR-CDCl3,
δ (ppm) 5.34-5.30 (2H, m, CH=CH), 5.20 (1H, m, CH-glycerol), 4.50
(1H, m, CH2O-acetate), 4.04 (1H, m, CH2O-acetate), 3.66 (1H, m, CH2O-alkyl), 3.44 (H, m, CH2O-alkyl),
3.33 (2H, m, CH2O-glycerol), 2.07–1.98 (10H, m, 2 x CH3 acetate and CH2-CH=CH-CH2), 1.55 (2H, m,
CH2CH2O), 1.47 (2H, m, CH2), 1.33–1.26 (16H, m, CH2), 0.89 (3H, t, J = 7 Hz, CH3). 13C NMR-CDCl3,
δ
(ppm) 171.8, 170.5, 130.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.3, 29.2, 29.0, 28.8, 28.0, 26.6, 23.2, 21.3,
20.7, 14.0.
3-(Heptadecyloxy)propane-1,2-diacetate (5d). This product was obtained from compound 4d by using
method A (0.36 g, 1.08 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 92%
1
(0.42 g). H NMR-CDCl3,
δ (ppm) 5.20 (1H, m, CH-glycerol), 4.49 & 4.09 (2H, m, CH2O-acetate), 3.67
& 3.46 (2H, s, CH2O-alkyl), 3.34–3.30 (2H, m, CH2O-glycerol), 2.07 (3H, s, CH3-acetate), 2.03 (3H, s,
CH3-acetate), 1.55–1.47 (4H, m, CH2-alkyl), 1.30–1.24 (26H, m, CH2), 0.89 (3H, t, J = 7 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 171.8, 170.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3, 20.7, 14.
3-(Octadecyloxy)propane-1,2-diacetate (5e). This product was obtained from compound 4e by using
method B (0.40 g, 1.16 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 93%
1
(0.46 g). H NMR-CDCl3,
δ (ppm) 5.20 (1H, m, CH-glycerol), 4.49 & 4.09 (2H, d, CH2O-acetate), 3.67
& 3.46 (2H, m, CH2O-alkyl), 3.34–3.30 (2H, m, CH2O-glycerol), 2.07 (3H, s, CH3-acetate), 2.03 (3H, s,
CH3-acetate), 1.55–1.47 (4H, m, CH2-alkyl), 1.30–1.24 (28H, m, CH2), 0.89 (3H, t, J = 7 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 171.8, 170.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3, 20.7, 14.
(9E)-3-(Octadec-9-enyloxy)propane-1,2-diacetate (5f). This product was obtained from compound
4f by using method B (0.48 g, 1.40 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77.
1
Yield = 96% (0.57 g). H NMR-CDCl3,
δ (ppm) 5.38–5.31 (m, 2H, CH=CH), 5.21–5.16 (m, 1H, CH), 4.33
(dd, J = 12.0, 3.6 Hz, 1H, CH), 4.16 (dd, J = 12.0, 6.4 Hz, 1H, CH), 3.55 (dd, J = 10.8, 5.4 Hz, 1H, CH),
3.54 (dd, J = 10.6, 5.1 Hz, 1H, CH), 3.50–3.38 (m, 2H, CH2), 2.09 (s, 3H, CH3 acetate), 2.07 (s, 3H, CH3
acetate), 2.01 (dd, J = 12.8, 6.8 Hz, 4H, CH2-CH=CH-CH2), 1.39–1.20 (m, 24H, CH2), 0.88 (t, J = 7.0 Hz,
3H, CH3). 13C NMR-CDCl3,
δ (ppm) 171.8, 170.5, 130.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.3, 29.2, 29.0,
28.8, 28.0, 26.6, 23.2, 21.3, 20.7, 14.0.
3-(Nonadecyloxy)propane-1,2-diacetate (5g). This product was obtained from compound 4g by using
method A (0.42 g, 1.17 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 95%
1
(0.49 g). H NMR-CDCl3,
δ (ppm) 5.20 (1H, m, CH-glycerol), 4.49 & 4.09 (2H, m, CH2O-acetate), 3.67
& 3.46 (2H, m, CH2O-alkyl), 3.34–3.30 (2H, m, CH2O-glycerol), 2.07 (3H, s, CH3-acetate), 2.03 (3H, s,

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CH3-acetate), 1.55–1.47 (4H, m, CH2-alkyl), 1.30–1.24 (30H, m, CH2), 0.89 (3H, t, J = 7 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 171.8, 170.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3, 20.7, 14.
3-(Eicosyloxy)propane-1,2-diacetate (5h). This product was obtained from compound 4h by using
method B (0.40 g, 1.07 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 95%
1
(0.47 g). H NMR-CDCl3,
δ (ppm) 5.16–5.21 (1H, m, CH-glycerol), 4.33 & 4.15 (2H, d, CH2O-acetate),
3.53 & 3.56 (2H, m, CH2O-alkyl), 3.38–3.48 (2H, m, CH2O-glycerol), 2.09 (3H, s, CH3-acetate), 2.07 (3H, s,
CH3-acetate), 1.61–1.50 (H, m, CH2-alkyl), 1.35–1.25 (34H, m, CH2), 0.87–0.89 (3H, t, J = 7 Hz, CH3). 13C
NMR-CDCl3, δ (ppm) 171.8, 170.5, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3, 20.7, 14.
3-(Docosyloxy)propane-1,2-diacetate (5i). This product was obtained from compound 4i by using
method A (0.46 g, 1.15 mmol). TLC performed in chloroform/acetone (96:4). Rf = 0.77. Yield = 95%
(0.53 g). 1H NMR-CDCl3,
δ (ppm) 5.18 (m, 1H, CH-glycerol), 4.33 (dd, J = 12.0, 3.6 Hz, 1H,
CH2O-acetate), 4.16 (dd, J = 12.0, 6.4 Hz, 1H, CH2O-acetate), 3.55 (dd, J = 10.8, 5.4 Hz, 1H, CH2O-alkyl),
3.52 (dd, J = 10.7, 5.3 Hz, 1H, CH2O-alkyl), 3.39–3.48 (2H, m, CH2O-glycerol), 2.07 (3H, s, CH3-acetate),
2.03 (3H, s, CH3-acetate), 1.55–1.47 (4H, m, CH2-alkyl), 1.30–1.24 (36H, m, CH2), 0.89 (t, J = 6.9 Hz, 3H,
CH3). 13C NMR-CDCl3,
δ (ppm) 171.1, 170.7, 71.8, 70.6, 70.0, 63.7, 31.7, 29.7, 29.2, 29.0, 26.6, 23.2, 21.3,
20.7, 14.
3.5. Analytical Chemistry
Analytical Biochemistry
The total lipids were extracted from 2 mg chimera and shark liver oil by Folch’s method [48].
Alkylglycerols were obtained by separation of lipid extracts by silica gel thin layer chromatography
with chloroform/acetone (90:10, v/v) as the developing solvent. Separated lipids were visualized under
ultraviolet light after spraying with 0.2% solution of 2,7-dichlorofluorescein in ethanol. Alkylglycerols
were scraped into glass tubes with Teflon screw caps and acetylated with a large excess (600 µL) of
acetyl chloride for 5 min at 140 ◦C. Then, the solution was neutralized with NaHCO3 and extracted
with diethyl ether. After evaporation, the sample was taken up with hexane.
For the chromatography procedure, the samples were analyzed in batches of 13 samples
comprising 10 oil samples, 3 samples comprising two blanks and one mixture of synthesized alkyl
lipids serving as a reference. The alkylglycerols were identified by comparison of their retention
times and their fragmentation peaks in mass spectroscopy with those of the synthesized standards
(see Table 1).
Table 1. Mass spectroscopy data.
Retention
Time (min)
Molecular Weight
Alkyl Chain R =
Most Characteristic Peaks (m/z, (%))
−1
(g. × mol
372.54
398.58
400.59
414.62
426.63
428.65
442.67
456.70
484.75
)
C14H29
(C14:0)
C16H31
(C16:1)
C16H33
(C16:0)
C17H35
(C17:0)
C18H35
(C18:1)
C18H37
(C18:0)
C19H39
(C19:0)
C20H41
(C20:0)
C22H45
(C22:0)
43 (base peak), 57 (60), 71 (35), 83 (30), 97 (26), 111 (13), 117
(19), 159 (119), 223 (4), 269 (6), 313 (35)
43 (base peak), 57 (63), 69 (44), 81 (75), 95(39), 117 (31), 159 (63),
222 (15), 281 (12), 339 (25)
43 (base peak), 57 (62), 71 (38), 83 (28), 97 (27), 111 (14), 159 (7),
219 (<5), 251 (5), 255 (<5), 297 (6), 341 (67)
43 (base peak), 57 (62), 83 (28), 97 (27), 111 (15), 117 (22) 159
(25), 209 (<5), 267 (8), 311 (12), 355 (54)
43 (base peak), 55 (52), 67 (48), 81(72), 95 (44), 117 (18), 159 (75),
191 (7), 219 (<5), 281 (12), 341 (7), 367 (25)
43 (base peak), 57 (70), 71(45), 85 (34), 97 (33), 111 (18), 117 (27),
159 (27), 191 (<5), 281 (10), 325 (9), 369 (37)
43 (base peak), 57 (65), 83 (38), 71 (38), 97 (36), 111 (20), 117(32),
159 (30), 233 (<5), 281 (7), 339 (6), 383 (47)
43 (base peak), 57 (70), 71 (42), 83 (36), 97 (36), 111 (18), 117
(32),159 (27), 125 (10), 207 (23), 295 (12), 355 (32), 398(40)
43 (base peak), 57 (70), 71 (38), 83 (28), 97 (38), 111 (22), 117
(12), 125 (12) 159 (30), 207 (22), 281 (8), 355 (10), 381 (6), 423 (27)
13.6
15.0
15.7
16.8
17.5
17.8
18.7
19.7
21.4

Mar. Drugs 2018, 16, 101
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3.6. Validation of the Analytical Procedure
The Table 2 represents the Statistical data for analytical procedure for GC and show the fiability
of the method used.
Table 2. Statistical data for analytical procedure for GC.
Parameters Studied
Results
Selectivity
Fidelity
Correct
Repeatability
Reproducibility
Correct (CVr = 3.73%)
Correct (CVR = 3.75%)
Correct (no deviation from linearity, R2 = 0.992)
2.9 pg (on-column injector, FID)
17.5 pg (on-column injector, FID)
Linearity
Detection limit LOD
Quantification limit LOQ
Stability
◦
1 year at −80 C
4. Conclusions
This work demonstrates that a series of 1-O-alkyl-glycerols featuring mainly saturated lipid chain
lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two mono-unsaturated lipid chains (16:1, 18:1) were easily
prepared and derivatized as diacetate compounds that were used as standards for GC analysis. These
standards were used to determine the molecular species of AEL present in shark oils. It would be
interesting to use these standards to also determine the AEL composition in tumors. Indeed, it is
known that the ether-lipids fraction is larger in tumors than non-tumoral tissues, but the molecular
species have not been precisely identified. Moreover, in a second time it would be interesting to
synthesize standards that would identify alkenyl-lipids. Indeed, alkenyl-lipids were found to be
highly present in the membrane phospholipids of some tissues such as the heart and brain [50]. The
determination of the composition of the alkenyl-lipids in tumors will help to understand the role of
these lipids in the biology of tumors cells.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/4/101/s1.
1
1
Figure SI1-1. H NMR (CDCl3) spectrum of compound
2
, Figure SI1-2. H NMR (CDCl3) spectrum of compound
1
3f, Figure SI1-3. H NMR (CDCl3) spectrum of compound 4f, Figure SI1-4. 2D NMR HSQC (CDCl3) spectrum of
1
compound 4f, Figure SI1-5. H NMR (CDCl3) spectrum of compound 5f, Figure SI1-6. 2D NMR HSQC (CDCl3)
spectrum of compound 5f, Figure SI1-7. 1H NMR (CDCl3) spectrum of compound 5h, Figure SI1-8. 2D NMR
HSQC (CDCl3) spectrum of compound 5h, Figure SI2-1. GC-MS chromatogram of a mixture of synthesized
alkyl-glycerolipids and Figure SI2-2. Mass spectroscopy data.
Acknowledgments: This work was funded by University of Tours, the “Région Centre-Val de Loire” (LIPIDS
project of ARD2020 Biomédicaments), “INSERM”, Canceropôle Grand Ouest and the Ion Channel and Cancer
Network, the “Ligue Nationale Contre le Cancer”, the Association “CANCEN”, and Tours’ Hospital oncology
association ACORT. We thank Bodimant of Phosphotec for providing chimera and shark liver oils. We also
thank Yves Frangin, Ludovic Sochard, Lydie Nadal-Desbarats, Aurore Lecaille and Violetta Guérin for technical
assistance, and Catherine Leroy for secretarial support.
Author Contributions: M.P., C.G., H.C., J.T., P.B., and P-A.J. designed the chemical synthesis. M.P. and C.G.
performed the chemical synthesis and the analytical analyses. D.F., A.C., and S.C. provided intellectual input. M.P.,
C.G., P-A.J., and C.V. wrote the manuscript. All the authors critically reviewed the final version of the manuscript.
Conflicts of Interest: The authors declare no competing financial interest.
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