Synthesis of specific deuterated derivatives of the long chained stratum corneum lipids [EOS] and [EOP] and characterization using neutron scattering

Article abstract of DOI:10.1002/jlcr.3504

The synthesis of specific deuterated derivatives of the long chained ceramides [EOS] and [EOP] is described. The structural differences with respect to the natural compounds are founded in the substitution of the 2 double bonds containing linoleic acid by a palmitic acid branched with a methyl group in 10-position. The specific deuteration is introduced both in the branched and in the terminal methyl group, which was realized by common methods of successive deuteration of carboxylic groups in 3 steps. These modified fatty acids resp. the corresponding ceramides [EOS] and [EOP] were prepared for neutron scattering investigations. First results of these investigations were presented in this manuscript showing that the deuterated compounds could be detected in the stratum corneum lipid model membranes. The deuterated ceramides [EOS] and [EOP] are valuable tools to investigate the influence of these long chained ceramide species on the nanostructure of stratum corneum lipid model membranes.

Full text of DOI:10.1002/jlcr.3504

Received: 28 February 2017
DOI: 10.1002/jlcr.3504
Accepted: 27 March 2017
R E S E A R C H A R T I C L E
Synthesis of specific deuterated derivatives of the long chained
stratum corneum lipids [EOS] and [EOP] and characterization
using neutron scattering
Stefan Sonnenberger1 | Adina Eichner1 | Thomas Schmitt1,3 | Thomas Hauß4 | Stefan Lange1,2
Andreas Langner1 | Reinhard H.H. Neubert3 | Bodo Dobner1
|
1 Institute of Pharmacy, Martin Luther
University Halle‐Wittenberg (MLU), Halle
The synthesis of specific deuterated derivatives of the long chained ceramides
[EOS] and [EOP] is described. The structural differences with respect to the natural
compounds are founded in the substitution of the 2 double bonds containing linoleic
acid by a palmitic acid branched with a methyl group in 10‐position. The specific
deuteration is introduced both in the branched and in the terminal methyl group,
which was realized by common methods of successive deuteration of carboxylic
groups in 3 steps. These modified fatty acids resp. the corresponding ceramides
[EOS] and [EOP] were prepared for neutron scattering investigations. First results
of these investigations were presented in this manuscript showing that the deuterated
compounds could be detected in the stratum corneum lipid model membranes. The
deuterated ceramides [EOS] and [EOP] are valuable tools to investigate the influ-
ence of these long chained ceramide species on the nanostructure of stratum
corneum lipid model membranes.
(Saale), Germany
2 Institute for Medical Physics and
Biophysics, University Leipzig, Leipzig,
Germany
3 Institute of Applied Dermatopharmacy,
Martin Luther University Halle‐Wittenberg,
Halle (Saale), Germany
4 Institute of Soft Matter and Functional
Materials, Helmholtz‐Zentrum Berlin für
Materialien und Energie, Berlin, Germany
Correspondence
Bodo Dobner, Institute of Pharmacy, Martin
Luther University Halle‐Wittenberg (MLU),
Wolfgang‐Langenbeck‐Str. 4, 06120 Halle
(Saale), Germany.
Email: bodo.dobner@pharmazie.uni‐halle.de
KEYWORDS
branched fatty acid, CER[EOP], CER[EOS], neutron scattering investigations, specific deuteration
Funding information
Deutsche Forschungsgemeinschaft, Grant/
Award Number: DO463/6‐1
1 | INTRODUCTION
the amino group under formation of amides. Regarding the fatty
acids bonded to the amino bases, the most common length is 16,
The skin as largest organ of the human body consists of 3 main
layers. The epidermis is the outermost of them, which is further
subdivided into 4 layers. The outside part of these 4 is the stra-
tum corneum (SC), which has to fulfill many functions. The SC
is formed by cornified cells embedded in a lipid matrix1 and
represents a protective barrier against water loss and the
invasion of toxic substances.2,3 This is based on the lipids of
the SC, which consist mainly of ceramides, free fatty acids,
and cholesterol,4,5 where the ceramides represent the
main and also the most important compounds.6 Ceramides
are not unique in their structure.7 The 4 amino bases
sphingosine, phytosphingosine, dehydrosphingosine, and 6‐
hydroxysphingosine are acylated with different fatty acids at
18, 22 and 24 carbon atoms. Beside these simple fatty acids also
(R)‐configurated α hydroxy fatty acids and a more complex, 48
carbon atoms containing a composed fatty acid was found. This
last acid contains a ω‐hydroxy acid with 30 carbon atoms
whereas the terminal hydroxy group is esterified with linoleic
acid. The long‐chain acid with the linoleoyl residue is
bound to sphingosine (CER[EOS]), to phytosphingosine
(CER[EOP]), and 6‐hydroxysphingosine (CER[EOH]).
CER[EOS] appears to play a key role in one model of the nano-
structure of the SC lipids because it is stated to form the long
periodicity phase of the SC lipids.8,9 This model is still an issue
of scientific debate.10-12 Therefore, additional studies are neces-
sary concerning the impact of CER[EOS].
J Label Compd Radiopharm. 2017;1–15.
wileyonlinelibrary.com/journal/jlcr
Copyright © 2017 John Wiley & Sons, Ltd.
1

2
SONNENBERGER ET AL.
The research of the nanostructure of the SC lipids was
of SC lipid model membranes with the new synthesized
compounds show that the deuterated long‐chain ceramides
are valuable tools to investigate the influence of these
ceramides on the nanostructure of SC lipid model membranes.
focused on model systems containing the main components
of skin in certain ratios. The investigative methods of choice
in this direction are neutron diffraction measurements and
the determination of neutron scattering density profile.12-16
However, this method has only a benefit when deuterated or
better partially deuterated ceramides are used. Main effort
was par for the course in the case of partial deuteration of the
linoleic acid in the molecules of CER[EOS] or CER[EOP],
however, this is associated with a large effort. In former inves-
tigation, we developed an alternative to the linoleic acid part of
the complex fatty acid11 by using a palmitic acid residue with
methyl branching in 10‐position of the chain. This is due to
very early papers in which we could show as the first that the
phase transition temperatures and enthalpies were strikingly
reduced through the introduction of a methyl branching in
the middle position of palmitic acid chains in phosphatidyl-
cholines.17,18 This effect was also found in phosphatidylcho-
lines with branched stearic acid residues.19 We have shown
the same tremendous effect of fluidization of lipids by methyl
branches at definite positions in the synthesis and investiga-
tion of lipid model compounds of archaea bacteria.20,21 In
the above mentioned paper, the CER[EOS] derivative with a
methyl group in 10‐position of the palmitoyl residue has
nearly identical physicochemical properties regarding the
natural CER[EOS]. On the other hand, the branched fatty acid
is stable against higher temperature and oxidative processes
especially during physicochemical investigations. Another
advantage of such modification of the fatty acid residue is
the simpler synthesis of the complex fatty acid with 46 carbon
atoms because of the complete abandonment of blocking
groups with exception of the THP group (see Section 2).
Therefore, the objective of this study was to synthesize 10‐
methylhexadecanoic acids with deuteration in the branched
methyl group and/or at the terminal methyl group of the fatty
acid. These different specific deuterated fatty acids were
sensitive with respect to neutron scattering experiments in
the corresponding ceramides and were already published.22
Here, we will present our results in the synthesis of CER[EOS]
and CER[EOP] with the specific deuterated branched fatty
acids (Figure 1). The results of neutron scattering investigation
2 | RESULTS AND DISCUSSION
2.1 | Synthesis of the branched deuterated
fatty acids
For the preparation of the branched and specifically deuter-
ated fatty acids as an alternative for the deuterated linoleic
acid, we used 2 different synthetic methods. These methods
are shown in Scheme 1. First, the synthetic way via a malonic
acid ester dialkylation reaction is classic. However, the yields
are very good especially in fatty acid chemistry. A shorter but
more labour‐intensive reaction pathway with respect to
synthetic equipment and also for the preparation of the
terminal deuterated compounds 12 is the α‐alkylation of an
acid dianion.23,24 The synthetic route to the branched and
deuterated compounds 12 via malonic acid ester alkylation
starts with the commercially available 6‐bromohexanoic acid
ethyl ester (1). This compound was transformed in 3 steps via
reduction of the ester group with lithium aluminum deuteride
at 0°C to the corresponding deuterated bromo alcohol,
mesylation, and repeated reduction of the corresponding
mesylate with lithium aluminum deuteride to yield
compound 2b.25,26 This compound was also commercially
available, however, since we needed bigger amounts with
respect to the following multistep procedure, we prepared it
in this way. Malonic acid ethyl ester was deprotonated with
sodium in ethanol in the case of hexylbromide 2a as
alkylating reagent but with sodium hydride in toluene in the
case of 6,6,6‐d3‐hexyl bromide 2b to avoid the deuterium
exchange especially in the during the alkylation step. The 2
mono alkyl malonic acid esters 4a,b were purified using
column chromatography. In a second alkylation, step 4a,b
were deprotonated using potassium hydride in dry toluene.
Then the 2‐(9‐bromonon‐1‐yloxy)tetrahydro‐2H‐pyrane (6)
reacted with the carbanion to realize the necessary chain
extension to 16 carbon atoms yielding compounds 7a,b.
Compounds 6 was prepared from nonane‐1,9‐diol (5) by
monobromination reaction according to Fockink et al.27 The
blocking of the free hydroxyl group that is necessary for the
following step was done with 3,4‐dihydro‐2H‐pyrane under
the common conditions.28 One of the 2 carboxylic ester
groups is the latent methyl group with or without deuteration.
Therefore, 7a,b were monosaponified using a modified
procedure of Breslow29 including decarboxylation of the
resulting free carboxyl group to the ester compounds 8a,b.
These substances were then transformed either to the normal
branched methyl group or to the CD3 group dependent on the
used reagents. Following the above described reaction,
FIGURE 1 Structure of synthesized ceramides

SONNENBERGER ET AL.
3
SCHEME 1 i: a) LiAlD4, Et2O, 0°C, CH3SO2Cl, TEA, CHCl3, 0°C, c) LiAlD4, Et2O, 0°C; ii: NaH, toluene, reflux; iii: a) 48% HBr, b) DHP,
CH2Cl2, PPTS; iv: KH, toluene, reflux; v: a) KOH, ethanol, 65°C, b) TEA, cumene, pyridine, reflux; vi: LiAlD4/LiAlH4, Et2O, reflux, CH3SO2Cl,
TEA, CHCl3, 0°C, c) LiAlD4/LiAlH4, Et2O, reflux; vii: NaH, DIPA, n‐BuLi, THF; viii: LiAlD4/LiAlH4, Et2O, reflux, CH3SO2Cl, TEA, CHCl3, 0°C,
c) LiAlD4/LiAlH4, Et2O, reflux; ix: a) MeOH, PPTS, reflux, b) CrO3, H2SO4, acetone, CHCl3, 0°C
sequence 8a,b were reduced with lithium aluminum hydride
2.2 | Synthesis of the branched and deuterated
in the case of the “normal” methyl group or with lithium
aluminum deuteride in the case of the deuterated methyl
branching. The resulting alcohols were transformed into the
methanesulfonic acid esters, which were reduced once more
with the corresponding hydride reduction reagents to com-
pounds 11a‐d.
CER[EOS] and CER[EOP]
In
Scheme
2,
the
syntheses
of
the
10‐
methylhexadecanoyloxytriacontanoic acids (15a‐c) and the
final ceramides I and II are shown. First, the preparation of
the ω‐acyl fatty acids 15a‐c starts with the acylation of
triacontan‐1,30‐diol (14), which was prepared by Grignard
coupling of suitable bromides.30 Therefore, the selective
deuterated 10‐methylpalmitic acids 12a‐c were transformed
into the corresponding acid chlorides using thionyl chloride.
According to reaction pathway B, starting material was
octanoic acid (9). First, 9 was transformed into the dianion
by double deprotonation in a modified way according to
Creger.24 This was realized by preparing the sodium salt of
the acid with sodium hydride in tetrahydrofuran/
diisopropylamine. After the hydrogen evolution was com-
plete at 60°C, the mixture was treated with the appropriate
amount of BuLi and the double deprotonation proceeds at
room temperature. This process is sensible with respect to
reaction time and the reaction temperature. The advantage
of dianions towards enolates in alkylation reactions is due
to the higher reaction temperatures. The dianion was then
reacted with the bromide 6. From resulting acid 10, com-
pounds 11a,b were prepared in the same way described
already in pathway A with the difference that the acid was
reduced without transformation into the corresponding ester.
However, synthetic success of the alkylation reaction is
connected with the full formation of the dianion as mentioned
already above. The final branched fatty acids 12a‐d were
received by elimination of the tetrahydropyranyl residue with
methanol and PPTS and oxidation of the resulting branched
fatty alcohol with Jones reagent.
SCHEME 2 i: a) Mg, THF; b) MeOH, PPTS, reflux; ii: a) SOCl2; b)
CHCl3, reflux; iii: CrO3, H2SO4, acetone, CHCl3, 0°C; iii: KOH, D2O,
Pd/C (10%), 195°C, 15 bar, 100 h; iv: PyBOP, DIPEA, CH2Cl2,
sphingosine, RT; v: PyBOP, DIPEA, CH2Cl2, phytosphingosine, RT

4
SONNENBERGER ET AL.
Regarding to the normal acylation procedure using acid chlo-
rides in our case, we dispense with a base and dropped the
acid chloride very slowly to the diol in boiling chloroform.
The procedure enables us to avoid to a great extent the double
acylation of the diol. While working below 5°C and using a
helper base, we isolated only the diester.11 This is due to
the bad solubility of the diol at that temperature. The acylated
alcohols 15a‐c were then transformed to the corresponding
acids 16a‐c by a modified Jones oxidation in good yields.
This is the advantage of the use of a branched fatty acid
instead of linoleic acid in the natural ceramides because of
the instability of the double bonds against the harsh oxidation
conditions. This means also a reduction of synthetic steps and
a reaction pathway without any blocking groups already men-
tioned above. The fatty acids 16a‐c were finally converted
into the corresponding ceramides. For the N‐acylation of
sphingobases like sphingosine or phytosphingosine PyBOP
was the reagent of choice.31 Other authors used various forms
of activated esters.32,33 We have studied this procedure with
all these activators. However, we find that the use of PyBOP
is the best reagent in this direction. To our knowledge, we are
the first to use this substance. The yield of the ceramides is
very high (>77%), and the workup is very simple especially
in the case of the ceramides containing phytosphingosine.
system is low enough to contribute sometimes very minor
differences to a specific component. Specifically, deuterated
CER as synthesized here furthermore allow determination
of the position of this label within the lamellar arrangement,
due to the difference in the neutron scattering cross‐section
of H and D. This allows to determine the position and chain
arrangement of the CER within the system. At the moment,
this is the most efficient way to get an insight into the details
of the nanostructure of a specific model system.
For the CER[NS]/CER[EOS]‐br–based system investi-
gated in this study, only a single phase was observed for all
4 investigated mixtures. This phase had a spacing of
3.94 0.1 nm, thus, no long periodicity phase (LPP) with
a spacing of 12 to 13 nm was observed.34 The observed spac-
ing however is also shorter than what would be expected for
an short periodicity phase (SPP) with 2 opposing CER[NS]
C18/18, which would be estimated as 4.42 nm.35 The differ-
ence of 0.48 nm cannot be explained by interdigitating
chains, temperature, or other minor influences. This differ-
ence is thus to be assumed, to be the result of a slightly tilted
CER chain arrangement. A chain tilt that would result in a
corresponding distance difference would have a tilt angle α
of about 26° (solely considering geometrical factors). The
position of the specific deuteration could be determined for
all 3 deuterated CER. A distinct difference between the
CER[EOS]‐br‐d3 with its terminal end deuterated and the
CER[EOS]‐d3‐br with its branching side chain deuterated
could be shown. For the terminally deuterated CER[EOS]‐
br‐d3, the difference is smaller than for the other CER but is
observed over the whole range of measurements for the con-
trast variation and thus can be confirmed. The positions of
the different deuterations also strongly support the assump-
tion of a tilted chain arrangement. Furthermore, to fit the
SPP arrangement, the CER[EOS] not including its ω‐acyl
group was determined to most likely span 2 whole lamellar
leaflets. Its ω‐acyl head group would then be located in the
2.3 | Neutron scattering measurements
Neutron scattering can be used as a very efficient tool to
determine the nanostructure of multilamellar CER‐based
mixtures. These models are used to model the properties of
the SC, the uppermost layer of the skin. To be able to access
specific properties of single CER species only distinguished
by a slightly different head group or chain length, pure
synthetic CER as synthesized here are necessary. Only using
very basic mixtures with these pure single CER or very basic
mixtures of only a few CER, the complexity of the model
FIGURE 2 Neutron scattering length density profiles for the CER([NS]/[EOS])/SA/CHOL system with a molar ratio of (0.8:0.2)/1/0.7 in
comparison to mixtures with deuterated CER. A, CER[EOS]‐br‐d3. B, CER[EOS]‐d3‐br. C, CER[NS]‐d3. The very long CER[EOS]‐d3‐br
spanning 2 lamellae is shown in 2 parts to fit a single profile

SONNENBERGER ET AL.
5
head group region of the next neighboring lamellar leaflet
and its ω‐hydroxy chain arranged parallel to the CER[NS]
in a third leaflet. A possible arrangement is shown in
Figure 2 for both the short CER[NS] and the very long
CER[EOS]‐br, which is split into 2 parts to fit a single
profile.
dried over Na2SO4, concentrated to dryness under reduced
pressure, and dried over P2O5 to give the deuterated alcohol
as colorless liquid.
To a solution of the liquid residue of the alcohol and TEA
(18.62 mL, 134 mmol) in CHCl3 (250 mL), a solution of
mesyl chloride (10.40 mL, 134 mmol) in CHCl3 (100 mL)
was added under ice cooling. Afterwards, the reaction was
stirred for further 12 hours at room temperature. For working
up, additional TEA (4.65 mL, 33 mmol) was given to the
mixture and ice (100 g) was added. The CHCl3 phase was
separated, and the water layer was extracted 3 times with
CHCl3 (100 mL). The combined organic layers were evapo-
rated to dryness under reduced pressure. The residue was taken
up in a mixture of heptane (400 mL) and ether (200 mL). The
organic layer was washed with water (150 mL). The water layer
was extracted with ether (100 mL). The combined organic
layers were evaporated to dryness under reduced pressure
and were dried over P2O5 to give the mesylate.
A solution of the mesylate in abs. ether (80 mL) was
added dropwise under argon atmosphere at a temperature of
−10°C to a stirred suspension of LiAlD4 (2.5 g, 60 mmol)
in abs. ether (100 mL). The reaction was stirred for further
5 hours at a temperature of −10°C. H2O (25 mL) was added
dropwise under cooling, and the mixture was stirred for fur-
ther 60 minutes. To remove the formed precipitate, the mix-
ture was filtered by suction and the frit was washed 3 times
with ether (20 mL). The combined ether phases were washed
with brine, dried over Na2SO4, and concentrated to dryness
under reduced pressure. For purification, the obtained liquid
was distilled under reduced pressure to give the 1‐bromo‐
6,6,6‐d3‐hexane (2b) as colorless oil (15.58 g, 77%). The
deuterated alkyl bromide was used without further
characterization.
3 | EXPERIMENTAL
3.1 | General procedures and materials
Lithium aluminum deuteride was purchased from ARMAR
(Europe), GmbH (Leipzig, Germany), and PyBOP from
Carbolution Chemicals GmbH (Saarbrücken, Germany).
The aminoalcohols sphingosine and phytosphingosine were
obtained from Evonik Goldschmidt GmbH (Essen,
Germany). From Sigma‐Aldrich (St Louis, Missouri) were
received all other chemicals. Before use, all solvents were
dried. Silica gel 60 (0.063‐0.200 mm) were used from Merck
(Darmstadt, Germany) for column chromatography. Thin‐
layer chromatography plates were obtained from Macherey‐
Nagel (Düren, Germany). For detection of the thin‐layer
chromatography plates, bromothymol blue solution was used.
Mass spectrometry data were recorded at a Finnigan MAT
710C (Thermoseparation Products, San Jose, CA) for ESI‐
MS and a LTQ‐Orbitrap XL hybrid mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany) for HR‐MS
(high‐resolution mass spectrometry). Nuclear magnetic reso-
nance (NMR) data were received on Varian Gemini 2000 and
Varian Inova 500 instruments. CDCl3, CD3OD, THF‐d8, and
mixtures were used. An HP 1100 Agilent (Agilent Technolo-
gies, Waldbronn, Germany) with ELSD 2000 Alltech
(Grace Davison, Columbia, Maryland) detector and a
Nucleodur 100‐5 125 × 2 column from Macherey‐Nagel
(Düren, Germany) were used for high‐performance liquid
chromatography (HPLC). Melting points were determined
on a Boetius apparatus and were not corrected.
3.2.2 | Monoalkylation of diethyl malonate
Diethyl hexylmalonate (4a)
To a solution of sodium ethanolate (2.30 g, 100 mmol
sodium in 50 mL abs. ethanol) was added under moderate
boiling diethyl malonate (3) (16.02 g, 100 mmol) and then
hexyl bromide (2a) (17.33 g, 105 mmol). The mixture was
heated under reflux for 12 hours; afterwards, ethanol (pre-
pared from 2,3 g sodium (100mmol) in 50 ml abs. ethanol)
was removed under reduced pressure. For working up, ice
water was added to the mixture and the organic layer was sep-
arated. The water layer was extracted 3 times with ether
(50 mL). The combined organic layers were dried over
Na2SO4 and filtered and evaporated to dryness under reduced
pressure. For purification, the crude residue was distilled
under reduced pressure to give 4a as colorless liquid
(18.75 g, 77%). Diethyl hexylmalonate (4a) was used without
further characterization.
3.2 | Synthesis of the ceramides
3.2.1 | Synthesis of 1‐bromo‐6,6,6‐d3‐
hexane (2b)
To a stirred suspension of LiAlD4 (2.77 g, 66 mmol) in
100 mL abs. ether (100 mL), a solution of methyl 6‐
bromohexanoate (1) (25.1 g, 120 mmol) in abs. ether
(150 mL) was dropwise added under argon atmosphere at a
temperature of −10°C. The mixture was stirred for further
4 hours at −10°C. For working up, D2O (10 mL) was added
dropwise under ice cooling and the reaction was stirred for
2 hours. To remove the formed precipitate, the mixture was
filtered by suction and the frit was washed 3 times with ether
(20 mL). The combined ether phases were washed with brine,

6
SONNENBERGER ET AL.
Diethyl 6,6,6‐d3‐hexylmalonate (4b)
evaporated to dryness under reduced pressure. The residue
was purified by column chromatography using gradient
technique with heptane/Et2O and TEA (0.5%) eluent to give
diethyl dialkylmalonate 7a,b.
To a stirred suspension of NaH (60% in paraffin, 5.45 g,
136 mmol) in abs. toluene (280 mL), a solution of 3
(21.83 g, 136 mmol) in abs. toluene (120 mL) was added
under argon atmosphere. The mixture was warmed to 70°C
for 2 hours, followed by dropwise adding of a solution of
2b (15.0 g, 0.09 mol) in abs. toluene (60 mL). The reaction
was heated under reflux for 24 hours. For working up, water
(50 mL) was added. The organic layer was separated, and the
water layer was extracted 3 times with ether (50 mL). The
combined organic layers were dried over Na2SO4 and filtered
and evaporated to dryness under reduced pressure. The resi-
due was purified by column chromatography using gradient
technique with heptane/Et2O eluent to give diethyl 6,6,6‐d3‐
hexylmalonate (4b) as colorless liquid (12.36 g, 56%): Rf
Diethyl
hexyl[9‐(tetrahydro‐2H‐pyran‐2‐yloxy)nonyl]
malonate (7a)
Colorless liquid (16.37 g, 85%): Rf 0.34 (CHCl3/heptane
6:4); 1H NMR (400 MHz, CDCl3)
δ
0.86 (t,
J = 6.6 Hz, 3H, H3C(CH2)5‐), 1.09‐1.38 (m, 26H,
H3C(CH2)4CH2C(COOCH2CH3)2CH2(CH2)6‐), 1.49‐1.62
(m, 6H, ‐CH2CH2OCH‐CH2(CH2)2‐), 1.67‐1.74 (m, 1H,
‐OCH‐CHH′‐), 1.78‐1.86 (m, 5H, ‐CH2CCH2‐, ‐OCH‐
CHH′‐), 3.37 (dt, J = 6.7, 9.5 Hz, 1H, ‐(CH2)8CHH′O‐),
3.46‐3.51 (m, 1H, ‐OCH‐(CH2)3CHH′‐), 3.71 (dt, J = 6.9,
9.4 Hz, 1H, ‐(CH2)8CHH′O‐), 3.82‐3.89 (m, 1H, ‐OCH‐
(CH2)3CHH′‐), 4.16 (q, J = 7.1 Hz, 4H, 2× ‐OCH2CH3),
4.55‐4.57 (m, 1H, ‐OCH‐O‐); 13C NMR (100 MHz, CDCl3)
δ 14.2, 14.3, 19.8, 22.7, 24.0, 24.1, 25.7, 26.4, 29.4, 29.6,
29.7, 29.9, 30.0, 30.9, 31.7, 32.3, 57.7, 61.0, 62.5, 67.8,
99.0, 172.2; ESI‐MS m/z: 493.3 (M + Na)+.
1
0.40 (CHCl3/heptane 6:4); H NMR (400 MHz, CDCl3) δ
1.26 (t, J = 7.1 Hz, 6H, 2× ‐OCH2CH3), 1.29‐1.32 (m, 8H,
D3C(CH2)4‐), 1.85‐1.91 (m, 2H, D3C(CH2)4CH2‐), 3.30 (t,
J = 7.5 Hz, 1H, ‐CH(COO‐)2), 4.19 (q, J = 7.1 Hz, 4H, 2×
‐OCH2CH3); 13C NMR (100 MHz, CDCl3) δ = 14.2, 22.4,
27.4, 28.9, 29.0, 31.6, 52.3, 61.4, 169.8; ESI‐MS m/z:
248.1 (M + H)+, 270.0 (M + Na)+, 516.8 (2M + Na)+.
Diethyl
6,6,6‐d3‐hexyl[9‐(tetrahydro‐2H‐pyran‐2‐yloxy)
Synthesis of 2‐(9‐bromononyloxy)tetrahydro‐2H‐pyran (6)
The preparation was following the procedure described in the
literature27 starting from nonan‐1,9‐diol (5) to give 6 as
colorless liquid: Rf 0.47 (CHCl3/heptane 6:4); 1H NMR
(400 MHz, CDCl3) δ 1.30‐1.43 (m, 10H, Br(CH2)2(CH2)5‐),
1.50‐1.61 (m, 6H, ‐CH2CH2OCH‐CH2(CH2)2‐), 1.68‐1.74
(m, 1H, ‐OCH‐CHH′‐), 1.78‐1.88 (m, 3H, BrCH2CH2‐,
‐OCH‐CHH′‐), 3.35‐3.41 (m, 3H, BrCH2(CH2)7CHH′‐),
3.47‐3.52 (m, 1H, ‐OCH‐(CH2)3CHH′‐), 3.72 (dt, J = 6.9,
9.5 Hz, 1H, Br(CH2)8CHH′‐), 3.84‐3.89 (m, 1H, ‐OCH‐
(CH2)3CHH′‐), 4.56‐4.57 (m, 1H, ‐OCH‐O‐); 13C NMR
(100 MHz, CDCl3) δ = 19.9, 25.7, 26.3, 28.3, 29.5, 29.9,
30.9, 33.0, 34.1, 62.5, 67.8, 99.0.
nonyl]malonate (7b)
Colorless liquid (15.90 g, 83%): Rf 0.40 (CHCl3/heptane
6:4); 1H NMR (400 MHz, CDCl3)
δ
1.09‐1.16
(m, 4H, 2× (‐OOC)2C(CH2CH2‐)2), 1.21 (m, 22H,
D3C(CH2)3(CH2)2C(COOCH2CH3)2(CH2)2(CH2)5‐), 1.48‐
1.62 (m, 6H, ‐CH2CH2OCH‐CH2(CH2)2‐), 1.68‐1.74 (m,
1H, ‐OCH‐CHH′‐), 1.79‐1.87 (m, 5H, ‐CH2CCH2‐, ‐OCH‐
CHH′‐), 3.37 (dt, J = 6.7, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐),
3.46‐3.52 (m, 1H, ‐OCH‐(CH2)3CHH′‐), 3.71 (dt, J = 6.9,
9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.83‐3.89 (m, 1H, ‐OCH‐
(CH2)3CHH′‐), 4.16 (q, J = 7.1 Hz, 4H, 2× ‐OCH2CH3),
4.55‐4.57 (m, 1H, ‐OCH‐O‐); 13C NMR (100 MHz, CDCl3)
δ 14.3, 19.8, 22.4, 24.0, 24.1, 25.7, 26.4, 29.4, 29.6, 29.7,
29.9, 30.0, 30.9, 31.6, 32.3, 57.7, 61.0, 62.5, 67.8, 99.0,
172.2; ESI‐MS m/z: 496.3 (M + Na)+.
3.2.3 | Second alkylation to diethyl
dialkylmalonate
Monosaponification and decarboxylation of diethyl
dialkylmalonate
A solution of 4a,b (10.0 g, 0.04 mol) in abs. toluene (40 mL)
was added dropwise under argon atmosphere to a stirred sus-
pension of KH (30% in paraffin, 6.03 g, 45 mmol) in abs. tol-
uene (50 mL). The mixture was stirred for further 12 hours at
room temperature. Afterwards, the reaction was warmed to
50°C before dropwise adding a solution of 6 (15.75 g,
51.25 mmol) in abs. toluene (60 mL). The mixture was
heated under reflux for 24 hours. For working up, ice water
(50 mL) was added, and the mixture was stirred for further
2 hours. Sat. NH4Cl (50 mL) was added, and the organic
layer was separated. The water phase was extracted 2 times
with ether (50 mL). The combined organic phases were
washed with brine, dried over Na2SO4, and filtered and
A mixture of 7a,b (15.0 g, 32 mmol) and KOH (2.53 g,
38.4 mmol) in 96% ethanol (80 mL) was stirred at a temper-
ature of 65°C for 24 hours. For working up, sat. NH4Cl were
added (80 mL) and the mixture was extracted 3 times with
ether (80 mL). The combined ether phases were washed with
brine, dried over Na2SO4, and filtered and evaporated to dry-
ness under reduced pressure. The crude residue was purified
by column chromatography using CHCl3 and TEA (1%) elu-
ent to remove the byproducts followed by CHCl3/MeOH
(9:1) to wash the acid from the column. To the crude acid res-
idue was given TEA (4.44 mL, 32 mmol), cumene (160 mL),
and 1 drop pyridine. The mixture was heated for 2 hours

SONNENBERGER ET AL.
7
under reflux. Afterwards, the solvent was evaporated to dry-
ness under reduced pressure. The crude residue was purified
by column chromatography using gradient technique with
heptane/Et2O and TEA (0.5%) eluent to give ethyl ester 8a,b.
After 30 minutes, a solution of 6 (4.61 g, 15 mmol) in abs.
THF (8 mL) was added and the mixture was stirred for fur-
ther 12 hours at 28°C. For working up, the reaction was
cooled to 0°C and cold sat. NH4Cl (40 mL) was added before
the mixture was extracted 3 times with ether (45 mL). The
combined ether phases were washed with water and brine.
The crude residue was purified by column chromatography
using CHCl3 and TEA (1%) eluent to remove the by‐products
followed by CHCl3/MeOH (9:1) to wash the acid 10 from the
column.
Ethyl
(2RS)‐2‐hexyl‐11‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]
undecanoate (8a)
Colorless liquid (9.40 g, 74%): Rf 0.44 (CHCl3/heptane 6:4);
1H NMR (500 MHz, CDCl3) δ 0.87 (t, J = 6.9 Hz, 3H,
H3C(CH2)5‐), 1.23‐1.45 (m, 25H, H3C(CH2)4CHH′CH‐
CHH′(CH2)6‐, ‐OCH2CH3), 1.50‐1.62 (m, 8H, ‐CHH′CH‐
CHH′(CH2)6CH2CH2OCH‐CH2(CH2)2‐), 1.68‐1.73 (m, 1H,
‐OCH‐CHH′‐), 1.79‐1.86 (m, 1H, ‐OCH‐CHH′‐), 2.26‐2.32
(m, 1H, ‐CH2CH‐CH2‐), 3.37 (dt, J = 6.7, 9.6 Hz, 1H,
‐(CH2)8CHH′O‐), 3.47‐3.51 (m, 1H, ‐OCH‐(CH2)3CHH′‐),
3.72 (dt, J = 6.9, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.84‐3.89
(m, 1H, ‐OCH‐(CH2)3CHH′‐), 4.13 (q, J = 7.1 Hz, 2H,
‐OCH2CH3), 4.56‐4.57 (m, 1H, ‐OCH‐O‐); 13C NMR
(100 MHz, CDCl3) δ 14.2, 14.5, 19.9, 22.7, 25.7, 26.4,
27.5, 27.6, 29.4, 29.6, 29.7, 29.9, 31.0, 31.8, 32.7, 45.9,
60.1, 62.5, 67.8, 99.0, 176.7; ESI‐MS m/z: 421.3 (M + Na)+.
Liquid (2,82 g, 51%): Rf 0.55 (CHCl3/Et2O 8:2); 1H
NMR (500 MHz, CDCl3) δ 0.87 (t, J = 6.9 Hz, 3H,
H3C(CH2)5‐), 1.27‐1.35 (m, 20H, H3C(CH2)4CH2CH‐
CH2(CH2)6‐), 1.40‐1.45 (m, 2H, ‐CHH′CH‐CHH′‐),
1.49‐ 1.62 (m, 8H, ‐CHH′CH‐CHH′(CH2)6CH2CH2OCH‐
CH2(CH2)2‐), 1.68‐1.74 (m, 1H, ‐OCH‐CHH′‐), 1.79‐1.86
(m, 1H, ‐OCH‐CHH′‐), 2.27‐2.32 (m, 1H, ‐CHH′CH‐
CHH′‐), 3.38 (dt, J = 6.7, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐),
3.48‐3.52 (m, 1H, ‐OCH‐(CH2)3CHH′‐), 3.72 (dt, J = 6.9,
9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.85‐3.89 (m, 1H, ‐OCH‐
(CH2)3CHH′‐), 4.57‐4.59 (m, 1H, ‐OCH‐O‐); 13C NMR
(100 MHz, CDCl3) δ 14.2, 19.8, 22.7, 25.6, 26.4, 27.6,
29.4, 29.6, 29.7, 29.9, 30.9, 31.9, 32.6, 44.8, 62.4, 67.8, 98.8,
181.9; ESI‐MS m/z: 369.5 (M − Na)−, 739.3 (2M − Na)−.
Ethyl (2RS)‐2‐(6,6,6‐d3‐hexyl‐11‐[(tetrahydro‐2H‐pyran‐2‐
yl)oxy]undecanoate (8b)
Colorless liquid (10.56 g, 83%): Rf 0.34 (CHCl3/heptane
1
6:4); H NMR (500 MHz, CDCl3) δ 1.23‐1.45 (m, 25H,
3.2.5 | Introduction of the branched methyl
D3C(CH2)4CHH′CH‐CHH′(CH2)6‐, ‐OCH2CH3), 1.48‐
1.61 (m, 8H, ‐CHH′CH‐CHH′(CH2)6CH2CH2OCH‐
CH2(CH2)2‐), 1.68‐1.74 (m, 1H, ‐OCH‐CHH′‐), 1.78‐1.86
(m, 1H, ‐OCH‐CHH′‐), 2.25‐2.32 (m, 1H, ‐CH2CH‐CH2‐),
3.37 (dt, J = 6.7, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.46‐3.52
(m, 1H, ‐OCH‐(CH2)3CHH′‐), 3.71 (dt, J = 6.9, 9.6 Hz,
1H, ‐(CH2)8CHH′O‐), 3.83‐3.89 (m, 1H, ‐OCH‐
(CH2)3CHH′‐), 4.12 (q, J = 7.1 Hz, 2H, ‐OCH2CH3), 4.55‐
4.57 (m, 1H, ‐OCH‐O‐); 13C NMR (100 MHz, CDCl3) δ
14.5, 19.9, 22.5, 25.7, 26.4, 27.5, 27.6, 29.4, 29.6, 29.7,
29.9, 30.9, 31.8, 32.7, 45.9, 60.0, 62.5, 67.8, 99.0, 176.7;
ESI‐MS m/z: 424.3 (M + Na)+.
group
The methyl‐branched THP‐protected alcohols 11a and 11b
were prepared from ethyl (2RS)‐2‐hexyl‐11‐[(tetrahydro‐2H‐
pyran‐2‐yl)oxy]undecanoate (8a) (method A) and from
(2RS)‐2‐hexyl‐11‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]undecanoic
acid (10) (method B) and the methyl‐branched THP‐protected
alcohols 11c and 11d from ethyl (2RS)‐2‐(6,6,6‐d3‐hexyl‐11‐
[(tetrahydro‐2H‐pyran‐2‐yl)oxy]undecanoate
(8b).
The
methyl‐branched THP‐protected alcohols 12a‐d were purified
by column chromatography using gradient technique with
heptane/Et2O and TEA (0.5%) eluent.
Method A: The ester 8a,b (4.50 g, 11 mmol) were reduced
using the procedure for 2b. For the preparation
of the compounds 11a and 11c, the reduction
agent LiAlH4 was used and for compounds 11b
and 11d LiAlD4. The reduction reactions were
heated under reflux until complete conversion.
Method B: The acid 10 (1.11 g, 3 mmol) were reduced
using the procedure for 2b, however, for the
first reduction, 0.9 equivalents were used of
the reducing agent. For the preparation of the
compound 11a, the reduction agent LiAlH4
was used and for compound 11b LiAlD4. The
reduction reactions were heated under reflux
until complete conversion.
3.2.4 | Synthesis of (2RS)‐2‐hexyl‐11‐
[(tetrahydro‐2H‐pyran‐2‐yl)oxy]undecanoic
acid (10)
To a stirred suspension of NaH (60% in paraffin, 0.90 g,
22.5 mmol) and DIPA (3.16 mL, 22.5 mL) in abs. THF
(22 mL), a solution of octanoic acid (9) (3.24 g, 22.5 mmol)
in abs. THF (8 mL) was added dropwise under argon atmo-
sphere at a temperature of 0°C. The mixture was warmed to
60°C until the gas development stopped. Afterwards, the
mixture was cooled to −10°C and n‐butyllithium (2.5 m in
hexane, 9 mL, 22.5 mmol) was added. The reaction was
warmed to 28°C within 45 minutes, followed by the warming
to 30°C for 3 minutes. Then the mixture was stirred at 28°C.

8
SONNENBERGER ET AL.
3.2.6 | 2‐[(10RS)‐10‐Methylhexadecyloxy]
tetrahydro‐2H‐pyran (11a)
3.2.7 | Deprotecting and oxidation to the
methyl‐branched fatty acid
Colorless liquid (method A: 2.88 g, 75%; method B: 0.43 g,
42%): the analytical spectra corresponds with the data
described in the literature.20
A solution of the THP‐protected alcohol 11a‐d (2.10 g,
6 mmol) and catalytic amounts of PPTS in methanol
(120 mL) were heated under reflux for 4 hours. Afterwards,
the solvent was removed under reduced pressure and the
obtained residue was purified by column chromatography
using gradient technique with heptane/CHCl3 eluent to give
the methyl‐branched alcohol.
A solution of the methyl‐branched alcohol (1.50 g,
5.8 mmol) in acetone (65 mL) was added dropwise over
90 minutes to ice‐cooled stirred suspension of CrO3
(1.45 g, 14.5 mmol) in 10 N H2SO4 (7.25 mL). The mixture
was stirred for further 2 hours under ice cooling. For working
up, the mixture was extracted 3 times with ether (50 mL). The
combined ether phases were extracted 4 times with 5% aque-
ous KOH solution (25 mL). The combined aqueous layers
were acidified with diluted H2SO4 before 3‐time ether
(50 mL) extraction. The combined ether phases were dried
over Na2SO4 and filtered and evaporated to dryness under
reduced pressure. The crude residue was purified by column
chromatography using gradient technique with heptane/
CHCl3/Et2O eluent to give the methyl‐branched acids 12a‐d.
2‐[(10RS)‐10‐(Methyl‐d3)hexadecyloxy]tetrahydro‐2H‐pyran
(11b)
Colorless liquid (method A: 2.69 g, 70%; method B: 0.40 g,
1
39%): Rf 0.43 (CHCl3/heptane 6:4); H NMR (500 MHz,
CDCl3) δ 0.87 (t, J = 6.9 Hz, 3H, H3C(CH2)5‐), 1.04‐1.38
(m, 25H, H3C(CH2)5CH‐(CH2)7‐), 1.48‐1.62 (m, 6H,
‐(CH2)7CH2CH2OCH‐CH2(CH2)2‐), 1.68‐1.74 (m, 1H,
‐OCH‐CHH′‐), 1.80‐1.86 (m, 1H, ‐OCH‐CHH′‐), 3.38 (dt,
J = 6.7, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.47‐3.52 (m, 1H,
‐OCH‐(CH2)3CHH′‐), 3.73 (dt, J = 6.9, 9.6 Hz, 1H,
‐(CH2)8CHH′O‐), 3.85‐3.89 (m, 1H, ‐OCH‐(CH2)3CHH′‐),
4.56‐4.58 (m, 1H, ‐OCH‐O‐); 13C NMR (125 MHz, CDCl3)
δ 14.3, 19.9, 22.9, 25.7, 26.4, 27.2, 29.7, 29.8, 29.9, 30.2,
31.0, 32.1, 32.7, 37.2, 62.5, 67.9, 99.0; ESI‐MS m/z: 366.2
(M + Na)+.
2‐[(10RS)‐16,16,16‐d3‐10‐Methylhexadecyloxy]tetrahydro‐
2H‐pyran (11c)
(10RS)‐10‐Methylhexadecanoic acid (12a)
Colorless liquid (method A: 3.04 g, 79%): Rf 0.38
(CHCl3/heptane 6:4); 1H NMR (400 MHz, CDCl3)
δ 0.83 (d, J = 6.5 Hz, 3H, ‐CH‐CH3), 1.04‐1.36
(m, 25H, D3C(CH2)5CH‐(CH2)7‐), 1.50‐1.63 (m, 6H,
‐(CH2)7CH2CH2OCH‐CH2(CH2)2‐), 1.68‐1.75 (m, 1H,
‐OCH‐CHH′‐), 1.79‐1.87 (m, 1H, ‐OCH‐CHH′‐), 3.38 (dt,
J = 6.7, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.47‐3.52 (m, 1H,
‐OCH‐(CH2)3CHH′‐), 3.73 (dt, J = 6.9, 9.6 Hz, 1H,
‐(CH2)8CHH′O‐), 3.84‐3.90 (m, 1H, ‐OCH‐(CH2)3CHH′‐),
4.56‐4.57 (m, 1H, ‐OCH‐O‐); 13C NMR (100 MHz, CDCl3)
δ 19.9, 22.9, 25.7, 26.4, 27.2, 29.7, 29.8, 29.9, 30.2, 31.0,
32.0, 32.9, 37.3, 62.5, 67.9, 99.0; ESI‐MS m/z: 366.2
(M + Na)+, 709.0 (2M + Na)+.
(10RS)‐10‐Methylhexadecanol: colorless liquid (1.53 g, 97%):
1
Rf 0.27 (CHCl3); H NMR (400 MHz, CDCl3) δ 0.83 (d,
J = 6.5 Hz, 3H, ‐CH‐CH3), 0.87 (t, J = 6.8 Hz, 3H,
H3C(CH2)5‐), 1.03‐1.35 (m, 25H, H3C(CH2)5CH‐(CH2)7‐),
1.52‐1.59 (m, 2H, ‐CH2CH2OH), 3.62 (t, J = 6.7 Hz, 2H,
‐CH2OH); 13C NMR (100 MHz, CDCl3) δ 14.2, 19.8, 22.8,
25.9, 27.2, 29.6, 29.8, 30.2, 32.1, 32.9, 37.2, 37.3, 63.1.
(10RS)‐10‐Methylhexadecanoic acid (12a): colorless
1
liquid (1.09 g, 69%): Rf 0.31 (CHCl3/Et2O 8:2); H NMR
(400 MHz, CDCl3) δ 0.84 (d, J = 6.5 Hz, 3H, ‐CH‐CH3),
0.88 (t,
J = 6.8 Hz, 3H, H3C(CH2)5‐), 1.03‐1.36
(m, 23H, H3C(CH2)5CH‐(CH2)6‐), 1.60‐1.67 (m, 2H,
‐CH2CH2COOH), 2.35 (t, J = 7.5 Hz, 2H, ‐CH2COOH);
13C NMR (100 MHz, CDCl3) δ 14.3, 19.9, 22.9, 24.9, 27.2,
29.2, 29.4, 29.6, 29.9, 30.1, 32.1, 32.9, 34.2, 37.2, 37.3,
179.9; ESI‐MS m/z: 269.4 (M − H)−, 539.0 (2M − H)−.
2‐[(10RS)‐16,16,16‐d3‐10‐(Methyl‐d3)hexadecyloxy]
tetrahydro‐2H‐pyran (11d)
Colorless liquid (method A: 2.80 g, 72%): Rf 0.35
1
(CHCl3/heptane 6:4); H NMR (400 MHz, CDCl3) δ 1.04‐
1.36 (m, 25H, D3C(CH2)5CH‐(CH2)7‐), 1.49‐1.63 (m, 6H,
‐(CH2)7CH2CH2OCH‐CH2(CH2)2‐), 1.68‐1.75 (m, 1H,
‐OCH‐CHH′‐), 1.79‐1.87 (m, 1H, ‐OCH‐CHH′‐), 3.38 (dt,
J = 6.7, 9.6 Hz, 1H, ‐(CH2)8CHH′O‐), 3.47‐3.52 (m, 1H,
‐OCH‐(CH2)3CHH′‐), 3.73 (dt, J = 6.9, 9.6 Hz, 1H,
‐(CH2)8CHH′O‐), 3.84‐3.90 (m, 1H, ‐OCH‐(CH2)3CHH′‐),
4.56‐4.58 (m, 1H, ‐OCH‐O‐); 13C NMR (100 MHz, CDCl3)
δ 19.9, 22.6, 25.7, 26.4, 27.2, 29.7, 29.8, 29.9, 30.2, 31.0,
32.0, 32.7, 37.2, 62.5, 67.9, 99.0; ESI‐MS m/z: 369.9
(M + Na)+, 715.6 (2M + Na)+.
(10RS)‐10‐(Methyl‐d3)hexadecanoic acid (12b)
(10RS)‐10‐(Methyl‐d3)hexadecanol: colorless liquid (1.52 g,
96%): Rf 0.28 (CHCl3); 1H NMR (400 MHz, CDCl3) δ
0.88 (t, J = 6.7 Hz, 3H, H3C(CH2)5‐), 1.04‐1.40 (m, 25H,
H3C(CH2)5CH‐(CH2)7‐), 1.53‐1.60 (m, 2H, ‐CH2CH2OH),
3.64 (t, J = 6.6 Hz, 2H, ‐CH2OH); 13C NMR (100 MHz,
CDCl3) δ 14.3, 22.9, 25.9, 27.2, 29.6, 29.8, 29.9, 30.2,
32.1, 32.7, 33.0 37.2, 37.2, 63.3.
(10RS)‐10‐(Methyl‐d3)hexadecanoic acid (12b): colorless
liquid (1.11 g, 70%); Rf 0.42 (CHCl3/Et2O 8:2); H NMR
1

SONNENBERGER ET AL.
9
(400 MHz, CDCl3) δ 0.88 (t, J = 6.8 Hz, 3H, H3C(CH2)5‐),
1.04‐1.36 (m, 23H, H3C(CH2)5CH‐(CH2)6‐), 1.60‐1.67 (m,
2H, ‐CH2CH2COOH), 2.35 (t, J = 7.5 Hz, 2H, ‐CH2COOH);
13C NMR (100 MHz, CDCl3) δ 14.3, 22.9, 24.9, 27.2, 29.2,
29.4, 29.6, 29.9, 30.1, 32.1, 32.7, 34.2, 37.2, 37.3, 180.2;
ESI‐MS m/z: 272.5 (M − H)−, 545.1 (2M − H)−.
atmosphere for 12 hours. The thionyl chloride excess was dis-
tilled off, and the residue was dried over KOH. The obtained
acid chloride was dissolved in CHCl3 (20 mL), and the solu-
tion was added dropwise over 90 minutes to a boiling solu-
tion of 14 (3.14 g, 6.9 mmol) in CHCl3 (140 mL). After
adding, the mixture was heated under reflux for further
6 hours. For a full crystallization of the diol excess, the mix-
ture was allowed to stand at room temperature for 3 hours. To
remove the diol excess, the mixture was filtrated. The filter
was washed 3 times with CHCl3 (20 mL). The filtrate was
evaporated to dryness under reduced pressure. The crude res-
idue was purified by column chromatography using gradient
technique with heptane/CHCl3 eluent to give the ω‐esterified
alcohols 15a‐d.
(10RS)‐16,16,16‐d3‐10‐Methylhexadecanoic acid (12c)
(10RS)‐16,16,16‐d3‐10‐Methylhexadecanol: colorless liquid
(1.53 g, 97%): Rf 0.33 (CHCl3); 1H NMR (500 MHz, CDCl3)
δ 0.83 (d, J = 6.6 Hz, 3H, ‐CH‐CH3), 1.03‐1.38 (m, 25H,
D3C(CH2)5CH‐(CH2)7‐), 1.54‐1.59 (m, 2H, ‐CH2CH2OH),
3.64 (t, J = 6.7 Hz, 2H, ‐CH2OH); 13C NMR (100 MHz,
CDCl3) δ 19.9, 22.6, 25.9, 27.2, 29.6, 29.8, 29.9, 30.2,
32.0, 32.9, 33.0, 37.3, 63.3.
(10RS)‐16,16,16‐d3‐10‐Methylhexadecanoic acid (12c):
colorless liquid (1.14 g, 72%): Rf 0.59 (CHCl3/Et2O 8:2);
1H NMR (400 MHz, CDCl3) δ 0.84 (d, J = 6.5 Hz, 3H,
‐CH‐CH3), 1.02‐1.36 (m, 23H, D3C(CH2)5CH‐(CH2)6‐),
1.60‐1.67 (m, 2H, ‐CH2CH2COOH), 2.35 (t, J = 7.5 Hz,
2H, ‐CH2COOH); 13C NMR (100 MHz, CDCl3) δ 19.9,
22.6, 24.8, 27.2, 29.2, 29.4, 29.6, 29.9, 30.1, 32.0, 32.9,
34.2, 37.2, 37.3, 180.0; ESI‐MS m/z: 272.9 (M − H)−,
545.5 (2M − H)−.
30‐Hydroxytriacontyl (10RS)‐10‐methylhexadecanoate (15a)
White solid (1.34 g, 82%): mp 70.5‐71°C; Rf 0.33 (CHCl3);
1H NMR (500 MHz, CDCl3) δ 0.84 (d, J = 6.6 Hz, 3H,
‐CH‐CH3), 0.88 (t, J = 6.9 Hz, 3H, H3C(CH2)5‐), 1.04‐1.44
(m, 75H, H3C(CH2)5CH‐(CH2)6‐, ‐(CH2)26(CH2)2OH),
1.54‐1.64 (m, 6H, ‐CH2CH2COOCH2CH2‐, ‐CH2CH2OH),
2.29 (t, J = 7.5 Hz, 2H, ‐CH2COO‐), 3.64 (t, J = 6.6 Hz,
2H, ‐CH2OH), 4.05 (t, J = 6.7 Hz, 2H, ‐COOCH2‐); 13C
NMR (100 MHz, CDCl3) δ 14.3, 19.9, 22.9, 25.2, 25.9,
26.1, 27.2, 28.8, 29.3, 29.4, 29.6, 29.7, 29.8, 29.9, 30.1,
32.1, 32.9, 33.0, 34.6, 37.3, 63.3, 64.6, 174.2; ESI‐MS m/z:
707.4 (M + H)+, 729.6 (M + H)+, 1435.6 (2M + Na)+.
(10RS)‐16,16,16‐d3‐10‐(Methyl‐d3)hexadecanoic acid (12d)
(10RS)‐16,16,16‐d3‐10‐(Methyl‐d3)hexadecanol: colorless
1
liquid (1.54 g, 97%): Rf 0.30 (CHCl3); H NMR (500 MHz,
CDCl3) δ 1.04‐1.36 (m, 25H, D3C(CH2)5CH‐(CH2)7‐),
1.53‐1.59 (m, 2H, ‐CH2CH2OH), 3.63 (t, J = 6.7 Hz, 2H,
‐CH2OH); 13C NMR (125 MHz, CDCl3) δ 22.6, 25.9, 27.2,
29.6, 29.8, 29.9, 30.2, 32.0, 32.7, 32.9, 37.2, 63.2.
30‐Hydroxytriacontyl (10RS)‐10‐(methyl‐d3)hexadecanoate
(15b)
White solid (1.36 g, 83%): mp 70‐70.5°C; Rf 0.37 (CHCl3); 1H
NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.7 Hz, 3H,
H3C(CH2)5‐), 1.04‐1.43 (m, 75H, H3C(CH2)5CH‐(CH2)6‐,
‐(CH2)26(CH2)2OH), 1.53‐1.64 (m, 6H, ‐CH2CH2COOCH2
CH2‐, ‐CH2CH2OH), 2.28 (t, J = 7.5 Hz, 2H, ‐CH2COO‐),
3.64 (t, J = 6.6 Hz, 2H, ‐CH2OH), 4.05 (t, J = 6.7 Hz, 2H,
‐COOCH2‐); 13C NMR (100 MHz, CDCl3) δ 14.3, 22.9,
25.2, 25.9, 26.1, 27.2, 28.8, 29.3, 29.4, 29.6, 29.7, 29.8, 29.9,
30.1, 32.1, 32.7, 33.0, 34.6, 37.2, 63.2, 64.6, 174.2; ESI‐MS
m/z: 732.8 (M + H)+.
(10RS)‐16,16,16‐d3‐10‐(Methyl‐d3)hexadecanoic
(12d): colorless liquid (1.18 g, 74%): Rf 0.53 (CHCl3/Et2O
8:2); 1H NMR (500 MHz, CDCl3)
1.04‐1.34
acid
δ
(m, 23H, D3C(CH2)5CH‐(CH2)6‐), 1.60‐1.66 (m, 2H,
‐CH2CH2COOH), 2.35 (t, J = 7.5 Hz, 2H, ‐CH2COOH);
13C NMR (125 MHz, CDCl3) δ 22.6, 24.9, 27.2, 29.2,
29.4, 29.6, 29.9, 30.1, 32.0, 32.7, 34.2, 37.2, 179.9; ESI‐MS
m/z: 275.7 (M − H)−.
30‐Hydroxytriacontyl
hexadecanoate (15c)
(10RS)‐16,16,16‐d3‐10‐methyl‐
3.2.8 | Synthesis of the 1,30‐triacontanediol
(14)
White solid (1.38 g, 84%): mp 70‐70.5°C; Rf 0.35 (CHCl3);
1H NMR (400 MHz, CDCl3) δ 0.83 (d, J = 6.5 Hz, 3H,
‐CH‐CH3), 1.04‐1.44 (m, 75H, D3C(CH2)5CH‐(CH2)6‐,
‐(CH2)26(CH2)2OH), 1.53‐1.65 (m, 6H, ‐CH2CH2COOCH2
CH2‐, ‐CH2CH2OH), 2.29 (t, J = 7.5 Hz, 2H, ‐CH2COO‐),
3.64 (t, J = 6.7 Hz, 2H, ‐CH2OH), 4.05 (t, J = 6.7 Hz, 2H,
‐COOCH2‐); 13C NMR (100 MHz, CDCl3) δ 19.9, 22.6,
25.2, 25.9, 26.1, 27.2, 28.8, 29.3, 29.4, 29.5, 29.6, 29.7,
29.8, 29.9, 30.1, 32.0, 32.9, 33.0, 34.6, 37.3, 63.3, 64.6,
174.2; ESI‐MS m/z: 711.0 (M + H)+, 732.8 (M + H)+.
The preparation was following the procedure described in the
literature30 starting from 15‐pentadecanolide (13) to give 14.
3.2.9 | Coupling of the methyl‐branched acid
with 1,30‐triacontandiol
A mixture of 12a‐d (0.63 mg, 2.3 mmol) and fresh distilled
thionyl chloride (1.67 mL, 23 mmol) was heated under reflux
for 4 hours. Afterwards, the mixture was stirred under argon

10
SONNENBERGER ET AL.
30‐Hydroxytriacontyl
hexadecanoate (15d)
(10RS)‐16,16,16‐d3‐10‐(methyl‐d3)
29.7, 29.8, 30.1, 32.1, 32.7, 34.1, 34.6, 37.2, 64.5, 174.2,
179.4; ESI‐MS m/z: 722.7 (M − H)−.
White solid (1.34 g, 82%): mp 69‐70.5°C; Rf 0.33 (CHCl3);
1H NMR (500 MHz, CDCl3) δ 1.04‐1.43 (m, 75H,
D3C(CH2)5CH‐(CH2)6‐, ‐(CH2)26(CH2)2OH), 1.54‐1.64 (m,
30‐{[(10RS)‐16,16,16‐d3‐10‐Methylhexadcanoyl]oxy}
triacontanoic acid (16c)
White solid (0.78 g, 64%): mp 76‐78°C; Rf 0.14 (CHCl3); 1H
NMR (400 MHz, CDCl3) δ 0.83 (d, J = 6.6 Hz, 3H, ‐CH‐
CH3), 1.04‐1.35 (m, 73H, D3C(CH2)5CH‐(CH2)6‐,
6H,
‐CH2CH2COOCH2CH2‐,
‐CH2CH2OH),
2.29
(t, J = 7.5 Hz, 2H, ‐CH2COO‐), 3.64 (t, J = 6.7 Hz, 2H,
‐CH2OH), 4.05 (t, J = 6.7 Hz, 2H, ‐COOCH2‐); 13C NMR
(100 MHz, CDCl3) δ 22.6, 25.2, 25.9, 26.1, 27.2, 28.8,
29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30.1, 32.0, 32.7,
33.0, 34.6, 37.2, 63.3, 64.6, 174.2; ESI‐MS m/z: 713.7
(M + H)+, 735.8 (M + H)+, 1447.4 (2M + Na)+.
‐(CH2)25(CH2)2COOH),
1.58‐1.66
(m,
6H,
‐CH2CH2COOCH2CH2‐, ‐CH2CH2COOH), 2.29 (t,
J = 7.5 Hz, 2H, ‐CH2COO‐), 2.35 (t, J = 7.5 Hz, 2H,
‐CH2COOH), 4.05 (t, J = 6.7 Hz, 2H, ‐COOCH2‐); 13C
NMR (100 MHz, CDCl3) δ 19.9, 22.6, 24.9, 25.2, 26.1,
27.2, 28.8, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9,
30.1, 32.0, 32.9, 34.0, 34.6, 37.3, 64.6, 174.2, 179.1; ESI‐MS
m/z: 722.8 (M − H)−.
3.2.10 | Oxidation to the ω‐esterified acid
To an ice‐cooled mixture of CrO3 (0.37 g, 3.7 mmol) in 10 N
H2SO4 (1.90 mL), a solution of 15a‐d (1.20 g, 1.7 mmol) in
acetone (20 mL) and CHCl3 (35 mL) was added dropwise
over a period of 90 minutes. The mixture was stirred for
further 60 minutes at a temperature of 0°C. For working up,
brine (25 mL) and aqua (25 mL) were added. The mixture
was extracted 3 times with CHCl3 (75 mL). The combined
organic layers were washed with brine, dried over Na2SO4,
and filtered and evaporated to dryness under reduced pres-
sure. The crude residue was purified by column chromatogra-
phy using gradient technique with heptane/CHCl3/Et2O
eluent to give the ω‐esterified acids 16a‐d.
30‐{[(10RS)‐16,16,16‐d3‐10‐(Methyl‐d3)hexadcanoyl]oxy}
triacontanoic acid (16d)
White solid (0.87 g, 71%): mp 76‐78.5°C; Rf 0.13 (CHCl3);
1H NMR (400 MHz, CDCl3) δ 1.04‐1.35 (m, 73H,
D3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2COOH), 1.58‐1.67
(m, 6H, ‐CH2CH2COOCH2CH2‐, ‐CH2CH2COOH), 2.29
(t, J = 7.5 Hz, 2H, ‐CH2COO‐), 2.35 (t, J = 7.5 Hz, 2H,
‐CH2COOH), 4.06 (t, J = 6.7 Hz, 2H, ‐COOCH2‐); 13C
NMR (100 MHz, CDCl3) δ 22.6, 24.9, 25.2, 26.1, 27.2,
28.8, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30.1,
32.0, 32.7, 34.0, 34.6, 37.2, 64.6, 174.2, 179.0; ESI‐MS
m/z: 727.2 (M − H)−.
30‐{[(10RS)‐10‐Methylhexadcanoyl]oxy}triacontanoic
acid (16a)
White solid (0.86 g, 70%): mp 77‐78°C; Rf 0.10 (CHCl3); 1H
NMR (400 MHz, CDCl3) δ 0.83 (d, J = 6.5 Hz, 3H, ‐CH‐
CH3), 0.88 (t, J = 6.8 Hz, 3H, H3C(CH2)5‐), 1.04‐1.35 (m,
73H, H3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2COOH), 1.58‐
1.67 (m, 6H, ‐CH2CH2COOCH2CH2‐, ‐CH2CH2COOH),
2.29 (t, J = 7.5 Hz, 2H, ‐CH2COO‐), 2.35 (t, J = 7.5 Hz,
3.2.11 | Coupling to the methyl‐branched
ceramide [mEOS]*
DIPEA (94 μL, 0.55 mmol) was added to a stirred suspension
of 16a‐d (200 mg, 0.28 mmol) and PyBOP (157 mg,
0.30 mmol) in dry CH2Cl2 (5 mL). After 15 minutes, sphin-
gosine (91 mg, 0.30 mmol) was added to the clear solution
and the mixture was stirred for further 16 hours. Afterwards,
the solvent was evaporated and the residue was purified by
column chromatography using gradient technique with
CHCl3/MeOH eluent to give the ceramides Ia‐d.
2H, ‐CH2COOH), 4.06 (t, J = 6.7 Hz, 2H, ‐COOCH2‐); 13
C
NMR (100 MHz, CDCl3) δ 14.3, 19.9, 22.9, 24.9, 25.2,
26.1, 27.2, 28.8, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9,
30.1, 32.1, 32.9, 33.9, 34.6, 37.3, 64.6, 174.2, 178.6; ESI‐MS
m/z: 719.7 (M − H)−.
N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐30‐
{[(10RS)‐10‐methylhexadecanoyl]oxy}‐triacontanamid
(CER[mEOS] Ia)
30‐{[(10RS)‐10‐(Methyl‐d3)hexadecanoyl]oxy}triacontanoic
acid (16b)
White solid (0.83 g, 68%): mp 76.5‐77°C; Rf 0.10 (CHCl3);
1H NMR (500 MHz, CDCl3) δ 0.88 (t, J = 6.9 Hz, 3H,
H3C(CH2)5‐), 1.04‐1.35 (m, 73H, H3C(CH2)5CH‐(CH2)6‐,
‐(CH2)25(CH2)2COOH), 1.59‐1.66 (m, 6H, ‐CH2CH2
COOCH2CH2‐, ‐CH2CH2COOH), 2.29 (t, J = 7.5 Hz, 2H,
‐CH2COO‐), 2.35 (t, J = 7.5 Hz, 2H, ‐CH2COOH), 4.06 (t,
J = 6.7 Hz, 2H, ‐COOCH2‐); 13C NMR (100 MHz, CDCl3)
δ 14.2, 22.8, 24.8, 25.2, 26.1, 27.2, 28.8, 29.3, 29.4, 29.6,
White waxy solid (245 mg, 88%): mp 84.5‐86°C; Rf 0.42
1
(CHCl3/MeOH 95:5); H NMR (400 MHz, CDCl3) δ 0.83
(d, J = 6.5 Hz, 3H, ‐CH‐CH3), 0.88 (t, J = 6.7 Hz, 6H, 2×
H3CCH2‐), 1.04‐1.38 (m, 95H, H3C(CH2)5CH‐(CH2)6‐,
‐(CH2)25(CH2)2CONH‐, ‐(CH2)11CH2CH═CH‐), 1.57‐1.64
*Differing to the IUPAC nomenclature, the name is based on the
amid‐bonding backbone of ceramides.

SONNENBERGER ET AL.
11
(m, 6H, ‐CH2CH2COOCH2CH2‐, ‐CH2CH2CONH‐), 2.02‐
2.07 (m, 2H, ‐CH2CH═CH‐), 2.22 (t, J = 7.6 Hz, 2H,
‐CH2CONH‐), 2.28 (t, J = 7.5 Hz, 2H, ‐CH2COO‐), 3.69
(dd, J = 2.5, 10.6 Hz, 1H, ‐CHH′OH), 3.88‐3.95 (m, 2H,
‐CH‐CHH′OH), 4.05 (t, J = 6.7 Hz, 2H, ‐COOCH2‐), 4.28‐
4.31 (m, 1H, ‐CH═CHCHOH‐), 5.52 (dd, J = 6.4, 15.4 Hz,
1H, ‐CH2CH═CH‐), 5.74‐5.81 (m, 1H, ‐CH2CH═CH‐),
6.28 (d, J = 7.2 Hz, 1 H, ‐CONH‐); 13C NMR (100 MHz,
CDCl3) δ 14.3, 19.9, 22.8, 25.2, 25.9, 26.1, 27.2, 28.8, 29.3,
29.4, 29.5, 29.7, 29.8, 29.9, 30.1, 32.1, 32.4, 32.9, 34.6,
37.0, 37.2, 37.3, 54.7, 62.6, 64.6, 74.7, 129.0, 134.3, 174.1,
174.2; HRMS [M + Na]+ m/z calcd for C65H127NO5Na
1024.9606, found 1024.9588; HPLC purity >99.5%.
1H, ‐CH2CH═CH‐), 6.33 (d, J = 4.5 Hz, 1H, ‐CONH‐);
13C NMR (125 MHz, CDCl3) δ 14.3, 19.9, 22.6, 22.9,
25.2, 25.9, 26.1, 27.2, 28.8, 29.3, 29.4, 29.5, 29.7, 29.8,
29.9, 30.1, 32.1, 32.5, 32.9, 34.6, 37.0, 37.2, 37.3, 54.7,
62.7, 64.6, 74.8, 129.0, 134.3, 174.0, 174.2; HRMS
[M + Na]+ m/z calcd for C65H124D3NO5Na 1027.9795,
found 1027.9779; HPLC purity 99.5%.
N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐30‐{[(10RS)‐
16,16,16‐d3‐10‐(methyl‐d3)‐hexadecanoyl]oxy}
triacontanamid (CER[mEOS] m,t‐d3 Id)
White waxy solid (257 mg, 93%): mp 84‐85.5°C; Rf 0.52
1
(CHCl3/MeOH 95:5); H NMR (500 MHz, CDCl3) δ 0.88
(t,
J = 7.0 Hz, 3H, H3C‐), 1.04‐1.38 (m, 95H,
N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐30‐{[(10RS)‐
10‐(methyl‐d3)hexadecanoyl]‐oxy}triacontanamid
(CER[mEOS] m‐d3 Ib)
D3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2CONH‐, ‐(CH2)11
CH2CH═CH‐), 1.58‐1.66 (m, 6H, ‐CH2CH2COO CH2CH2‐,
‐CH2CH2CONH‐), 2.03‐2.07 (m, 2H, ‐CH2CH═CH‐), 2.22
(t, J = 7.7 Hz, 2H, ‐CH2CONH‐), 2.28 (t, J = 7.6 Hz, 2H,
‐CH2COO‐), 3.70 (dd, J = 3.3, 11.2 Hz, 1H, ‐CHH′OH),
3.90 (dt, 2H, J = 3.6, 11.2 Hz, 1H, ‐CH‐CH2OH), 3.95
(dd, 2H, J = 3.8, 11.2 Hz, 1H, ‐CHH′OH), 4.05 (t,
White waxy solid (233 mg, 84%): mp 83‐84°C; Rf 0.46
1
(CHCl3/MeOH 95:5); H NMR (500 MHz, CDCl3) δ 0.87‐
0.89 (m, 6H, 2× H3CCH2‐), 1.04‐1.38 (m, 95H,
H3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2CONH‐, ‐(CH2)11
CH2CH═CH‐), 1.58‐1.67 (m, 6H, ‐CH2CH2COOCH2CH2‐,
‐CH2CH2CONH‐), 2.04‐2.08 (m, 2H, ‐CH2CH═CH‐), 2.23
(t, J = 7.6 Hz, 2H, ‐CH2CONH‐), 2.29 (t, J = 7.4 Hz, 2H,
‐CH2COO‐), 3.71 (dd, J = 3.3, 11.2 Hz, 1H, ‐CHH′OH),
3.91 (dt, 2H, J = 3.7, 11.2 Hz, 1H, ‐CH‐CH2OH), 3.96 (dd,
J = 3.8, 11.2 Hz, 2H, CHH′OH), 4.05 (t, J = 6.7 Hz, 2H,
‐COOCH2‐), 4.31‐4.33 (m, 1H, ‐CH═CHCHOH‐), 5.53 (dd,
J = 6.4, 15.5 Hz, 1H, ‐CH2CH═CH‐), 5.76‐5.81 (m, 1H,
‐CH2CH═CH‐), 6.23 (d, J = 7.3 Hz, 1 H, ‐CONH‐); 13C
NMR (125 MHz, CDCl3) δ 14.3, 22.9, 25.2, 25.9, 26.1,
27.2, 28.8, 29.3, 29.4, 29.5, 29.7, 29.8, 29.9, 30.1, 32.1,
32.4, 32.7, 34.6, 37.0, 37.2, 37.3, 54.7, 62.7, 64.6, 74.9,
129.0, 134.5, 174.0, 174.2; HRMS [M + Na]+ m/z calcd for
C65H124D3NO5Na 1027.9795, found 1027.9777; HPLC
purity 98.5%.
J
= 6.7 Hz, 2H, ‐COOCH2‐), 4.30‐4.32 (m, 1H,
‐CH═CHCHOH‐), 5.53 (dd, J = 6.4, 15.4 Hz, 1H,
‐CH2CH═CH‐), 5.75‐5.81 (m, 1H, ‐CH2CH═CH‐), 6.33 (d,
J = 7.1 Hz, 1H, ‐CONH‐); 13C NMR (125 MHz, CDCl3) δ
14.3, 22.6, 22.9, 25.2, 25.9, 26.1, 27.2, 28.8, 29.3, 29.4,
29.5, 29.7, 29.8, 29.9, 30.1, 32.0, 32.1, 32.4, 32.7, 34.6,
37.0, 37.2, 54.6, 62.7, 64.6, 74.8, 129.0, 134.4, 174.1,
174.2; HRMS [M + Na]+ m/z calcd for C65H121D6NO5Na
1030.9983, found 1030.9970; HPLC purity 99.5%.
3.2.12 | Coupling to the methyl‐branched cer-
amide [mEOP]
DIPEA (94 μL, 0.55 mmol) was added to a stirred suspension
of 16a‐d (200 mg, 0.28 mmol) and PyBOP (157 mg,
0.30 mmol) in dry CH2Cl2 (5 mL). After 15 minutes,
phytosphingosine (96 mg, 0.30 mmol) was added to the clear
solution and the mixture was stirred for further 16 hours.
Afterwards, the formed precipitate was filtered and the filter
was washed 2 times with CH2Cl2 (2 mL). For purification,
the residue was adsorbed on silica gel before column chroma-
tography using gradient technique with CHCl3/MeOH and
NH3 (0.5%) eluent to give the ceramides IIa‐d.
N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐30‐{[(10RS)‐
16,16,16‐d3‐10‐methyl‐hexadecanoyl]oxy}triacontanamid
(CER[mEOS] t‐d3 Ic)
White waxy solid (235 mg, 84%): mp 82.5‐84°C; Rf 0.53
1
(CHCl3/MeOH 95:5); H NMR (500 MHz, CDCl3) δ 0.83
(d, J = 6.6 Hz, 3H, ‐CH‐CH3), 0.88 (t, J = 7.0 Hz, 3H,
H3CCH2‐), 1.04‐1.38 (m, 95H, D3C(CH2)5CH‐(CH2)6‐,
‐(CH2)25(CH2)2CONH‐, ‐(CH2)11CH2CH═CH‐), 1.58‐1.67
(m, 6H, ‐CH2CH2COOCH2CH2‐, ‐CH2CH2CONH‐), 2.03‐
2.07 (m, 2H, ‐CH2CH═CH‐), 2.23 (t, J = 7.6 Hz, 2H,
‐CH2CONH‐), 2.28 (t, J = 7.6 Hz, 2H, ‐CH2COO‐), 3.70
(dd, J = 3.3, 11.7 Hz, 1H, ‐CHH′OH), 3.90 (dt, 2H,
J = 3.7, 11.3 Hz, 1H, ‐CH‐CH2OH), 3.96 (dd, 2H, J = 3.5,
11.2 Hz, 1H ‐CHH′OH), 4.05 (t, J = 6.7 Hz, 2H,
‐COOCH2‐), 4.30‐4.32 (m, 1H, ‐CH═CHCHOH‐), 5.53
(dd, J = 6.3, 15.5 Hz, 1H, ‐CH2CH═CH‐), 5.75‐5.81 (m,
N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐30‐{[(10RS)‐10‐
methylhexadecanoyl]oxy}‐triacontanamid (CER[mEOP] IIa)
White waxy solid (227 mg, 80%): mp 102‐103°C; Rf 0.21
1
(CHCl3/MeOH/NH3 95:5:1); H NMR (400 MHz, CDCl3,
40°C) δ 0.84 (d, J = 6.5 Hz, 3H, ‐CH‐CH3), 0.89 (t,
J
=
6.7 Hz, 6H, 2× H3CCH2‐), 1.05‐1.36
(m, 97H, H3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2CONH‐,
‐(CH2)12CH2CHOH‐), 1.48‐1.78 (m, 8H,

12
SONNENBERGER ET AL.
‐CH2CH2COOCH2CH2‐, ‐CH2CH2CONH‐, ‐CH2CHOH‐),
2.24 (t, J = 7.5 Hz, 2H, ‐CH2CONH‐), 2.29 (t, J = 7.5 Hz,
2H, ‐CH2COO‐), 3.58‐3.68 (m, 2H, ‐CH2CHOHCHOH‐),
3.76 (dd, J = 5.3, 11.6 Hz, 1H, ‐CHH′OH), 3.95 (dd,
J = 2.7, 11.6 Hz, 1H, ‐CHH′OH), 4.06 (t, J = 6.7 Hz, 2H,
‐COOCH2‐), 4.12‐4.16 (m, 1H, ‐CH‐CH2OH), 6.32 (d,
J = 7.6 Hz, 1H, ‐CONH‐); 13C NMR (100 MHz, THF‐d8,
40°C) δ 14.6, 20.3, 23.7, 26.8, 26.9, 27.1, 28.1, 28.2, 29.9,
30.2, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 33.0,
34.0, 34.5, 34.9, 37.0, 38.2, 54.0, 62.7, 64.7, 73.4, 77.4,
173.4; HRMS [M + Na]+ m/z calcd for C65H129NO6Na
1042.9712, found 1042.9694; HPLC purity >99.5%.
173.4; HRMS [M + H]+ m/z calcd for C65H127D3NO6
1024.0081, found 1024.0099; HPLC purity >99.5%.
N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐30‐{[(10RS)‐
16,16,16‐d3‐10‐(methyl‐d3)‐hexa‐decanoyl]oxy}
triacontanamid (CER[mEOP] m,t‐d6 IId)
White waxy solid (217 mg, 77%): mp 100‐103°C; Rf 0.18
1
(CHCl3/MeOH/NH3 95:5:1); H NMR (400 MHz, CDCl3,
40°C) δ 0.89 (t, J = 6.9 Hz, 3H, H3CCH2‐), 1.05‐1.36 (m,
97H, D3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2CONH‐,
‐(CH2)12CH2CHOH‐), 1.47‐1.82 (m, 8H, ‐CH2CH2
COOCH2CH2‐, ‐CH2CH2CONH‐, ‐CH2CHOH‐), 2.24 (t,
J = 7.5 Hz, 2H, ‐CH2CONH‐), 2.29 (t, J = 7.5 Hz, 2H,
‐CH2COO‐), 3.58‐3.67 (m, 2H, ‐CH2CHOHCHOH‐), 3.76
(dd, J = 5.6, 11.5 Hz, 1H, ‐CHH′OH), 3.96 (dd, J = 2.4,
11.5 Hz, 1H, ‐CHH′OH), 4.06 (t, J = 6.7 Hz, 2H,
‐COOCH2‐), 4.12‐4.16 (m, 1H, ‐CH‐CH2OH), 6.32 (d,
J = 7.3 Hz, 1H, ‐CONH‐); 13C NMR (100 MHz, THF‐d8,
40°C) δ 14.6, 23.4, 23.7, 26.8, 26.9, 27.1, 28.1, 28.2, 29.9,
30.2, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 33.0,
33.7, 34.5, 34.9, 37.0, 38.2, 54.0, 62.7, 64.7, 73.4, 77.4,
173.4; HRMS [M + H]+ m/z calcd for C65H124D3NO6
1027.0269, found 1027.0261; HPLC purity >99.5%.
N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐30‐{[(10RS)‐10‐
(methyl‐d3)‐hexadecanoyl]‐oxy}triacontanamid
(CER[mEOP] m‐d3 IIb)
White waxy solid (223 mg, 79%): mp 101‐103°C; Rf 0.25
1
(CHCl3/MeOH/NH3 95:5:1); H NMR (400 MHz, CDCl3,
40°C) δ 0.89 (t, J = 6.7 Hz, 6H, 2× H3CCH2‐), 1.05‐1.36
(m, 97H, H3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2CONH‐,
‐(CH2)12CH2CHOH‐),
1.45‐1.82
(m,
8H,
‐CH2CH2COOCH2CH2‐, ‐CH2CH2CONH‐, ‐CH2CHOH‐),
2.24 (t, J = 7.6 Hz, 2H, ‐CH2CONH‐), 2.29 (t, J = 7.5 Hz,
2H, ‐CH2COO‐), 3.59‐3.67 (m, 2H, ‐CH2CHOHCHOH‐),
3.76 (dd, J = 5.43, 11.3 Hz, 1H, ‐CHH′OH), 3.95 (dd,
J = 2.2, 11.4 Hz, 1H, ‐CHH′OH), 4.06 (t, J = 6.8 Hz, 2H,
‐COOCH2‐), 4.13‐4.17 (m, 1H, ‐CH‐CH2OH), 6.32 (d,
J = 7.8 Hz, 1H, ‐CONH‐); 13C NMR (100 MHz, THF‐d8,
40°C) δ 14.6, 23.7, 26.8, 27.0, 27.1, 28.1, 28.2, 29.9, 30.2,
30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 33.0, 33.7,
34.5, 34.9, 37.0, 38.2, 54.1, 62.7, 64.7, 73.4, 77.4, 173.4;
3.3 | Sample preparation
The SC model membranes were prepared on quartz glass
slides. For the sample application, the pure substances were
solved in a 2:1 mixture of chloroform and methanol (analyti-
cal grade, by Sigma‐Aldrich) with a concentration of
10 mg/mL. These solutions were then combined, to generate
CER[NS]/CER[EOS]‐br/CHOL/FFA mixtures with a molar
ratio of 0.8/0.2/0.7/1, see Table 1. For deposition, the
mixtures were sprayed onto the surface, using an airbrush
instrument (Harder & Steenbeck, Norderstedt, Germany).
HRMS [M
+
Na]+ m/z calcd for C65H129NO6Na
1042.9712, found 1042.9694; HPLC purity >99.5%.
N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐30‐{[(10RS)‐
16,16,16‐d3‐10‐methylhexa‐decanoyl]oxy}triacontanamid
(CER[mEOP] t‐d3 IIc)
White waxy solid (215 mg, 76%): mp 103‐105°C; Rf 0.22
(CHCl3/MeOH/NH3 95:5:1); H NMR (400 MHz, CDCl3,
3.4 | Neutron diffraction experiments
1
40°C) δ 0.84 (d, J = 6.5 Hz, 3H, ‐CH‐CH3), 0.89 (t,
Diffraction data were collected at the membrane diffractome-
ter V1 located at the Hahn‐Meitner campus of the Helmholtz‐
Zentrum Berlin. Neutrons generated by the research reactor
BER II were cooled to a thermal level, using an H2 cold
source. A gallium monochromator was used, to filter
neutrons with a wavelength of λ = 4.567 Å. The distance
between sample and detector for this device was 101.8 cm.
The intensity of the scattered neutrons was recorded as a
function of the detector angle 2ϴ. For contrasting against
the water background, the 3 different H2O/D2O ratios of
0:100, 50:50, and 92:8 (w/w) were applied. A higher D2O
content also generates a higher signal‐to‐background ratio,
which allows for detection of peaks of higher diffraction
orders with high precision. These higher order peaks are very
J
=
6.8 Hz, 3H, H3CCH2‐), 1.05‐1.37 (m,
97H, D3C(CH2)5CH‐(CH2)6‐, ‐(CH2)25(CH2)2CONH‐,
‐(CH2)12CH2CHOH‐), 1.44‐1.81 (m, 8H,
‐CH2CH2COOCH2CH2‐, ‐CH2CH2CONH‐, ‐CH2CHOH‐),
2.24 (t, J = 7.5 Hz, 2H, ‐CH2CONH‐), 2.29 (t, J = 7.5 Hz,
2H, ‐CH2COO‐), 3.58‐3.67 (m, 2H, ‐CH2CHOHCHOH‐),
3.77 (dd, J = 5.3, 11.2 Hz, 1H, ‐CHH′OH), 3.96 (dd,
J = 1.9, 11.2 Hz, 1H, ‐CHH′OH), 4.06 (t, J = 6.7 Hz, 2H,
‐COOCH2‐), 4.12‐4.16 (m, 1H, ‐CH‐CH2OH), 6.32 (d,
J = 7.3 Hz, 1 H, ‐CONH‐); 13C NMR (100 MHz, THF‐d8,
40°C) δ 14.6, 20.3, 23.5, 23.7, 26.8, 27.0, 27.1, 28.2, 29.9,
30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31.0, 31.1, 33.0,
34.0, 34.5, 34.9, 37.0, 38.2, 54.0, 62.7, 64.7, 73.4, 77.4,

SONNENBERGER ET AL.
13
d
2
TABLE 1 Measurement conditions for the neutron scattering
measurements
maximum position x ¼ within the hydrophilic head group
region allows determination of the Fn by application of
different H2O/D2O contrast, representing an isomorphous
replacement.36,37 The centrosymmetric lipid arrangement
that is to be expected of CER membranes leads to a phase
of either + or – for each Fn. A 3‐point measurement with 3
different H2O/D2O contrasts is thus sufficient for the calcula-
tion of the phase of each structure factor. A detailed
description of this procedure (contrast variation) and of the
neutron diffraction data evaluation can be assessed
elsewhere.36,38,39
Mixture
x, mol/mol ϑ, °C R.H., % D2O, %
NS(18)/EOS‐br/CHOL/SA 0.8/0.2/0.7/1 32
98
98
98/50/8
98/50/8
NS(18)‐D3/EOS‐br/
CHOL/SA
0.8/0.2/0.7/1 32
0.8/0.2/0.7/1 32
0.8/0.2/0.7/1 32
NS(18)/EOS‐br‐D3/
CHOL/SA
98
98
98/50/8
98/50/8
NS(18)/EOS‐D3‐br/
CHOL/SA
important for calculation of a detailed scattering length
density profile.
4 | CONCLUSION
Conditions according to Table 1 were applied for the
measurements. For the base system and the sample contain-
ing the CER[EOS]‐d3‐br, an additional measurement at a
hydration of 57%, which is about the natural moisture content
of the skin, and 100% D2O, which gives the best signal, was
performed to investigate possible swelling under excess
hydration. Temperature was kept at 32°C, which is about
the average temperature of the human skin. Since it could
be observed that no more changes in the diffraction signal
occurred after about 6 hours of equilibration, samples were
given a time of at least 7 hours after any change of tempera-
ture, humidity, or H2O/D2O ratio. To determine the mem-
brane spacing, the scattering vector Q was calculated. Q is
Long chained ceramides [EOS] and [EOP] containing a
methyl‐branched fatty acid instead of linoleic acid were syn-
thesized. Besides, the full protonated ceramides, a specifi-
cally deuteration in the branched methyl group and/or at the
end of the fatty acid, were introduced. Two methods for the
preparation of the methyl‐branched fatty acid were presented,
a classic method by dialkylation of diethyl malonate and a
shorter method by α‐alkylation of an acid dianion, which
needs some higher demands to the equipment. For the ester
formation of the branched fatty acid with a diol, a modifica-
tion of the “normal acylation” procedure was necessary
because of the insolubility of the diol at low reaction temper-
atures. Regarding the synthesis of natural CER[EOS] or
CER[EOP] with linoleic acid, the preparation of the deriva-
tives is possible in a shorter way without a complex blocking
group strategy. For the amid formation of the methyl‐
branched ω‐acyl ceramides, PyBOP was used as coupling
reagent, which ensures high yield and a simple workup. First
results for the neutron scattering of a CER[NS]‐based system,
containing the 2 variants of the synthesized deuterated
CER[EOS]‐br, were already obtained. These results clearly
show that the CER[EOS]‐br is incorporated into the SPP of
CER‐based model membranes. It can be used to obtain
further information on the lamellar nanostructure. It was even
possible to distinguish the 2 differently deuterated long‐chain
ceramides.
dependent of the scattering angle by Q ¼ 4π sinðϴÞ; it is a result
λ
→
→
of the incoming wave vector ki and the scattering vector ks,
with λ being the neutron wavelength. The repeat distance d
of a series of equidistant peaks Qn is then calculated as d ¼
2nπ
, with n being the diffraction order. The neutron scattering
Qn
length density profile as function ps(x) was calculated by
Fourier synthesis of the structure factors Fh according to
ꢀ
ꢁ
2 n
2πnx
d
max
psðxÞ ¼ a þ b ∑ Fn
:
d n¼1
The coefficients a and b are for relative normalisation of
ps(x), d is the lamellar periodicity, and n the order of diffrac-
tion. The absolute values of the structure factors could be cal-
pffiffiffiffiffiffiffiffiffiffiffi
ACKNOWLEDGEMENTS
culated as jFnj ¼ hAnIn, with h as the Lorentz correction,
An as absorption correction,36 and In as intensity of the nth
peak. For calculation of ps(x), at least 3 diffraction orders
have to be determined. Contrasting with D2O as mentioned
above can be used, to determine diffractions of a higher order.
Necessary Bragg peak determination and integration were
performed using the IGOR Pro 6.1 software (WaveMetrics
Inc., Portland, Oregon). For the Fourier transform, both the
absolute value and the phase of each structure factor Fn are
required. Assuming a Gaussian water distribution with a
This work was supported by grants of the Deutsche
Forschungsgemeinschaft (DFG DO 463/6‐1 and NE
427/30‐1) and Evonik Industries, AG. The authors thank Prof
Dr Andrea Sinz and Dr Christian Ihling (Institute of
Pharmacy, Martin Luther University, Halle (Saale),
Germany) for performing the high‐resolution mass spectrom-
etry data and Manuela Woigk (Institute of Pharmacy,
Martin‐Luther‐University, Halle (Saale), Germany) for the
HPLC data.

14
SONNENBERGER ET AL.
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the structure and hydration of a stratum corneum lipid model mem-
brane by neutron diffraction. Eur Biophys J. 2005;34(8):1030‐1040.
https://doi.org/10.1007/s00249‐005‐0488‐6
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