Synthesis of ceramides NS and NP with perdeuterated and specifically ω deuterated N-acyl residues

Article abstract of DOI:10.1002/jlcr.3443

The synthesis of 12 deuterated ceramides with either a deuteration at the last carbon atom of the amide bound fatty acid or a perdeuterated fatty acid chain is described. The ceramides were prepared starting from sphingosine or phytosphingosine and ω deuterated or perdeuterated fatty acids with PyBOP as activating agent in high yields. For the synthesis of the specifically deuterated fatty acids, dicarboxylic acids were transformed into ω deuterated alkyl bromide, which was chain elongated with blocked ω bromo alcohols by copper catalyzed Grignard coupling. Oxidation of regenerated alcohol function yields the ω deuterated fatty acids.

Full text of DOI:10.1002/jlcr.3443

Received: 31 March 2016
DOI 10.1002/jlcr.3443
Revised: 11 August 2016
Accepted: 16 August 2016
R E S E A R C H A R T I C L E
Synthesis of ceramides NS and NP with perdeuterated and
specifically ω deuterated N‐acyl residues
Stefan Sonnenberger1 | Stefan Lange2 | Andreas Langner1 | Reinhard H.H. Neubert3 |
1
*
Bodo Dobner
1 Institute of Pharmacy, Martin‐Luther‐University
Halle‐Wittenberg (MLU), Halle/Saale, Germany
2 Institute for Medical Physics and Biophysics,
The synthesis of 12 deuterated ceramides with either a deuteration at the last carbon
atom of the amide bound fatty acid or a perdeuterated fatty acid chain is described.
The ceramides were prepared starting from sphingosine or phytosphingosine and ω
deuterated or perdeuterated fatty acids with PyBOP® as activating agent in high
yields. For the synthesis of the specifically deuterated fatty acids, dicarboxylic acids
were transformed into ω deuterated alkyl bromide, which was chain elongated with
blocked ω bromo alcohols by copper catalyzed Grignard coupling. Oxidation of
regenerated alcohol function yields the ω deuterated fatty acids.
Leipzig University, Leipzig, Germany
3 Institute of Applied Dermatopharmacy, Halle
Technology and Founder’s Centre GmbH, (TGZ),
Halle/Saale, Germany
Correspondence
Bodo Dobner, Institute of Pharmacy, Martin‐
Luther‐University Halle‐Wittenberg (MLU), Halle/
Saale, Germany.
Email: bodo.dobner@pharmazie.uni‐halle.de.
KEYWORDS
ceramides, Grignard coupling reactions, perdeuteration, PyBOP® coupling, stratum
corneum, ω deuterated fatty acids synthesis
1
|
INTRODUCTION
requires specifically deuterated ceramides. In literature the
use of deuterated compounds in skin research is described
rather marginally. Some authors used fully or partially
deuterated fatty acids in the mixture with different ceramides
and cholesterol or isopropyl myristate.1–3 Also ceramides
containing perdeuterated fatty acids as N‐acyl residues have
been described and investigated.4–6 Only a few publications
were found using specifically deuterated acyl chains.
However, the sphingosine moiety was specifically deuterated
in the head region by H‐D exchange.7 To the best of our
knowledge, there was only 1 publication using ceramides
comprising an ω deuterated fatty acid.8
The objective of this study was the synthesis of the SC
model ceramides with 3 terminally deuterated fatty acids with
chain lengths of 18, 22, and 24 carbon atoms for neutron
scattering experiments. For this we used the copper catalyzed
coupling of deuterated Grignard reagents with ω functional-
ized bromides. In addition, for the solid‐state NMR investiga-
tions, ceramides with 3 perdeuterated fatty acids with 12, 18,
and 24 carbon atoms were used, in which the tetracosanoic
acid was prepared by hydrothermal Pd‐catalyzed H/D
exchange.9 The synthesized fatty acids were efficiently bound
to the amino group of sphingosine and phytosphingosine
with PyBOP® as activating reagent.
The skin is the largest organ of the human body and consists
of 3 different layers, wherein the outermost, the epidermis, is
again split into 4 layers. Stratum corneum (SC) as the outside
part of the epidermis is necessary to protect the organism.
Some significant functions are connected with this skin layer,
for instance, the barrier function against various outside
influences and the protection against water loss. On the other
hand, the SC is the main barrier for dermally administered
drugs. The SC consists of corneocytes embedded in a lipid
matrix. The lipids in the SC are ceramides, cholesterol, and
free fatty acids, the ceramides representing the main
component. The distribution of the ceramides is not homoge-
neous in the lipid matrix. The most important representatives
of this lipid class are the amides of sphingosine and
phytosphingosine with normal fatty acids of varying chain
lengths (CER [NS], CER [NP]). To understand the function
of ceramides and manipulate dermal and transdermal drug
delivery, it is necessary to understand the molecular
superlattice of the lipid matrix. This can be achieved by
neutron scattering investigations, especially the determina-
tion of the neutron scattering length density profiles of model
SC preparations. However, the application of this method
J. Label Compd Radiopharm 2016; 59: 531–542
wileyonlinelibrary.com/journal/jlcr
Copyright © 2016 John Wiley & Sons, Ltd.
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SONNENBERGER ET AL.
In Figure 1, the synthesized ceramides with partially and
or fatty acids. Dicarboxylic acid 1 was transformed into
diester 2 using Dean‐Stark apparatus followed by a
monosaponification step yielding the monoester 3 according
to a method described first by Ställberg‐Stenhagen11 with
barium hydroxide. Afterwards, compound 3 was selectively
reduced to the corresponding methyl 9‐hydroxynonanoate
(4a) by 2 methods. First, we used borane/DMS as reagent.12
However, beside the displeasing odor of DMS, the reaction in
our hands was not satisfying because of low yield and too
many by‐products. Therefore, we used another method
according to Soai et al.13 The free acid group of the monoes-
ter was transformed into the mixed anhydride with ethyl
chloroformate. The obtained ω hydroxy ester 4a was in turn
protected by 3,4‐dihydro‐2H‐pyran to yield compound 5a.
This substance is one suitable building block for the 2
compounds 7a and 13 as already mentioned above. The
transformation of 5a into 7a was realized by a high yield
reduction with LiAlH4 to the mono protected diol 6a, which
gives 7a in 2 steps via the corresponding methanesulfonate
and nucleophilic displacement of the mesylate residue against
bromine by lithium bromide.
The synthesis of fatty acids with 22 carbon atoms requires
compound 7b (Scheme 2). 7b is commercially available,
however, at a high price. Because we needed larger amounts,
we developed a short synthesis protocol starting from the
inexpensive ethylene brassylate (8). This compound was
reduced to diol 9 with LiAlH4. After that, diol 9 was
transformed with HBr using a Dean‐Stark apparatus into
bromotridecanol 10. For the following Grignard reactions,
the free hydroxyl group had to be blocked. This was achieved
by a reaction with 3,4‐dihydro‐2H‐pyran yielding the corre-
sponding acetal 6b. Any resultant dibromide and the output
product were separated by column chromatography.
fully deuterated fatty acids are shown. Whereas type I repre-
sents ceramides with sphingosine as amino base, the structure
type II is constituted by ceramides with phytosphingosine.
2
| RESULTS AND DISCUSSION
The structures of different partially deuterated and
perdeuterated synthesized CER [NS] and [NP] are shown in
Figure 1, as already mentioned above. The syntheses of the
precursors are shown in Schemes 1 to 4; those for the
deuterated fatty acids and for the final ceramides are given
in Scheme 5.
Perdeuterated lauric acid 15d and stearic acid 15e were
commercially acquired, while perdeuterated lignoceric acid
15f was prepared according to a modified procedure from
the literature.9 For that, the normal tetracosanoic acid 16
was heated in a hydrogenation apparatus under hydrothermal
conditions at 195°C and 15 bar in D2O for 100 hours. The
crude perdeuterated fatty acid was then purified by column
chromatography. The deuteration grade was determined to
be 96% by high‐resolution mass spectrometry (HR MS).10
For the construction of the corresponding terminally deuter-
ated fatty acids 15a to c (Scheme 5), our synthesis strategy
is composed of 2 separate structures, which were connected
by a coupling reaction. These 2 structures are on the one hand
THP blocked ω bromo alcohols 7a to c and on the other hand
terminally deuterated nonanylbromide 13. The coupling of
the protected deuterated fatty alcohols and the further trans-
formation of these alcohols into deuterated fatty acids and
the final ceramides I and II are shown in Scheme 5. The
starting materials for the free acids were 2 dicarboxylic acids,
or a derivative and a macrocyclic lactone. However, some
intermediates in our synthetic process are commercially
available, for instance, 9‐bromononanol as precursor of 7a.
Because 5a is not only a building block for 7a but also for
compound 13, we decided to start from azelaic acid, which
is inexpensive.
In Scheme 3, the preparation pathway of compound 7c as
non‐deuterated building block for the coupling reaction to the
fatty acid is described. Therefore, cyclopentadecanoate (11)
was transformed into ω hydroxy ester 4b by reaction with
methanol according to the literature.14 4b was converted to
7c following the same procedure described for the prepara-
tion of 7a starting from 4a. All bromides 7a to c were care-
fully purified by column chromatography before use in the
Grignard reaction.
Following Scheme 1, azelaic acid was used in the synthe-
sis of compounds 7a and 13 as 2 intermediates for the
coupling reactions to the terminally deuterated fatty alcohols
FIGURE
1
Synthesized
ceramides
with
perdeuterated and terminally deuterated labelled
N‐acyl residues

SONNENBERGER ET AL.
533
mesylation of the originated alcohol function. The mesylate
was not cleaned in between but immediately reacted further
to give the deuterated methyl group with lithium aluminum
deuteride.19 For the conversion of compound 12 into
nonylbromide 13, the method of Meyer‐Schwartz et al was
used.20
The terminally protected bromides 7a to c were
transformed into the Grignard‐reagents in THF. Below −5°C
these Grignard compounds were coupled with bromide 13
yielding 3 protected long chain alcohols with a triple deutera-
tion at the terminal carbon atom.21,22 To our knowledge, this
coupling reaction was not used for the synthesis of ω deuter-
ated fatty acids before. After removal of the protecting group,
the resulting fatty alcohols 14a to c have been oxidized with-
out further purification to the deuterated fatty acids 15a to c
(Scheme 5). For that, the corresponding alcohols were
oxidized in a different mode. The oxidation with pyridinium
dichromate seems to be a very mild method, and therefore,
we tested this reaction.23 However, as a result of this proce-
dure, we found a peak indicating the loss of 1 CH2 group in
the mass spectrum. This is a hint for α oxidation depending
on the solvent used during oxidation. The side reaction was
significant when chloroform was used in excess, but the use
of chloroform beside DMF was necessary to avoid solvation
problems. However, pure fatty acids were isolated with a
modified jones oxidation procedure.
The further reactions to the ceramides I and II are shown
in Scheme 5. While the perdeuterated fatty acids 15d,e were
acquired commercially, 15f was synthesized from the
corresponding acid 16 by reaction in D2O at 195°C on Pd/C
contact.9 The perdeuterated as well as the partially deuterated
fatty acid 15a to f were then linked to sphingosine or
phytoshpingosine. For the activation of the fatty acid, some
methods have been reported in the literature. The most
commonly used coupling agent is EEDQ.24,25 Other authors
have utilized EDC to form the anhydrides as reactive fatty acid
derivative4 or activated esters of the acids.26,27 A conventional
condensing agent in peptide synthesis is BOP® or PyBOP® as
the non‐toxic form.28 Because ceramides contain also an
amide bond, we used this reagent successfully and obtained
high to very high yields of compounds I and II. To our
knowledge, the use of PyBOP® in the ceramide synthesis
has not been previously described. The ceramides of the
sphingosines (Ia‐f) were precipitated with heptane, while the
ceramides from phytosphingosine (IIa‐f) precipitated virtu-
ally completely during the reaction.
SCHEME 1 Synthesis of 2‐[(9‐bromononyl)oxy]tetrahydro‐2H‐pyran (7a)
SCHEME
2
Synthesis of 2‐[(13‐bromotridecyl)oxy]tetrahydro‐2H‐pyran
(7b)
As mentioned above, compound 5a serves as starting
material for the preparation of the terminally deuterated
nonylbromide as coupling partner for compound 7a to c
(Scheme 4). For the introduction of the terminal CD3‐group
in deuterated compounds, some methods have been
described. Beside the reduction of a terminal trichloromethyl
group using zinc,15 the anodic coupling of deuterated acetic
acid with mono esters of dicarboxylic acids is described.16,17
Terminally deuterated fatty acids were also prepared by
alkylating the silver salts of carboxylic esters with a terminal
triple bond with CD3I followed by reduction of the triple
bond.15 Other authors used the reaction of acid chlorides with
enamines (Hünig method) or with organocadmium
reagents.18 However, the most common method for the intro-
duction of a terminal CD3 group is the successive reduction
of the ester group by lithium aluminum deuteride. Thus, ester
5a is reduced with lithium aluminum deuteride followed by
SCHEME
3
Synthesis of 2‐[(15‐bromopentadecyl)oxy]tetrahydro‐2H‐
3
| EXPERIMENTAL
pyran (7c)
3.1 | General
Perdeuterated lauric acid‐d23 and stearic acid‐d35 were
purchased from Sigma‐Aldrich (St. Louis, MO, USA).
Lithium aluminium deuteride was obtained from ARMAR
SCHEME 4 Synthesis of 1‐bromo‐9,9,9‐d3‐nonane (13)

534
SONNENBERGER ET AL.
SCHEME
5
Synthesis of the deuterated fatty
acids and ceramides NS and NP
(Europe) GmbH (Leipzig, Germany). PyBOP® was pur-
chased from Carbolution Chemicals GmbH (Saarbrücken,
Germany). Sphingosine and phytosphingosine were obtained
from Evonik Goldschmidt GmbH (Essen, Germany) and
were purified before use. All other chemicals and solvents
were purchased from Sigma‐Aldrich (St. Louis, MO, USA),
and the solvents were dried before use. For column chroma-
tography, silica gel 60 (0.063‐0.200 mm) was obtained from
Merck (Darmstadt, Germany). TLC plates were received
from Macherey‐Nagel (Düren, Germany) and were pre‐
the organic layer was washed with water (100 mL), with 5%
aqueous KOH (100 mL) and with brine (100 mL). The ether
layer was dried over Na2SO4 and concentrated to dryness
under reduced pressure. The crude residue was purified by
column chromatography using gradient technique with
heptane/Et2O gradient to give diester 2 (116.6 g, 95%) as a
colorless oil. Rf = 0.50 (CHCl3); 1H NMR (400 MHz,
CDCl3): δ (ppm) 1.28‐1.32 (m, 6H, –(CH2)2(CH2)3(CH2)
2–), 1.56‐1.64 (m, 4H, 2× –CH2CH2COO–), 2.28 (t,
J = 7.5 Hz, 4H, 2× –CH2COO–), 3.65 (s, 6H, 2× –CH3);
13C NMR (100 MHz, CDCl3): δ (ppm) 25.0, 29.0, 29.1,
34.2, 51.6, 174.3; EI MS (m/z): 217 [M]+, 185
[M − OCH3]+; elemental analysis calcd (%) for C11H20O4
(216.27): C 61.09, H 9.32; found: C 60.93, H 9.26.
coated aluminium sheets ALUGRAM® Xtra SIL G/UV254
.
The TLC detection was obtained by using bromothymol blue
solution. Melting points were determined on a Boetius appa-
ratus and were not corrected. For mass spectrometry, a
Finnigan MAT 710C (Thermoseparation Products, San Jose,
CA, USA) for ESI MS, an LTQ‐Orbitrap XL hybrid mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany)
for HR MS, and an MS 5971 A (Hewlett‐Packard) for GC–
MS were used. 1H NMR and 13C NMR spectra were obtained
on Varian Gemini 2000 and Varian Inova 500 instruments.
CDCl3, CD3OD, THF‐d8, and mixtures thereof were used.
On a CHNS‐932 (Leco Corporation, St. Joseph, MI, USA)
the elemental analyses were obtained. For HPLC an HP
1100 Agilent (Agilent Technologies, Waldbronn, Germany)
was used with an ELSD 2000 Alltech (Grace Davison,
Columbia, MD, USA) detector and a Nucleodur 100‐5
125 × 2 column from Macherey‐Nagel (Düren, Germany).
3.2.2
| 9‐Methoxy‐9‐oxononanoic acid (3)
Dimethyl azelate (2) (116.6 g, 0.54 mol) was dissolved in
methanol (300 mL). Under stirring a solution of Ba(OH)2
(85 g, 0.27 mol, 0.5 eq.) in methanol (500 mL) was added.
The mixture was allowed to stand for 12 hours at room
temperature. Then, the formed precipitate was filtered by
suction and washed 3 times with diethyl ether (150 mL).
The precipitate was taken up with a solution of diluted HCl
(20%) (500 mL). The water layer was extracted 3 times with
diethyl ether (250 mL). The combined ether layers were
washed twice with 5% aqueous K2CO3 solution
(150 mL), with water (150 mL), with saturated NH4Cl,
dried over Na2SO4, and concentrated under reduced pres-
sure. The crude residue was purified by column chromatog-
raphy using gradient technique with CHCl3/Et2O gradient
to give monoester 3 (64.4 g, 59%) as a colorless oil.
3.2 | Synthetic procedures
3.2.1
| Dimethyl azelate (2)
A mixture of azelaic acid (1) (106.7 g, 0.57 mol), methanol
(85.2 mL, 2.1 mol, 4 eq.), CH2Cl2 (100 mL), and conc.
sulfuric acid (5 g) was heated in a Dean‐Stark apparatus to
remove the produced water. When no more water was sepa-
rated, most of the solvent was evaporated under reduced pres-
sure. The crude diester was taken up in Et2O (500 mL). Then,
1
Rf = 0.38 (CHCl3/Et2O 8/2); H NMR (400 MHz, CDCl3):
δ (ppm) 1.27‐1.34 (m, 6H, –(CH2)2(CH2)3(CH2)2–), 1.56‐
1.64 (m, 4H, 2× –CH2CH2COO–), 2.26‐2.33 (m, 4H, 2×
–CH2COO–), 3.64 (s, 3H, –CH3); 13C NMR (100 MHz,
CDCl3): δ (ppm) 24.7, 24.9, 28.9‐29.0, 34.1, 51.6, 174.4,

SONNENBERGER ET AL.
535
180.1; ESI MS (m/z): 201.2 [M − H]−; elemental analysis
calcd (%) for C10H18O4 (202.24): C 59.38, H 8.97; found:
C 59.27, H 8.98.
3.2.4 | Methyl 15‐hydroxypentadecanoate (4b)
Compound 4b was prepared from the 15‐pentadecanolide
11 in accordance with literature.14 The methyl 15‐
hydroxypentadecanoate 4b was used without further charac-
terization and purification. ESI MS (m/z): 273.0 [M − H]−.
3.2.3
|
Methyl 9‐hydroxynonanoate (4a)
3.2.3.1
|
Method A
3.2.5
| Methyl 9‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]nonanoate
Compound 3 (22.6 g, 0.11 mol) was dissolved in abs.
THF (60 mL). The solution was cooled to −20°C, and a
solution of borane/dimethylsulfide (10.4 mL, 0.11 mol,
1 eq.) in abs. THF (100 mL) was added dropwise over
30 minutes under stirring. The stirring was continued at
room temperature for 4 hours. For working up, water
(200 mL) was added over 10 minutes. K2CO3 (26.2 g,
0.19 mol) and Et2O (200 mL) were added. The organic
layer was separated, and the water phase was extracted
twice with Et2O (150 mL). The combined organic layers
were washed with brine (200 mL), dried over Na2SO4,
and concentrated to dryness under reduced pressure. The
crude residue was purified by column chromatography
using gradient technique with CHCl3/Et2O gradient to give
the methyl 9‐hydroxynonanoate 4a (10.1 g, 48%) as a
colorless oil.
(5a)29
A solution of methyl 9‐hydroxynonanoate (4a) (24.5 g,
130 mmol), DHP (21.2 mL, 234 mmol, 1.8 eq.), and catalytic
amounts of PPTS in CH2Cl2 (300 mL) was stirred at room
temperature. For working up, water (150 mL) was added,
and the organic layer was separated. The water phase was
extracted twice with CH2Cl2 (50 mL). The combined organic
layers were washed with brine (150 mL), dried over Na2SO4,
and concentrated to dryness under reduced pressure. The
crude residue was purified by column chromatography using
gradient technique with heptane/Et2O and TEA (10 drops per
100 mL) eluent to give the methyl 9‐[(tetrahydro‐2H‐pyran‐
2‐yl)oxy]nonanoate (5a) (35.1 g, 99%) as a colorless oil.
1
Rf = 0.41 (CHCl3/Et2O 8/2); H NMR (400 MHz, CDCl3):
δ
(ppm) 1.26‐1.36 (m, 8H, –(CH2)4(CH2)2COO–),
1.48‐1.63 (m, 8H, –(CH2)2CH2CH–OCH2CH2(CH2)6–,
–CH2CH2COO–), 1.68‐1.74 (m, 1H, –OCH–CHH′–), 1.78‐
1.88 (m, 1H, –OCH–CHH′–), 2.29 (t, J = 7.5 Hz, 2 H,
–CH2COO–), 3.37 (dt, J = 9.6 Hz, J = 6.7 Hz, 1H,
–OCHH′(CH2)6–), 3.46‐3.52 (m, 1H, –OCH–(CH2)3CHH
3.2.3.2
|
Method b
Compound 3 (20.73 g, 0.1 mol) and TEA (14.3 mL, 0.1 mol,
1 eq.) were dissolved in abs. THF (150 mL). The solution was
cooled to −10°C, and ethyl chloroformate (9.8 mL, 0.1 mol,
1 eq.) was added dropwise over 45 minutes. The stirring was
continued at −10°C for additional 30 minutes. The precipi-
tate was removed by inert filtration. The precipitate was
washed twice with abs. THF (30 mL). The combined THF
phases were cooled down to −5°C, and NaBH4 (14.73 g,
0.39 mol, 3.8 eq.) was added. Over a period of 2 hours, meth-
anol (61.5 mL, 1.5 mol) was added dropwise. The stirring
was continued at −5°C for 1 hour and then at room tempera-
ture for 12 hours. For working up, 5 N HCl (100 mL) was
added dropwise, followed by adding water (100 mL). The
water layer was extracted 3 times with CHCl3 (200 mL).
The combined organic layers were washed with 5%
aqueous KOH (100 mL), aqua (100 mL), brine (100 mL),
dried over Na2SO4, and concentrated to dryness under
reduced pressure. The crude residue was purified
by column chromatography using gradient technique
with CHCl3/Et2O gradient to give the methyl 9‐
hydroxynonanoate 4a (15.7 g, 81%) as a colorless oil.
′–), 3.66 (s, 3H, –CH3), 3.71 (dt,
J = 9.6 Hz,
J = 6.9 Hz, 1H, –OCHH′(CH2)6–), 3.83‐3.89 (m, 1H,
–OCH–(CH2)3CHH′–), 4.55‐4.57 (m, 1H, –OCH–O–); 13C
NMR (100 MHz, CDCl3): δ (ppm) 19.9, 25.1, 25.7, 26.3,
29.2‐29.9, 30.9, 34.2, 51.6, 62.5, 67.8, 99.0, 174.4; ESI MS
(m/z): 295.1 [M + Na]+; elemental analysis calcd (%) for
C15H28O4 (272.37): C 66.14, H 10.36; found: C 66.14,
H 10.64.
3.2.6
| Methyl 15‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]
pentadecanoate (5b)29
Compound 5b was obtained following the procedure for
compound 5a. Methyl 15‐hydroxypentadecanoate (4b)
(81.4 g, 0.30 mol) was converted into methyl 15‐
[(tetrahydro‐2H‐pyran‐2‐yl)oxy]pentadecanoate
(5b)
1
(103.6 g, 97%) as a colorless oil. Rf = 0.43 (CHCl3); H
NMR (400 MHz, CDCl3): δ (ppm) 1.23‐1.34 (m, 20H, –
(CH2)10(CH2)2COO–), 1.48‐1.63 (m, 8H, –(CH2)2CH2CH–
OCH2CH2(CH2)12–, –CH2CH2COO–), 1.66‐1.72 (m, 1H, –
OCH–CHH′–), 1.77‐1.85 (m, 1H, –OCH–CHH′–), 2.28 (t,
J = 7.5 Hz, 2H, –CH2COO–), 3.36 (dt, J = 9.4 Hz,
J = 6.7 Hz, 1H, –OCHH′(CH2)13–), 3.45‐3.50 (m, 1H, –
OCH–(CH2)3CHH′–), 3.66 (s, 3H, –CH3), 3.71 (dt,
J = 9.6 Hz, J = 6.9 Hz, 1H, –OCHH′(CH2)13–), 3.82‐3.87
(m, 1H, –OCH–(CH2)3CHH′–), 4.54‐4.56 (m, 1H, –OCH–
O–); 13C NMR (100 MHz, CDCl3): δ (ppm) 19.8, 25.1,
25.6, 26.4, 29.3‐29.9, 30.9, 34.2, 51.5, 62.4, 67.8, 98.9,
1
Rf = 0.32 (CHCl3/Et2O 8/2); H NMR (400 MHz, CDCl3):
δ (ppm) 1.21‐1.32 (m, 8H, HO(CH2)2(CH2)4(CH2)2–),
1.48‐1.62 (m, 4H, HOCH2CH2–, –CH2CH2COO–), 2.26
(t, J = 7.5 Hz, 2 H, –CH2COO–), 3.58 (t, J = 6.6 Hz,
2H, HOCH2–), 3.63 (s, 3H, –CH3); 13C NMR (100 MHz,
CDCl3): δ (ppm) 25.0, 25.7, 29.1‐29.3, 32.8, 34.1, 51.5,
62.9, 174.4; ESI MS (m/z): 189.0 [M + H]+; elemental
analysis calcd (%) for C10H20O3 (188.26): C 63.80, H
10.71; found: C 63.59, H 10.72.

536
SONNENBERGER ET AL.
174.4; ESI MS (m/z): 379.2 [M + Na]+; elemental analysis
calcd (%) for C21H40O4 (356.53): C 70.74, H 11.31; found:
C 70.78, H 11.43.
C20H40O3 (328.52): C 73.12, H 11.27; found: C 73.11,
H 11.54.
3.2.9
| Tridecane‐1,13‐diol (9)
Compound 9 was prepared from ethylene brassylate (8) by
reduction with LiAlH4. The tridecane‐1,13‐diol (9) was used
without further characterization and purification.
3.2.7
| 9‐[(Tetrahydro‐2H‐pyran‐2‐yl)oxy]nonan‐1‐ol (6a)
A solution of compound 6a (13.62 g, 0.05 mol) in Et2O
(150 mL) was added dropwise to a suspension of LiAlH4
(1.85 g, 0.05 mol, 1 eq.) in Et2O (100 mL). The reaction
was heated under reflux until complete reduction. For
quenching of the reaction, water (150 mL) was added
dropwise under cooling. The mixture was stirred for further
2 hours. The organic layer was separated, and the water phase
was extracted twice with Et2O (100 mL). The combined ether
layers were washed with brine (150 mL), dried over Na2SO4,
and concentrated to dryness under reduced pressure. The
crude residue was purified by column chromatography using
gradient technique with heptane/Et2O and TEA (10 drops per
100 mL) eluent to give the 9‐[(tetrahydro‐2H‐pyran‐2‐yl)
oxy]nonan‐1‐ol (6a) (11.2 g, 92%) as a colorless oil. Rf = 0.36
3.2.10
| 13‐Bromotridecan‐1‐ol (10)
Compound 10 was prepared from tridecane‐1,13‐diol (9) in
accordance with the literature.30 13‐Bromotridecan‐1‐ol
(10) was used without further characterization and
purification.
3.2.11
|
2‐[(9‐Bromononyl)oxy]tetrahydro‐2H‐pyran (7a)31
Compound 6a (10.51 g, 0.043 mol) and TEA (6.26 mL,
0.045 mol, 1.05 eq.) were dissolved in CHCl3 (100 mL).
The mixture was cooled to 0°C, and a solution of
methanesulfonyl chloride (3.5 mL, 0.045 mol, 1.05 eq.) in
CHCl3 (25 mL) was added dropwise under strong stirring.
The mixture was warmed to room temperature and was
stirred for further 12 hours. For working up, TEA
(6.26 mL, 0.045 mol) was added before ice (100 g) was
added. The mixture was stirred for further 2 hours. The
organic layer was separated, and the water phase was
extracted 3 times with CHCl3 (50 mL). The combined
organic layers were washed with brine (150 mL), dried over
Na2SO4, concentrated to dryness under reduced pressure,
and were dried over P2O5. The crude residue was dissolved
in acetone (80 mL). Dry LiBr (9.34 g, 0.108 mol) was added,
and the mixture was heated under reflux for 6 hours. For
working up, most of the solvent was evaporated. The residue
was poured into ice. The precipitate was extracted 3 times
with Et2O (100 mL). The combined ether phases were
washed twice with brine (100 mL), dried over Na2SO4, and
concentrated to dryness under reduced pressure. The crude
residue was purified by column chromatography using
gradient technique with heptane/Et2O and TEA (10 drops
per 100 mL) eluent to give the 2‐[(9‐bromononyl)oxy]
tetrahydro‐2H‐pyran (7a) (10.6 g, 80%) as a colorless oil.
Rf = 0.47 (CHCl3/heptane 6/4); 1H NMR (400 MHz, CDCl3):
δ (ppm) 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 = 9.5 Hz,
J = 6.9 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 (ppm) 19.9, 25.7, 26.3, 28.3,
28.3, 29.5, 29.9, 30.9, 33.0, 34.1, 62.5, 67.8, 99.0; EI‐MS
(m/z): 305/307 [M − H]+; elemental analysis calcd (%)
for C14H27BrO2 (307.25): C 54.72, H 8.86; found: C
54.91, H 9.06.
1
(CHCl3/Et2O 8/2); H NMR (400 MHz, CDCl3): δ (ppm)
1.30‐1.35 (m, 10H, HO(CH2)2(CH2)5–), 1.48‐1.61 (m, 8H,
HOCH2CH2–, –CH2CH2OCH–CH2(CH2)2–), 1.67‐1.73
(m, 1H, –OCH–CHH′–), 1.77‐1.88 (m, 1H, –OCH–CHH
′–), 3.37 (dt, J = 9.6 Hz, J = 6.7 Hz, 1H, –(CH2)8CHH′
O–), 3.46‐3.52 (m, 1H, –OCH–(CH2)3CHH′–), 3.61 (t,
J = 6.7 Hz, 2H, HOCH2–), 3.71 (dt, J = 9.6 Hz,
J = 6.9 Hz, 1H, –(CH2)8CHH′O–), 3.83‐3.88 (m, 1H,
–OCH–(CH2)3CHH′–), 4.55‐4.57 (m, 1H, –OCH–O–); 13C
NMR (100 MHz, CDCl3): δ (ppm) 19.8, 25.6, 25.9, 26.3,
29.5‐29.9, 30.9, 32.9, 62.5, 63.1, 67.8, 99.0; ESI MS (m/z):
267.1 [M + Na]+; elemental analysis calcd (%) for
C14H28O3 (244.36): C 68.81, H 11.55; found: C 68.69,
H 11.56.
3.2.8
(6b)
| 15‐[(Tetrahydro‐2H‐pyran‐2‐yl)oxy]pentadecan‐1‐ol
Compound 6b was obtained following the procedure for
compound 6a. Methyl 15‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]
pentadecanoate (5b) (17.83 g, 0.05 mol) was converted into
15‐[(tetrahydro‐2H‐pyran‐2‐yl)oxy]pentadecan‐1‐ol
(6b)
(14.46 g, 88%) as a white waxy solid. Rf = 0.38 (CHCl3/Et2O
8/2); mp 29°C; 1H NMR (400 MHz, CDCl3): δ (ppm)
1.26‐1.38 (m, 22H, HO(CH2)2(CH2)11–), 1.48‐1.62 (m, 8H,
HOCH2CH2–, –CH2CH2OCH–CH2(CH2)2–), 1.68‐1.74 (m,
1H, –OCH–CHH′–), 1.80‐1.87 (m, 1H, –OCH–CHH′–),
3.38 (dt, J = 9.6 Hz, J = 6.7 Hz, 1H, –(CH2)14CHH′O–),
3.47‐3.52 (m, 1H, –OCH–(CH2)3CHH′–), 3.64 (t,
J = 6.7 Hz, 2H, HOCH2–), 3.72 (dt, J = 9.6 Hz,
J = 6.9 Hz, 1H, –(CH2)14CHH′O–), 3.85‐3.89 (m, 1H,
–OCH–(CH2)3CHH′–), 4.57‐4.58 (m, 1H, –OCH–O–); 13C
NMR (100 MHz, CDCl3): δ (ppm) 19.9, 25.7, 25.9, 26.4,
29.6‐29.9, 31.0, 33.0, 62.5, 63.3, 67.9, 99.0; ESI MS (m/z):
351.2 [M + Na]+; elemental analysis calcd (%) for

SONNENBERGER ET AL.
537
3.2.12
|
2‐[(13‐Bromotridecyl)oxy]tetrahydro‐2H‐pyran
gradient technique with heptane/Et2O and TEA (10 drops
per 100 mL) eluent to give the 2‐(9,9,9‐d3‐nonyloxy)
tetrahydro‐2H‐pyran (12) (8.83 g, 64%) as a colorless oil.
(7b)29
Compound 7b was prepared from 13‐bromotridecan‐1‐ol
(10) in accordance with literature. The analytical data of 2‐
[(13‐bromotridecyl)oxy]tetrahydro‐2H‐pyran (7b) are in
accordance with the reported in the literature.32 2‐[(13‐
Bromotridecyl)oxy]tetrahydro‐2H‐pyran (7b) was used with-
out further characterization. ESI MS (m/z): 385.0/387.1
[M + Na]+.
1
Rf = 0.61 (CHCl3); H NMR (400 MHz, CDCl3): δ (ppm)
1.25‐1.36 (m, 12H, D3C(CH2)6–), 1.48‐1.62 (m, 6H, –
CH2CH2OCH–CH2(CH2)2–), 1.67‐1.74 (m, 1H, –OCH–
CHH′–), 1.78‐1.86 (m, 1H, –OCH–CHH′–), 3.37 (dt,
J = 9.6 Hz, J = 6.7 Hz, 1H, D3C(CH2)7CHH′–), 3.46‐
3.54 (m, 1H, –OCH–(CH2)3CHH′–), 3.72 (dt, J = 9.6 Hz,
J = 6.9 Hz, 1H, D3C(CH2)7CHH′–), 3.83‐3.89 (m, 1H, –
OCH–(CH2)3CHH′–), 4.55‐4.57 (m, 1H, –OCH–O–); 13C
NMR (100 MHz, CDCl3): δ (ppm) 19.8, 22.5, 25.7, 26.4,
29.4, 29.6, 29.7, 29.9, 30.9, 31.9, 62.4, 67.8, 99.0; ESI MS
(m/z): 254.2 [M + Na]+; GC‐MS: tR = 12.2 minutes,
purity > 99.5%, (m/z) 230 [10%, M − H+].
3.2.13
| 2‐[(15‐Bromopentadecyl)oxy]tetrahydro‐2H‐pyran
(7c).31
Compound 7c was obtained following the procedure for
compound 7a. 15‐[(Tetrahydro‐2H‐pyran‐2‐yl)oxy]
pentadecan‐1‐ol (6b) (13.80 g, 0.042 mol) was converted
into 2‐[(15‐bromopentadecyl)oxy]tetrahydro‐2H‐pyran (7c)
(12.5 g, 76%) as a colorless oil. The analytical data of
compound 7c are in accordance with the reported in the liter-
ature.33 ESI MS (m/z): 413.1/415.1 [M + Na]+.
3.2.15
|
1‐Bromo‐9,9,9‐d3‐nonane (13)20
To a stirred solution of triphenylphosphine (21.35 g,
0.082 mol) in CH2Cl2 (200 mL), a solution of bromine
(4.2 mL, 0.082 mol) in CH2Cl2 (40 mL) was added at a
temperature of 0°C. The white colored mixture was stirred
for further 30 minutes before a solution of compound 12
(8.83 g, 0.038 mol) was added. The reaction was stirred for
a 12 hours at room temperature. The CH2Cl2 phase was
washed twice with water (50 mL), dried over Na2SO4, and
concentrated to dryness under reduced pressure. The residue
was absorbed on silica gel before purifying by column chro-
matography with heptane eluent to give the 1‐bromo‐9,9,
9‐d3‐nonane (13) (8.02 g, 89%) as a colorless oil. Rf = 0.60
(CHCl3/heptane 2/8); 1H NMR (400 MHz, CDCl3): δ
(ppm) 1.27‐1.32 (m, 10H, D3C(CH2)5–), 1.39‐1.44 (m, 2H,
–CH2(CH2)2Br), 1.82‐1.89 (m, 2H, –CH2CH2Br), 3.40 (t,
J = 6.9 Hz, 2H, –CH2Br); 13C NMR (100 MHz, CDCl3): δ
(ppm) 22.5, 28.3, 28.9, 29.4, 29.6, 31.9, 33.0, 34.2; GC‐MS:
tR = 7.5 minutes, purity > 99.5%, (m/z) 209/211 [2%, M+],
135/137 [100%, Br(CH2)4+].
3.2.14
|
2‐(9,9,9‐d3‐Nonyloxy)tetrahydro‐2H‐pyran (12)19
A solution of compound 5a (15.62 g, 0.057 mol) in Et2O
(65 mL) was added dropwise to a suspension of LiAlD4
(1.44 g, 0.034 mol, 0.6 eq.) in Et2O (50 mL). The mixture
was heated until complete conversion. For working up, D2O
(3.5 mL) was added dropwise under ice cooling. The mixture
was stirred for 2 hours. The precipitate was removed via fil-
tering by suction. The filter was washed 3 times with Et2O
(10 mL). The combined ether phases were washed with water
(70 mL), brine (70 mL), dried over Na2SO4, concentrated to
dryness under reduced pressure, and dried over P2O5. The
crude residue of the alcohol and TEA (8.6 mL, 0.062 mol)
were dissolved in CHCl3 (100 mL). The mixture was cooled
to 0°C, and a solution of methanesulfonyl chloride (4.8 mL,
0.062 mol) in CHCl3 (50 mL) was added dropwise under
stirring. The mixture was warmed to room temperature and
was stirred for further 12 hours. For working up, TEA
(8.6 mL, 0.062 mol) was added before ice water (50 mL)
was added. The mixture was stirred for further 2 hours. The
organic layer was separated, and the water phase was
extracted 3 times with CHCl3 (50 mL). The combined organic
layers were washed with brine (150 mL), dried over Na2SO4,
concentrated to dryness under reduced pressure, and dried
over P2O5. A solution of the dried mesylate in Et2O (75 mL)
was dropped into a suspension of LiAlD4 (1.34 g, 32 mmol)
in Et2O (25 mL). The mixture was heated until complete
conversion. For working up, the reaction was cooled to 0°C.
Water (3.5 mL) was added dropwise, and the mixture was
stirred for further 2 hours. The precipitate was removed via
filtering by suction. The filter was washed 3 times with
Et2O (10 mL). The combined ether phases were washed with
water (50 mL), brine (50 mL), dried over Na2SO4, and
concentrated to dryness under reduced pressure. The crude
residue was purified by column chromatography using
3.3 | General procedure for the synthesis of the ω,ω,ω‐
d3‐alkan‐1‐ol (14a,b,c).21,22
A solution of compound 7a,b,c (8 mmol) in abs. THF
(20 mL) was dropped to magnesium turnings (0.40 g,
16.4 mmol) under argon atmosphere. The mixture was
warmed at 50°C for 3 hours. The magnesium excess was
removed. The Grignard solution was cooled to −5°C before
a solution of compound 13 (1.47 g, 7 mmol) in abs THF
(50 mL) and a solution of freshly prepared Li2CuCl4 in abs
THF (1.4 mL, 0.1M) were added under stirring. The mixture
was stirred for 3 hours at −10°C. For working up, a cold sat-
urated NH4Cl solution (50 mL) was added, and the mixture
was extracted 3 times with ether (50 mL). The combined
ether phases were washed with brine, dried over Na2SO4,
and concentrated to dryness under reduced pressure. The
residue was dissolved in 30 mL MeOH. Catalytic amounts
of PPTS were added. The mixture was heated under reflux

538
SONNENBERGER ET AL.
for 3 hours. Afterwards, the mixture was cooled to −20°C for
full crystallization. The formed precipitated was filtered off
to give the ω,ω,ω‐d3‐alkan‐1‐ols (14a,b,c).
24.9, 29.2‐29.9, 32.0, 34.1, 179.6; ESI MS (m/z): 286.6
[M − H]−, 573.3 [2M − H]−, 595.7 [2M − 2H − Na]−.
3.4.2
| 22,22,22‐d3‐Docosanoic acid (15b)
Yield: (0.54 g, 79%); white crystalline solid; Rf = 0.51
(CHCl3/Et2O 8/2); mp 76°C; H NMR (400 MHz, CDCl3):
δ (ppm) 1.26‐1.35 (m, 36H, D3C(CH2)18–), 1.61‐1.66 (m,
3.3.1
| 18,18,18‐d3‐Octadecan‐1‐ol (14a)
1
Yield: (1.05 g, 55%); white crystalline solid; Rf = 0.27
(CHCl3); mp 54°C; H NMR (400 MHz, CDCl3): δ (ppm)
1
2H, –CH2CH2COOH), 2.35 (t,
J = 7.5 Hz, 2H,
1.26‐1.43 (m, 30H, D3C(CH2)15–), 1.53‐1.60 (m, 2H,
–CH2CH2OH), 3.44 (t, J = 6.6 Hz, 2H, –CH2OH); 13C
NMR (100 MHz, CDCl3): δ (ppm) 22.6, 25.9, 29.5‐29.9,
32.0, 33.0, 63.3; GC‐MS: tR = 17.24 minutes, purity > 99.5%,
(m/z) 273 [1%, M+], 227 [5%, M–C3H4D3+].
–CH2COOH); 13C NMR (100 MHz, THF‐d8): δ (ppm)
23.5, 26.0, 30.3‐30.8, 33.0, 34.4, 174.6; ESI MS (m/z):
685.4 [2M − H]−.
3.4.3
| 24,24,24‐d3‐Tetracosanoic acid (15c)
Yield: (0.60 g, 84%); white crystalline solid; Rf = 0.51
(CHCl3/Et2O 8/2); mp 80.5°C; 1H NMR (400 MHz, CDCl3):
δ (ppm) 1.26‐1.36 (m, 40H, D3C(CH2)20–), 1.60‐1.67 (m,
3.3.2
| 22,22,22‐d3‐Docosan‐1‐ol (14b)
Yield: (1.11 g, 48%); white crystalline solid; Rf = 0.37
(CHCl3); mp 68°C; H NMR (400 MHz, CDCl3): δ (ppm)
1.26‐1.45 (m, 38H, D3C(CH2)19–), 1.53‐1.60 (m, 2H,
–CH2CH2OH), 3.64 (t, J = 6.6 Hz, 2H, –CH2OH); 13C
NMR (100 MHz, CDCl3): δ (ppm) 22.6, 25.9, 29.5‐29.9,
32.0, 33.0, 63.3; GC–MS: tR = 17.03 minutes, purity > 95%,
(m/z) 328 [2%, M − H+].
1
2H, –CH2CH2COOH), 2.35 (t,
J = 7.5 Hz, 2H,
–CH2COOH); 13C NMR (100 MHz, THF‐d8): δ (ppm)
23.5, 26.0, 30.3‐30.8, 33.0, 34.4, 174.6; ESI MS (m/z):
370.8 [M − H]−, 763.9 [2M + Na − 2H]−.
3.4.4
|
Tetracosanoic acid‐d47 (15f)9
A mixture of tetracosanoic acid 16 (3 g, 8.1 mmol), KOH
(530 mg, 9.4 mmol), D2O (100 mL), and Pd/C (10%)
(300 mg) was stirred under argon atmosphere at 195°C and
15 bar for 100 hours. For working up, the mixture was
acidified with conc. DCl (1 mL), and CHCl3 (200 mL) was
added. The mixture was filtrated to remove Pd/C. Afterwards,
the organic layer was separated, and then the aqueous phase
was extracted twice with CHCl3 (100 mL). The combined
organic layers were dried over Na2SO4 and concentrated to
dryness under reduced pressure. The crude residue was
purified by column chromatography using CHCl3/MeOH
eluent to give the tetracosanoic acid‐d47 (15d) (2.81 g, 83%)
as a white, crystalline solid.
3.3.3
| 24,24,24‐d3‐Tetracosan‐1‐ol (14c)
Yield: (1.28 g, 51%); white crystalline solid; Rf = 0.29
(CHCl3); mp 73°C; H NMR (400 MHz, CDCl3): δ (ppm)
1.26‐1.43 (m, 42H, D3C(CH2)21–), 1.53‐1.60 (m, 2H,
–CH2CH2OH), 3.64 (t, J = 6.6 Hz, 2H, –CH2OH); 13C
NMR (100 MHz, CDCl3): δ (ppm) 22.6, 25.9, 29.5‐29.9,
32.0, 33.0, 63.3; ESI MS (m/z): 735.7 [2M + Na]+;
1
3.4 | General procedure for the synthesis of the ω,ω,ω‐
d3‐alkanoic acid (15a,b,c)
CrO3 (4.4 mmol) was suspended in 10 N H2SO4 (2.22 mL).
The mixture was cooled to 0°C. A solution of the ω,ω,ω‐d3‐
alkan‐1‐ols (14a,b,c) (2 mmol) in a mixture of acetone
(50 mL) and CHCl3 (30 mL) was added dropwise over a
period of 2 hours. Afterwards, the mixture was stirred for
an additional hour under cooling. For working up, water
(20 mL) and brine (20 mL) were added, and the organic
phase was separated. The aqueous phase was extracted 3
times with CHCl3 (50 mL). The combined organic phases
were dried over Na2SO4 and concentrated to dryness under
reduced pressure. The crude residue was purified by column
chromatography using gradient technique with heptane/
CHCl3/Et2O eluent to give the ω,ω,ω‐d3‐alkanoic acids
(15a,b,c).
Rf = 0.48 (CHCl3/Et2O 8/2); mp 75°C; HR MS (m/z)
−
[M
H]− C24H1D46O2: calculated 413.6469; found
413.6483.
3.5 | General procedure for coupling of the fatty acids
and sphingoid bases to the ceramides28
The fatty acid (15a‐f) (1 eq.) and PyBOP® (1.1 eq.) were
given to CH2Cl2 (5 mL/0.5 mmol acid). DIPEA (2 eq.) was
added under stirring to the mixture. After 15 minutes, sphin-
gosine or phytosphingosine (1.1 eq.) was added to the clear
solution. The mixture was stirred for 12 hours.
For working up of the sphingosine containing ceramides
(Ia‐f), heptane (10 mL/0.5 mmol acid) was added to the sus-
pension for a full precipitation before filtration. The precipi-
tate was washed twice with heptane. The separated residue
was purified by column chromatography using gradient tech-
nique with CHCl3/MeOH and NH3 (0.5%) eluent.
3.4.1
| 18,18,18‐d3‐Octadecanoic acid (15a)
Yield: (0.47 g, 81%); white crystalline solid; Rf = 0.34
(CHCl3/Et2O 8/2); mp 67°C; H NMR (400 MHz, CDCl3):
δ (ppm) 1.26‐1.35 (m, 28H, D3C(CH2)14–), 1.60‐1.67 (m,
1
2H, –CH2CH2COOH), 2.35 (t,
J
=
7.5 Hz, 2H,
For working up of the phytosphingosine containing
ceramides (IIa‐f), the mixtures were evaporated to dryness.
–CH2COOH); 13C NMR (100 MHz, CDCl3): δ (ppm) 22.6,

SONNENBERGER ET AL.
539
The crude residues were absorbed on silica gel for purifying
by column chromatography using gradient technique with
CHCl3/MeOH and NH3 (0.5%) eluent.
3.71 (dd, J = 11.2 Hz, J = 3.4 Hz, 1H, –CHH′OH),
3.89‐3.93 (m, 1H, –CH–CH2OH), 3.96 (dd, J = 11.3 Hz,
J = 3.8 Hz, 1H, –CHH′OH), 4.31‐4.34 (m, 1H, –
CH=CHCHOH–), 5.53 (dd, J = 15.4 Hz, J = 6.5 Hz, 1H,
–CH=CHCHOH–), 5.76‐5.82 (m, 1H, –CH=CHCHOH–),
6.20 (d, J = 7.1 Hz, 1H, –CONH–); 13C NMR (125 MHz,
CDCl3, 40°C): δ (ppm) 14.2, 22.6, 22.8, 25.9, 29.3‐29.9,
32.0, 32.1, 32.4, 37.0, 54.8, 62.7, 74.9, 129.1, 134.5, 174.0;
ESI MS (m/z): 651.8 [M − H]−; HR MS (m/z) [M + H]+
C42H81D3NO3: calculated 653.6634; found 653.6644; HPLC
ELSD purity >98%.
3.5.1
| N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐
18,18,18‐d3‐octadecanamid (Ia)
Compound 15a (200 mg, 0.7 mmol) was converted into the
product Ia (0.32 g, 81%); white waxy solid; Rf = 0.27
(CHCl3/MeOH/NH3 95/5/1); mp 98°C; 1H NMR
(500 MHz, CDCl3): δ (ppm) 0.88 (t, J = 6.9 Hz, 3H,
–CH3), 1.26‐1.39 (m, 50H, D3C(CH2)14–, H3C(CH2)11–),
1.61‐1.67 (m, 2H, –CH2CH2CONH–), 2.04‐2.08 (m, 2H,
–CH2CH=CH–), 2.23 (t, J = 7.6 Hz, 2H, –CH2CONH–),
3.71 (dd, J = 11.3 Hz, J = 3.3 Hz, 1H, –CHH′OH),
3.89‐3.93 (m, 1H, –CH–CH2OH), 3.96 (dd, J = 11.2 Hz,
J = 3.8 Hz, 1H, –CHH′OH), 4.31‐4.33 (m, 1H, –
CH=CHCHOH–), 5.53 (dd, J = 15.4 Hz, J = 6.4 Hz, 1H,
–CH=CHCHOH–), 5.76‐5.82 (m, 1H, –CH=CHCHOH–),
6.22 (d, J = 7.3 Hz, 1H, –CONH–); 13C NMR (125 MHz,
CDCl3): δ (ppm) 14.3, 22.6, 25.9, 29.3‐29.9, 32.0, 32.1,
32.4, 37.0, 54.7, 62.7, 74.9, 129.0, 134.5, 174.0; ESI MS
(m/z): 591.6 [M + Na]+, 1159.4 [2M + Na]+, 567.7
[M − H]–; HR MS (m/z) [M + H]+ C36H69D3NO3: calculated
569.5695; found 569.5696; HPLC ELSD purity >99.5%.
3.5.4
dodecanamid (Id)
| N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐d23‐
Lauric acid‐d23 (15d) (100 mg, 0.45 mmol) was converted into
the product Id (0.17 g, 71%); white waxy solid; Rf = 0.44
(CHCl3/MeOH/NH3 95/5/1); mp 49°C; 1H NMR (500 MHz,
CDCl3): δ (ppm) 0.87 (t, J = 6.7 Hz, 3H, –CH3), 1.19‐1.37
(m, 22H, H3C(CH2)11–), 2.01‐2.05 (m, 2H, –CH2CH=CH–),
3.67 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H, –CHH′OH), 3.86‐3.93
(m,
2H,
–CH–CHH′OH),
4.25‐4.28
(m,
1H,
–CH=CHCHOH–), 5.51 (dd, J = 15.4 Hz, J = 6.4 Hz, 1H, –
CH=CHCHOH–), 5.72‐5.78 (m, 1H, –CH=CHCHOH–),
6.36‐6.40 (m, 1H, –CONH–); 13C NMR (125 MHz, CDCl3):
δ (ppm) 14.2, 22.8, 29.3‐29.8, 32.0, 32.5, 54.8, 62.5, 74.4,
129.1, 134.1, 174.2; ESI MS (m/z): 527.6 [M + Na]+; 503.7
[M − H]−, HR MS (m/z) [M + H]+ C30H37D23NO3: calculated
505.6011; found 505.6012; HPLC ELSD purity >99.5%.
3.5.2
| N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐
22,22,22‐d3‐docosanamid (Ib)
Compound 15b (200 mg, 0.58 mmol) was converted into the
product Ib (0.30 g, 83%); white waxy solid; Rf = 0.31
(CHCl3/MeOH/NH3 95/5/1); mp 97°C; 1H NMR
(400 MHz, CDCl3): δ (ppm) 0.88 (t, J = 6.8 Hz, 3H,
–CH3), 1.10‐1.39 (m, 58H, D3C(CH2)18–, H3C(CH2)11–),
1.60‐1.68 (m, 2H, –CH2CH2CONH–), 2.03‐2.09 (m, 2H,
–CH2CH=CH–), 2.23 (t, J = 7.6 Hz, 2H, –CH2CONH–),
3.71 (dd, J = 11.1 Hz, J = 3.1 Hz, 1H, –CHH′OH),
3.89‐3.93 (m, 1H, –CH–CH2OH), 3.96 (dd, J = 11.2 Hz,
J = 3.8 Hz, 1H, –CHH′OH), 4.31‐4.34 (m, 1H, –
CH=CHCHOH–), 5.53 (dd, J = 15.4 Hz, J = 6.4 Hz, 1H,
–CH=CHCHOH–), 5.75‐5.82 (m, 1H, –CH=CHCHOH–),
6.22 (d, J = 7.1 Hz, 1H, –CONH–); 13C NMR (125 MHz,
CDCl3, 40°C): δ (ppm) 14.2, 22.6, 22.8, 25.9, 29.3‐29.9,
32.0, 32.1, 32.4, 37.0, 54.8, 62.7, 74.8, 129.1, 134.5, 174.0;
ESI MS (m/z): 623.8 [M − H]−; HR MS (m/z) [M + H]+
C40H77D3NO3: calculated 625.6321; found 625.6353; HPLC
ELSD purity >99.5%.
3.5.5
| N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐d35‐
octadecanamid (Ie)
Stearic acid‐d35 (15d) (100 mg, 0.31 mmol) was converted into
product Ie (0.15 g, 79%); white waxy solid; Rf = 0.46 (CHCl3/
MeOH/NH3 95/5/1); mp 94°C; 1H NMR (500 MHz, CDCl3):
δ (ppm) 0.89 (t, J = 6.7 Hz, 3H, –CH3), 1.24‐1.41 (m, 22H,
H3C(CH2)11–), 2.02‐2.10 (m, 2H, –CH2CH=CH–), 3.72 (dd,
J = 11.0 Hz, J = 2.8 Hz, 1H, –CHH′OH), 3.89‐3.93 (m, 1H, –
CH–CH2OH), 3.96 (dd, J = 11.2 Hz, J = 3.8 Hz, 1H, –CHH′
OH), 4.31‐4.34 (m, 1H, –CH=CHCHOH–), 5.54 (dd,
J = 15.4 Hz, J = 6.4 Hz, 1H, –CH=CHCHOH–), 5.75‐5.83
(m, 1H, –CH=CHCHOH–), 6.19 (d, J = 6.5 Hz, 1H, –CONH–);
13C NMR (125 MHz, CDCl3): δ (ppm) 14.1, 22.7, 28.0‐29.7,
31.9, 32.3, 54.5, 62.5, 74.6, 128.8, 134.3, 174.0; ESI MS (m/
z): 623.6 [M + Na]+; 1223.8 [2M − Na]+; 599.2 [M − H]−;
HR MS (m/z) [M + Na]+ C36H36D35NO3Na: calculated
623.7523; found 623.7522; HPLC ELSD purity >99.5%.
3.5.3
| N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐
24,24,24‐d3‐tetracosanamid (Ic)
3.5.6
| N‐[(2S,3R,4E)‐1,3‐Dihydroxyoctadec‐4‐en‐2‐yl]‐D47‐
tetracosanamid (If)
Compound 15c (200 mg, 0.54 mmol) was converted into the
product Ic (0.27 g, 77%); white waxy solid; Rf = 0.34
(CHCl3/MeOH/NH3 95/5/1); mp 95.5°C; 1H NMR
(500 MHz, CDCl3): δ (ppm) 0.88 (t, J = 7.0 Hz, 3H,
–CH3), 1.26‐1.39 (m, 60H, D3C(CH2)20–, H3C(CH2)11–),
1.61‐1.67 (m, 2H, –CH2CH2CONH–), 2.04‐2.08 (m, 2H,
–CH2CH=CH–), 2.23 (t, J = 7.6 Hz, 2H, –CH2CONH–),
Tetracosanoic acid‐d47 (15f) (100 mg, 0.24 mmol) was
converted into the product If (0.14 g, 81%); white waxy solid;
Rf = 0.56 (CHCl3/MeOH/NH3 95/5/1); mp 93°C; H NMR
(500 MHz, CDCl3): δ (ppm) 0.89 (t, J = 6.7 Hz, 3H,
–CH3), 1.20‐1.40 (m, 22H, H3C(CH2)11–), 2.04‐2.09 (m,
2H, –CH2CH=CH–), 3.71 (dd, J = 10.9 Hz, J = 2.8 Hz,
1

540
SONNENBERGER ET AL.
1H, –CHH′OH), 3.89‐3.92 (m, 1H, –CH–CH2OH), 3.95 (dd,
J = 11.1 Hz, J = 3.8 Hz, 1H, –CHH′OH), 4.31‐4.33 (m, 1H,
–CH=CHCHOH–), 5.54 (dd, J = 15.4 Hz, J = 6.4 Hz, 1H, –
CH=CHCHOH–), 5.76‐5.83 (m, 1H, –CH=CHCHOH–),
6.18 (d, J = 7.2 Hz, 1H, –CONH–); 13C NMR (125 MHz,
CDCl3, 40°C): δ (ppm) 14.2, 22.8, 29.3‐29.9, 32.1, 32.4,
54.8, 62.7, 74.9, 129.1, 134.5, 173.9; ESI MS (m/z): 718.8
[C42H37D46NO3 + Na]+; 695.0 [C42H37D46NO3 − H]−; HR
3.5.9
|
N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐24,24,24‐
d3‐tetracosanamid (IIc)
Compound 15c (200 mg, 0.54 mmol) was converted into the
product IIc (0.32 g, 89%); white waxy solid, Rf = 0.15
(CHCl3/MeOH/NH3 95/5/1); mp 114°C; 1H NMR
(500 MHz, CDCl3, 40°C): δ (ppm) 0.89 (t, J = 6.9 Hz, 3H,
–CH3), 1.14‐1.32 (m, 64H, D3C(CH2)20–, H3C(CH2)12–),
1.44‐1.56 (m, 1H, –CHH′CHOH–), 1.61‐1.67 (m, 2H,
–CH2CH2CONH–), 1.76‐1.82 (m, 1H, –CHH′CHOH–),
2.34 (t, J = 7.4 Hz, 2H, –CH2CONH–), 3.58 (dd,
J = 7.0 Hz, J = 2.6 Hz, 1H, –CH2CHOHCHOH–), 3.61‐
3.65 (m, 1H, –CH2CHOH–), 3.76 (dd, J = 11.5 Hz,
J = 5.3 Hz, 1H, –CHH′OH), 3.95 (dd, J = 11.6 Hz,
J = 2.5 Hz, 1H, –CHH′OH), 4.12‐4.16 (m, 1H, –CH–
CH2OH), 6.29 (d, J = 5.7 Hz, 1H, –CONH–); 13C NMR
(125 MHz, CDCl3/CD3OD, 40°C): δ (ppm) 13.6, 22.2, 22.5,
25.6, 25.7 29.1‐29.6, 31.6, 31.7, 32.7, 36.3, 51.9, 61.1, 72.4,
75.4, 174.6; ESI MS (m/z): 693.7 [M + Na]+, 669.8
[M − H]−; HR MS (m/z) [M + H]+ C42H83D3NO4: calculated
671.6740; found 671.6733; HPLC ELSD purity >99.5%.
MS (m/z) [M
718.9152; found 718.9144; HPLC ELSD purity >99.5%.
+
Na]+ C42H37D46NO3Na: calculated
3.5.7
| N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐18,18,18‐
d3‐octadecanamid (IIa)
Compound 15a (200 mg, 0.7 mmol) was converted into the
product IIa(0.35 g, 85%); white waxy solid. Rf = 0.13
(CHCl3/MeOH/NH3 95/5/1); mp 125.5°C; 1H NMR
(500 MHz, CDCl3, 40°C): δ (ppm) 0.89 (t, J = 6.9 Hz, 3H,
–CH3), 1.14‐1.32 (m, 52H, D3C(CH2)14–, H3C(CH2)12–),
1.45‐1.52 (m, 1H, –CHH′CHOH–), 1.61‐1.67 (m, 2H,
–CH2CH2CONH–), 1.76‐1.82 (m, 1H, –CHH′CHOH–, over-
laid by H2O), 2.24 (t, J = 7.6 Hz, 2H, –CH2CONH–), 3.58
(dd, J = 6.7 Hz, J = 2.9 Hz, 1H, –CH2CHOHCHOH–),
3.61‐3.65 (m, 1H, –CH2CHOH–), 3.75 (dd, J = 11.6 Hz,
J = 5.3 Hz, 1H, –CHH′OH), 3.94 (dd, J = 11.5 Hz,
3.5.10
dodecanamid (IId)
| N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐d23‐
Lauric acid‐d23 (15d) (100 mg, 0.34 mmol) was converted
into the product IId (0.19 g, 82%); white waxy solid; Rf = 0.20
(CHCl3/MeOH/NH3 95/5/1); mp 117°C; 1H NMR (500 MHz,
CDCl3, 40°C): δ (ppm) 0.89 (t, J = 6.8 Hz, 3H, –CH3), 1.20‐
1.37 (m, 24H, H3C(CH2)12–), 1.44‐1.55 (m, 1H, –CHH′
CHOH–), 1.73‐1.80 (m, 1H, –CHH′CHOH–), 3.58 (dd,
J = 6.7 Hz, J = 2.9 Hz, 1H, –CH2CHOHCHOH–), 3.60‐
3.67 (m, 1H, –CH2CHOH–), 3.75 (dd, J = 11.5 Hz,
J = 5.3 Hz, 1H, –CHH′OH), 3.94 (dd, J = 11.5 Hz,
J
= 2.4 Hz, 1H, –CHH′OH), 4.12‐4.16 (m, 1H,
–CH–CH2OH), 6.30 (d, J = 7.2 Hz, 1H, –CONH–); 13C
NMR (125 MHz, CDCl3/CD3OD, 45°C): δ (ppm) 13.6,
22.1, 22.4, 25.6, 29.1‐29.4, 31.6, 31.7, 32.8, 36.3, 51.9,
61.1, 72.4, 75.5, 174.5; ESI MS (m/z): 609.5 [M + Na]+,
585.6 [M − H]−; HR MS (m/z) [M + H]+ C36H71D3NO4:
calculated 587.5801; found 587.5790; [M
+
Na]+
C36H70D3NO4Na: calculated 609.5620; found 609.5606;
HPLC ELSD purity >99.5%.
J
= 2.5 Hz, 1H, –CHH′OH), 4.10‐4.14 (m, 1H,
–CH–CH2OH), 6.29 (d, J = 7.0 Hz, 1H, –CONH–); 13C
NMR (125 MHz, CDCl3, 27°C): δ (ppm) 14.3, 23.1, 26.3,
29.8‐30.2, 32.4, 33.2, 52.5, 61.6, 72.9, 75.9, 175.4; ESI MS
(m/z): 521.7 [M − H]−, 1043.8 [2M − H]−; HR MS (m/z)
[M + H]+ C30H39D23NO4: calculated 523.6117; found
3.5.8
| N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐22,22,22‐
d3‐docosanamid (IIb)
Compound 15b (200 mg, 0.58 mmol) was converted into the
product IIb (0.34 g, 91%); white waxy solid; Rf = 0.14
(CHCl3/MeOH/NH3 95/5/1); mp 120°C; 1H NMR
(400 MHz, CDCl3, 40°C): δ (ppm) 0.89 (t, J = 6.7 Hz, 3H,
–CH3), 1.27‐1.35 (m, 60H, D3C(CH2)18–, H3C(CH2)12–),
1.48‐1.53 (m, 1H, –CHH′CHOH–), 1.61‐1.67 (m, 2H,
–CH2CH2CONH–), 1.75‐1.871 (m, 1H, –CHH′CHOH–,
overlaid by H2O), 2.24 (t, J = 7.6 Hz, 2H, –CH2CONH–),
3.59 (dd, J = 6.8 Hz, J = 2.8 Hz, 1H, –CH2CHOHCHOH–),
3.62‐3.66 (m, 1H, –CH2CHOH–), 3.76 (dd, J = 11.5 Hz,
J = 4.9 Hz, 1H, –CHH′OH), 3.94 (dd, J = 11.4 Hz,
523.6118; [M
545.5937; found 545.5936; HPLC ELSD purity >99.5%.
+
Na]+ C30H38D23NO4Na: calculated
3.5.11
octadecanamid (IIe)
| N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐d35‐
Stearic acid‐d35 (15e) (100 mg, 0.31 mmol) was converted into
the product IIe (0.16 g, 81%); white waxy solid; Rf = 0.25
(CHCl3/MeOH/NH3 95/5/1); mp 121°C; 1H NMR
(500 MHz, CDCl3, 40°C): δ (ppm) 0.89 (t, J = 6.8 Hz, 3H, –
CH3), 1.26‐1.34 (m, 24H, H3C(CH2)12–), 1.46‐1.53 (m, 1H,
–CHH′CHOH–), 1.73‐1.80 (m, 1H, –CHH′CHOH–, overlaid
by H2O), 3.58 (dd, J = 6.7 Hz, J = 2.9 Hz, 1H,
–CH2CHOHCHOH–), 3.61‐3.66 (m, 1H, –CH2CHOH–),
3.75 (dd, J = 11.5 Hz, J = 5.2 Hz, 1H, –CHH′OH), 3.93
(dd, J = 11.7 Hz, J = 1.9 Hz, 1H, –CHH′OH), 4.11‐4.15 (m,
1H, –CH–CH2OH), 6.41 (d, J = 5.7 Hz, 1H, –CONH–);13C
J
= 2.0 Hz, 1H, –CHH′OH), 4.12‐4.16 (m, 1H,
–CH–CH2OH), 6.32 (d, J = 5.9 Hz, 1H, –CONH–); 13C
NMR (100 MHz, CDCl3/CD3OD, 40°C): δ (ppm) 13.8,
22.3, 22.6, 25.7, 25.8, 29.1‐29.4, 31.8, 31.9, 33.0, 36.5,
52.0, 61.2, 72.5, 75.7, 174.6; ESI MS (m/z): 665.6
[M + Na]+, 641.7 [M − H]−; HR MS (m/z) [M + H]+
C40H79D3NO4: calculated 643.6427; found 643.6401; HPLC
ELSD purity >99.5%.

SONNENBERGER ET AL.
541
REFERENCES
NMR (125 MHz, THF‐d8, 40°C): δ (ppm) 13.4, 22.5, 25.8,
29.3‐29.8, 31.8, 33.2, 52.8, 61.5, 72.2, 76.2, 172.3; ESI MS
(m/z): 641.7 [M + Na]+, 1259.8 [2M + Na]+, 617.8 [M − H]
−; HR MS (m/z) [M + H]+ C36H29D35NO5: calculated
619.7809; found 619.7790; HPLC ELSD purity >98%.
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3.5.12
tetracosanamid (IIf)
| N‐[(2S,3S,4R)‐1,3,4‐Trihydroxyoctadec‐2‐yl]‐d47‐
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Tetracosanoic acid‐d47 (15f) (100 mg, 0.24 mmol) was
converted into the product IIf (0.15 g, 84%); white waxy
solid; Rf = 0.29 (CHCl3/MeOH/NH3 95/5/1); mp 114°C;
1H NMR (500 MHz, CDCl3, 40°C): δ (ppm) 0.89 (t,
J = 6.7 Hz, 3H, –CH3), 1.20‐1.35 (m, 24H, H3C(CH2)12–),
1.42‐1.59 (m, 1H, –CHH′CHOH–), 1.75‐1.81 (m, 1H,
–CHH′CHOH–), 3.58 (dd, J = 6.6 Hz, J = 2.9 Hz, 1H,
–CH2CHOHCHOH–), 3.60‐3.66 (m, 1H, –CH2CHOH–),
3.76 (dd, J = 11.5 Hz, J = 5.3 Hz, 1H, –CHH′OH), 3.95
(dd, J = 11.4 Hz, J = 2.2 Hz, 1H, –CHH′OH), 4.11‐4.16
(m, 1H, –CH–CH2OH), 6.28 (d, J = 8.0 Hz, 1 H, –CONH–);
13C NMR (125 MHz, THF‐d8, 35°C): δ (ppm) 14.6, 23.7,
27.0, 29.1‐31.0, 33.0, 34.5, 54.0, 62.7, 73.4, 77.4, 173.5;
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4
| CONCLUSION
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Six ceramides [NS] and six ceramides [NP] with
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specifically deuterated acids were prepared for the first time
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acids with the sphingoid bases, PyBOP® was successfully
used as the activator, and the corresponding ceramides could
be isolated in high yields. The specifically deuterated
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study
was
supported
by
the
Deutsche
Forschungsgemeinschaft (DFG DO 463/6‐1and NE 427/30‐
1) and Evonik Industries, AG. The authors thank Dr. Christian
Ihling and Prof. Dr. Andrea Sinz (Institute of Pharmacy,
Martin‐Luther‐University, Halle (Saale), Germany) for
obtaining the HRMS data and Manuela Woigk (Institute of
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CONFLICT OF INTEREST
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thesis. 1971;6:303–305.
There is no conflict of interest.

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