Synthesis of a panel of carbon-13-labelled (glyco)sphingolipids

FULL PAPER

DOI: 10.1002/ejoc.201500025

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

Patrick Wisse,^[a][‡]Henrik Gold,^[a][‡]Mina Mirzaian,^[b]Maria J. Ferraz,^[b]Ginger Lutteke,^[a]

Richard J. B. H. N. van den Berg,^[a]Hans van den Elst,^[a]Johan Lugtenburg,^[a]

Gijsbert A. van der Marel,^[a]Johannes M. F. G. Aerts,^[a,b]Jeroen D. C. Codée,*^[a]and

Herman S. Overkleeft*^[a]

Keywords: Sphingolipids / Glycolipids / Ceramides / Metathesis / Isotopic labeling

The synthesis of a focussed library of sphingolipids differing

in the number and position of ¹³C labels is described. The

synthesised sphingolipids differ in substitution at both the

sphingosine amine (either palmitoylated or unmodified) and

the sphingosine primary hydroxyl (unmodified or glycos-

ylated). Moreover, ¹³C atoms are incorporated into either the

sphingosine or the palmitate moiety, or both. This set of com-

pounds is intended for use in relative quantitative lipidomics

studies to gain insight into sphingolipid metabolism in heal-

thy and diseased (lysosomal storage disorders) patients and

animal models.

Gaucher and globotriaosylceramide in Fabry), the occur-

rence of alternative metabolic pathways.^[1–3]We also ob-

tained evidence that metabolites produced by these alterna-

tive pathways – lysoglycosphingolipids in both cases – may

be involved in or perhaps even causative in the onset and

development of the disease. We made these discoveries

thanks in part to stable-isotope-labelled (¹³C₅) sphingolip-

ids, which we synthesised for this purpose. Based on these

findings, we reasoned that a comprehensive set of sphingo-

lipids differing both in structure and in the number of ¹³C

atoms embedded in both the sphingosine and the N-acyl

(palmitate) moieties, as represented by the general structure

in the insert of Figure 1, would be a very useful set of re-

search tools. A selection of the sphingolipid biosynthetic

pathways are shown in Figure 1. At the basis of the biosyn-

thesis of all sphingolipids is sphinganine 1, itself the con-

densation product of serine and palmitate. In a reaction cat-

alysed by sphinganine acyl transferase (SAT), the free

amine in 1 is condensed with a fatty acid, here shown as

palmitate but in reality one of a number of saturated or

partially unsaturated fatty acids of varying size. In the next

step, the resulting dihydroceramide (i.e., 2) is dehydroge-

nated through the action of dihydroceramide dehydroge-

nase (DCD) to produce ceramide 3. At this stage, a number

of different pathways can take place, giving rise to a wide

variety of sphingolipids featuring different polar head

groups. Glucosylceramide (4) is the product of the glucosyl-

ceramide synthase (GCS) catalysed condensation of 3 with

UDP-glucose. Glucosylceramide (4) in turn is the starting

point for the synthesis of a wide variety of glycosphingolip-

ids and gangliosides featuring oligosaccharides of different

sizes and natures, and including branched oligosaccharides.

After its synthesis, glucosylceramide is modified to more

Introduction

Sphingolipids and their derivatives (glycosphingolipids,

phosphosphingolipids, sphingomyelins) are important

structural components of mammalian cell membranes. The

biosynthesis of sphingolipids is a tightly controlled process,

and disruption of a specific metabolic step can lead to dis-

ease. A variety of genetic disorders linked to sphingolipid

metabolism occur in man. Often, these diseases are charac-

terised by mutations in genes that encode for enzymes or

chaperones involved in a specific metabolic step in the lyso-

somal degradation of sphingolipids. Prominent examples of

such lysosomal storage disorders are Gaucher disease (in-

herited defect in acid glucocerebrosidase, GBA1 – the en-

zyme responsible for the hydrolysis of glucosylceramide to

glucose and ceramide) and Fabry disease (inherited defect

in lysosomal α-galactosidase – the enzyme responsible for

the hydrolysis of globotriaosylceramide to galactose and

lactosylceramide).^[1]

In the past decade, we have studied both diseases in

molecular detail, and we have found that both are charac-

terised by, in addition to storage of the substrate of the

genetically impaired enzyme (i.e., glucosylceramide in

[a] Leiden Institute of Chemistry, Gorleaus Laboratories,

Einsteinweg 55, 2300 RA Leiden, The Netherlands

E-mail: jcodee@chem.leidenuniv.nl

h.s.overkleeft@chem.leidenuniv.nl

http://biosyn.lic.leidenuniv.nl

[b] Department of Medical Biochemistry, Academic Medical

Center,

Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

[‡] Patrick Wisse and Henrik Gold contributed equally to the

work, and both should be considered as first authors.

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201500025.

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Figure 1. Partial overview of sphingolipid metabolism in man, and the target structures (insert) of the studies presented here.

complex glycosphingolipids by the sequential action of ful for the diagnosis of both diseases and for monitoring

glycosyltransferases. As a representative example, globo- their treatment, with corrections for glycolipid metabolism

triaosylceramide (5) emerges after sequential β-galactosyl- being reflected by lowered levels of lysolipids in tissue sam-

ation and α-galactosylation of glucosylceramide (4) effected ples.^[5–7]With this reasoning in mind, we set out to con-

by two independent glycosyltransferases.^[4]In time, sphing- struct a focussed library of stable-isotope (glyco)sphingo-

olipids are internalised by endocytosis, and transported to sine and (glyco)sphingolipid derivatives. In our design, we

the lysosomal compartments, where they are degraded. The chose to incorporate five ¹³C atoms into the sphingosine

degradation of glycosphingolipids is commonly viewed to base, and three into the palmitate, to obtain compounds

take place in a stepwise manner, with the product of one that would be easily detected, together with their unlabelled

enzyme acting as the substrate of the next enzyme of the counterparts, from complex biological lipid fractions. The

disassembly line. In this fashion, globotriaosylceramide (5) details of their synthesis, relying on a cross-metathesis reac-

is transformed by the action of lysosomal α-galactosidase tion to give stable-isotope-labelled sphingosine for further

into lactosylceramide. Lysosomal β-galactosidase next re- elaboration into a library of 24 compounds, are reported

moves the β-galactose residue to deliver glucosylceramide, here.

which in turn is deglucosylated by GBA1 to give ceramide

as the penultimate degradation product. Finally, acid

ceramidase (ACase) hydrolyses the amide bond to produce

Results and Discussion

sphingosine (6; Figure 1) and palmitate for reuptake into

the cytoplasm as new building blocks for catabolism.

Ready access to [¹³C₅]-sphingosine, the common back-

bone of all of the target structures, is crucial to the synthesis

In contrast to common belief, a few years ago, we found of the panel of [¹³C_n]-sphingolipids. To this end, and based

that in tissue from Fabry patients, as well as in animal mod- on literature precedent,^[8,9]we designed a synthetic route

els, which are characterised by elevated levels of globotri- based on the cross-metathesis of [¹³C₅]-pentadeca-1-ene

aosylceramide due to genetically and partially disabled lyso- (20) with aminodiol 21.^[10–16]The insertion of the labels into

somal α-galactosidase, the N-acyl chain of a portion of the 20 was achieved using [¹³C]-potassium cyanide and [¹³C₂]-

accumulated globotriaosylsphingosine is removed, resulting acetic acid, which was converted into Horner–Wadsworth–

in the formation of the lysoglycosphingolipid, globotriaos- Emmons (HWE) reagent 12 in a four-step procedure as

ylsphingosine (8).^[2]Later, we discovered the existence of a shown in Scheme 1. Transformation of acetic acid 9 into

related alternative pathway that occurs in Gaucher patients: bromoacetic acid 10 by a Hell–Volhard–Zelinsky reaction^[8]

accumulated glucosylceramide, caused by partially dysfunc- was followed by treatment of 10 with oxalyl chloride and

tional GBA1, is partially deacylated to produce glucosyl- addition of N,O-dimethylhydroxylamine in an one-pot fash-

sphingosine (7).^[3]These alternative pathways are probably ion to give a mixture of bromo- and chloro-N-methoxy-N-

occurring through the action of ACase, although this needs methylacetamides (11). Subjection of this mixture of Wein-

to be confirmed. The generation of stable-isotope-labelled reb amides to Arbuzov reaction conditions gave the target

(¹³C₅) globotriaosylsphingosine (8) and glucosylsphingos- HWE reagent (i.e., 12) in 74% yield over four steps.

ine (7) allows the detailed study of such alternative meta-

bolic pathways. Stable-isotope analogues are also very use- sium cyanide to give nitrile 14, which was partially reduced

Next, 1-bromononane (13) was treated with [¹³C]-potas-

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Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

Scheme 1. Reagents and conditions: (a) (i) TFAA (trifluoroacetic acid anhydride), Br₂, room temp., 20 h; (ii) water 88%; (b) (i) oxalyl

chloride, DMF, CH₂Cl₂, 0 °C to r.t., 2 h; (ii) N,O-dimethylhydroxylamine, –78 °C to r.t., 2 h, 97%; (c) triethylphosphite, 150 °C, 3 h, 95%.

to aldehyde 15 using DIBAL-H (diisobutylaluminium been reported as side-reactions in (cross) metathesis studies

hydride) (87% over two steps; Scheme 2). This aldehyde was unrelated to the synthesis of sphingosine. These events are

treated with reagent 12 and nBuLi to give unsaturated thought to be the result of alkene-isomerisation of terminal

[¹³C₃]-Weinreb amide 16, the C=C double bond in which alkenes while bound to the ruthenium metal centre.^[17–19]

was reduced to give 17 in 82% yield. A similar sequence of This isomerisation can be prevented by the addition of

events – reduction of the Weinreb amide in 17 to the alde- acetic acid to the cross-metathesis reaction mixture.^[20]In-

hyde, followed by HWE olefination with 12, and C=C re- deed, we found that the addition of acetic acid (20 mol-%

duction – provided the corresponding Weinreb amide (i.e., relative to 21) to an otherwise unchanged reaction mixture

19), which was transformed in two steps (reduction to the led to a clean cross-metathesis reaction to give 22 as the

aldehyde, followed by Wittig reaction with in-situ-generated major product in 81% yield. Sphingosine 22 was trans-

Ph₃P=CH₂) into [¹³C₅]-pentadeca-1-ene (20) in 93% yield.

formed into a suitable substrate for the ensuing glycosyl-

With the [¹³C₅]-pentadeca-1-ene in hand, we went on to ation by protecting-group manipulation. Benzoylation of

investigate the cross-metathesis of 20 with alkene 21 under the secondary alcohol in 22 and removal of the isopropylid-

the conditions advocated in the literature (i.e., Grubbs 2^ndene with a catalytic amount of pTsOH in methanol/ethanol

generation catalyst, dichloromethane, 20:21 = 1:2).^[9]How- to suppress unwanted Boc (tert-butoxycarbonyl) cleavage

ever, close examination of the metathesis product revealed led to the isolation of the key building block.

the partial elimination of one or two methylene units, lead-

[¹³C₃]-Palmitoyl chloride (30) was obtained starting from

ing to truncated cross-metathesis products. This came as a commercially available [¹³C₃]-myristic acid (24; Scheme 3).

surprise, since there are several literature reports that de- Labelled acid 24 was converted into the corresponding

scribe the synthesis of unlabelled sphingosine using essen- Weinreb amide (i.e., 25) by treatment with oxalyl chloride,

tially the same procedure as described here, and none and subsequent addition of N,O-dimethylhydroxylamine.

of these report the formation of truncated (C₁₇or C₁₆) The two-carbon elongation of 25 to give 27 was realised by

sphingosines.^[11–16]Methylene eliminations have, however, reduction with DIBAL-H, and subsequent subjection of the

Scheme 2. Reagents and conditions: (a) K¹³CN, EtOH/H₂O, 80 °C, 20 h, 95%; (b) DIBAL-H, THF, 0 °C to room temp., 2.5 h, acidic

work up, 92%; (c) (i) 12, nBuLi, THF, 0 °C, 10 min; (ii) [¹³C₁]-decanal (15), THF, 0 °C to r.t., 20 h, 87%; (d) Pd/C, H₂(g), EtOAc, r.t.,

20 h, 82%; (e) LiAlH₄, THF, 0 °C, 45 min, to give crude [¹³C₃]-dodecanal, which was added to a solution of (12, nBuLi, THF, 0 °C,

10 min), 0 °C to r.t., 20 h, 77%; (f) Pd/C, H₂(g), EtOAc, 93%; (g) LiAlH₄, THF, 0 °C, 45 min, then transfer to a solution of (MePh₃PBr,

nBuLi, THF, 0 °C, 10 min), 0 °C to r.t., 20 h, 93%; (h) 21, Grubbs 2nd generation catalyst, AcOH, CH₂Cl₂, reflux, 48 h, 81%; (i) BzCl,

DMAP [4-(dimethylamino)pyridine], CH₂Cl₂/pyridine, room temp., 20 h, 92%; (ii) MeOH/EtOH, pTsOH, r.t., 20 h, 63%.

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Scheme 3. Reagents and conditions: (a) (i) oxalyl chloride, DMF, CH₂Cl₂, 0 °C to room temp., 2 h; (ii) N,O-dimethylhydroxylamine,

–78 °C to r.t., 2 h, 98%; (b) DIBAL-H, THF, –78 °C, 30 min, to give crude [¹³C₃]-tetradecanal, which was added to a solution of (26,

nBuLi, THF, 0 °C, 10 min), 0 °C to r.t., 20 h, 81%; (c) Pd-C, H₂(g), EtOAc, 20 h, 95%; (d) LiOH, THF/EtOH/H₂, 20 h, 95%; (e) oxalyl

chloride, DMF, CH₂Cl₂, 0 °C to r.t., 2 h, 100%.

resulting aldehyde to HWE olefination with reagent 26. Re- removal gave stable-isotope sphinganine pair 33a and 33b,

duction of the double bond in 27, saponification, and treat- which were used as starting materials to produce dihydro-

ment with oxalyl chloride gave [¹³C₃]-palmitoyl chloride ceramides 34a–34d.

(30).

The glycosylated sphingolipids were assembled by cou-

The synthesis of sphingolipids and glycosphingolipids in pling the labelled sphingosine alcohols with the appropriate

various ¹³C-labelled forms based on 23 is shown in glycosyl donors. Thus, N-phenyltrifluoroacetimidate gluc-

Scheme 4. Debenzoylation of 23b with sodium methoxide in ose donor 35 (see Experimental Section for its synthesis;

methanol, followed by TFA (trifluoroacetic acid) mediated Scheme 5) and sphingosine 23a/b were condensed in a reac-

removal of the Boc group provided [¹³C₅]-sphingosine (31b; tion promoted by boron trifluoride diethyl etherate to give

59% yield). Both [¹³C₀]-31a and [¹³C₅]-31b were condensed fully protected glucosylsphingosines 36a/b. The moderate

with either [¹³C₀]-palmitoyl chloride or [¹³C₃]-palmitoyl yield of the glycosylation reaction can be explained by the

chloride (30) to give the panel of labelled ceramides 32a– concomitant cleavage of the Boc group, which took place

32d. Alternatively, debenzoylation of 23a/b, reduction of the under the Lewis acidic reaction conditions. It is interesting

alkene moiety with Adams catalyst, and TFA-mediated Boc to note that attempted glucosylation of 23a/b using the cor-

Scheme 4. Reagents and conditions: (a) (i) NaOMe, MeOH, room temp., 20 h; (ii) KOH, H₂O, r.t., 20 h; (iii) TFA, H₂O, 0 °C, 30 min,

59 %; (b) palmitoyl chloride, satd. aq. NaOAc, THF, r.t., 3 h, 50–70 %; (c) (i) NaOMe, MeOH, r.t., 20 h; (ii) KOH, H₂O, r.t., 20 h;

(iii) PtO₂, H₂(g), EtOAc, r.t., 20 h; (iv) TFA, H₂O, 0 °C, 30 min, 52%; (d) Glucosyl donor (35 or 39), BF₃·OEt₂, CH₂Cl₂, 0 °C, 1 h, 49–

61%; (e) (i) HF/pyridine, THF/pyridine, r.t., 2 h; (ii) NaOMe, MeOH, r.t., 20 h; (iii) KOH, H₂O, r.t., 20 h; (iv) TFA, H₂O, 0 °C, 30 min,

48–53%.

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Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

responding perbenzoylated N-phenyltrifluoroacetimidate mass spectra and their ¹H and ¹³C NMR spectra. As a rep-

1

donor and boron trifluoride diethyl etherate was unproduc- resentative example, Figure 2 shows the H and ¹³C NMR

tive, and led only to the isolation of the product of Boc spectra of ¹³C-labelled globotriasylsphingosine 41b (Fig-

removal from 23a/b. Global deprotection of 36 by treatment ure 2a, b and d), and the ¹³C NMR spectrum of its unlab-

with HF/pyridine, sodium methoxide, and trifluoroacetic elled counterpart 41a (Figure 2c). In Figure 2b, the ¹³C-de-

1

acid provided stable-isotope glucosylsphingosine pair 37a/ coupled H NMR spectrum of ¹³C-labelled 41b is shown,

b in 53% yield. Both [¹³C₀]-glucosylsphingosine (37a) and which is identical in all respects to the spectrum of unlab-

[¹³C₅]-glucosylsphingosine (37b) were condensed with either elled 41a. Integration of the peaks due to the ¹³C labels in

[¹³C₀]-palmitoyl chloride or [¹³C₃]-palmitoyl chloride (30) 41b clearly shows the ratio of the incorporated atoms.

to give the panel of labelled glucosylceramide derivatives

38a–38d.

Conclusions

Finally, the syntheses of globotriaosylsphingosines 41a/b

and globotriaosylceramides 42a–42d were undertaken. To

this end, sphingosine 23 was condensed with trisaccharide

donor 39^[21]in a reaction promoted by boron trifluoride

diethyl etherate to give fully protected globotriaosylsphin-

gosines 40a/b. Subsequent global deprotection by the same

procedure described above gave 41a/b in 48% yield. Stan-

dard palmitoylation with either [¹³C₀]-palmitoyl chloride or

[¹³C₃]-palmitoyl chloride gave the panel of globotriaosylcer-

amides 42a–42d in an average yield of 59% to complete the

library of labelled (glyco)sphingosines.

In conclusion, a comprehensive library of stable-isotope-

enriched sphingolipids has been constructed by straightfor-

ward synthetic routes taking into consideration that the

synthesis of ¹³C-enriched lipids with the carbons introduced

at specific predetermined sites can be executed with only a

limited number of reagents available from commercial

sources. The key step in the assembly of the sphingosine

backbone, the cross-metathesis reaction between the sphin-

gosine head-group alkene and the long-chain alkene, was

optimised to minimise truncation of the long-chain alkene

before the cross-metathesis event. Elimination of one or two

methylene units, leading to the loss of ¹³C labels, was ob-

served during this reaction under conditions previously de-

scribed. The addition of acetic acid to the reaction mixture

effectively prevented the truncation of the alkene chain.

With this work we believe we have obtained a valuable set

of molecular probes to study sphingolipid metabolism in

healthy and disease states in a chemical metabolomics set-

ting. The route is also flexible, and is thus amenable for the

production of other sphingolipid metabolites, with respect

to both the polar head group, such as for instance phos-

phate and phosphate diesters, and also the N-acyl-substi-

tuted fatty acid moiety.

The physical properties of all the labelled compounds

matched those of their ¹²C counterparts, apart from their

Experimental Section

General Remarks: [¹³C₂]-Acetic acid (99.95% isotopically pure,

product code CLM-105), potassium [¹³C]-cyanide (99% isotopi-

cally pure, product code CLM-297), and [1,2,3-¹³C₃]-myristic acid

(99% isotopically pure, product code CLM-3665) were purchased

from Cambridge Isotope Laboratories, Inc., and were used as re-

ceived. Commercially available reagents and solvents (Acros,

Fluka, or Merck) were used as received, unless otherwise stated.

CH₂Cl₂and THF were freshly distilled before use, over P₂O₅and

Na/benzophenone, respectively. Triethylamine was distilled from

calcium hydride and stored over potassium hydroxide. Traces of

water were removed from starting compounds by coevaporation

with toluene. All moisture-sensitive reactions were carried out un-

der an argon atmosphere. Molecular sieves (3 Å) were flame-dried

before use. Column chromatography was carried out using forced

flow of the indicated solvent systems on Screening Devices Silica

gel 60 (40–63 μm mesh). Size-exclusion chromatography was car-

ried out on Sephadex LH20 (MeOH/CH₂Cl₂, 1:1). Analytical TLC

was carried out on aluminium sheets (Merck, silica gel 60, F₂₅₄).

Compounds were visualised by UV absorption (254 nm), or by

spraying with ammonium molybdate/cerium sulphate solution

[(NH₄)₆Mo₇O₂₄·4H₂O (25 g/L), (NH₄)₄Ce(SO₄)₆·2H₂O (10 g/L),

Figure 2. ¹H and ¹³C NMR spectra of the synthesised labelled and

unlabelled globotriaosylsphingsosine. (a) 400 MHz H NMR spec-

1

trum ([D₄]methanol) of labelled globotriaosylsphingosine 41b, in

which the ¹³C,¹H coupling of the double-bond proton is apparent.

1

(b) 400 MHz ¹³C-decoupled H NMR spectrum ([D₄]methanol) of

labelled globotriaosylsphingosine 41b. (c) 151.1 MHz ¹³C NMR

spectrum ([D₄]methanol) of unlabelled globotriaosylsphingosine

41a. (d) 151.1 MHz ¹³C NMR spectrum ([D₄]methanol) of labelled

globotriaosylsphingosine 41b, with integration of the ¹³C labels.

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10% sulphuric acid in ethanol] or phosphormolybdic acid in EtOH

(150 g/L), followed by charring (ca. 150 °C). IR spectra were re-

corded with a Shimadzu FTIR-8300 instrument and are reported

The stirred mixture was allowed to reach room temperature over

2 h. The reaction mixture was then stirred at room temperature for

30 min. The solids were removed by filtration through a Whatmann

in cm^–1. Optical rotations were measured with a Propol automatic paper, and washed with CH₂Cl₂. The filtrate was concentrated in

polarimeter (sodium D-line, λ = 589 nm). ¹H and ¹³C NMR spectra vacuo, and resulting residue was purified by column chromatog-

were recorded with a Bruker AV 400 MHz spectrometer at 400.2

(¹H) and 100.6 (¹³C) MHz, or with a Bruker AV 600 MHz spec-

trometer at 600.0 (¹H) and 151.1 (¹³C) MHz. Chemical shifts are

reported as δ values (ppm), and were referenced to tetramethylsil-

ane (δ = 0.00 ppm) directly in CDCl₃, or using the residual solvent

peak (D₂O). Coupling constants (J) are given in Hz, and all ¹³C

spectra were proton decoupled. NMR assignments were made

using COSY and HSQC, and in some cases TOCSY experiments.

LC–MS analysis was carried out with an LCQ Advantage Max

(Thermo Finnigan) instrument equipped with a Gemini C₁₈

column (Phenomenex, 50ϫ4.6 mm, 3 μm), using the following

buffers: A: H₂O, B: acetonitrile, and C: aq. TFA (1.0%). HPLC–

MS purifications were carried out with an Agilent Technologies

1200 Series automated HPLC system with a Quadrupole MS 6130,

equipped with a semi-preparative Gemini C₁₈column (Phe-

nomenex, 250ϫ10.00, 5 μm). Products were eluted using the fol-

lowing buffers: A: aq. TFA (0.2%), B: acetonitrile (HPLC-grade),

5 mL/min. Purified products were lyophilised with a CHRIST

ALPHA 2–4 LD_PLUSapparatus to remove water and traces of

buffer salts.

raphy (10–40% EtOAc in petroleum ether) to give a mixture of

[1,2-¹³C₂]-2-bromo-N-methoxy-N-methylacetamide and [1,2-¹³C₂]-

2-chloro-N-methoxy-N-methylacetamide (4:1 ratio, as determined

1

by H and ¹³C NMR spectroscopy) (10.25 g, 58.3 mmol, 97%) as

a colourless oil. R_f= 0.35 (30% EtOAc in petroleum ether).

Data for [1,2-¹³C₂]-2-Bromo-N-methoxy-N-methyl-acetamide: ¹H

NMR (400 MHz, CDCl₃): δ = 4.01 (dd, J = 154.0, 3.6 Hz, 2 H),

3.80 (s, 3 H), 3.24 (s, 3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ

= 167.5 (d, J = 58.5 Hz), 61.6, 32.5, 25.1 (d, J = 58.4 Hz) ppm.

HRMS: calcd for [C₂¹³C₂H₈NO₂Br

183.9877.

+

H]⁺183.9878; found

Data for [1,2-¹³C₂]-2-Chloro-N-methoxy-N-methylacetamide: ¹H

NMR (400 MHz, CDCl₃): δ = 4.25 (dd, J = 152.3, 4.4 Hz, 2 H),

3.76 (s, 3 H), 3.24 (s, 3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ

= 167.5 (d, J = 57.2 Hz), 61.6, 40.7 (d, J = 57.7 Hz), 32.5 ppm.

HRMS: calcd. for [C₂¹³C₂H₈NO₂Cl

140.0381.

+

H]⁺140.0383; found

Diethyl-([1,2-¹³C₂]-N-methoxy-N-methylcarbamoylmethyl)

Phos-

phonate (12): [1,2-¹³C₂]-2-Bromo/chloro-N-methoxy-N-methylacet-

amide (11; 10.25 g, 58.3 mmol, 1.0 equiv.) and triethylphosphite

(10.5 mL, 60 mmol, 1.05 equiv.) were put in a round-bottomed

flask equipped with a 15 cm air-cooled condenser, and the mixture

was heated for 3 h at 150 °C. The crude mixture was cooled down,

and directly purified by column chromatography (30–50% acetone

in petroleum ether) to give compound 12 (13.7 g, 56.8 mmol, 95%)

as a colourless oil. R_f= 0.20 (40% acetone in petroleum ether). ¹H

NMR (400 MHz, CDCl₃): δ = 4.24–4.13 (m, 4 H), 3.79 (s, 3 H),

3.22 (s, 3 H), 3.16 (ddd, J = 129.8, 21.9, 6.6 Hz, 2 H), 1.35 (t, J =

7.1 Hz, 6 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 165.5 (dd, J

= 53.1, 4.5 Hz), 62.0, 61.9, 60.9, 31.57, 30.9 (dd, J = 136.1,

General Procedure for the Synthesis of Ceramides from Sphingosine:

Sphingosine (0.1 mmol) was dissolved in THF (12 mL), and satd.

aq. NaOAc (10 mL) was added. Palmitoyl chloride (0.13 mmol,

1.3 equiv.) was added, and the reaction mixture was stirred vigor-

ously at room temperature for 3 h. The mixture was diluted with

THF (20 mL), and washed with water (10 mL). The aqueous layer

was extracted with THF (3ϫ 20 mL), and the combined organic

extracts were dried (Na₂SO₄), filtered, and concentrated in vacuo.

The ceramides were purified by column chromatography (chloro-

form/MeOH) and HPLC–MS, using a C₄column. Products were

eluted using the following buffers: A: NH₄OAc [25 nM in MeOH/

H₂O (3:1)], B: acetonitrile (HPLC grade). Purified products were

lyophilised to remove water and traces of buffer salts.

53.1 Hz), 15.82, 15.76 ppm. IR (neat): ν = 2984, 1658, 1423, 1381,

˜

1253, 1018, 961, 789 cm^–1. HRMS: calcd. for [C₆¹³C₂H₁₈NO₅P +

H]⁺242.1063; found 242.1064.

[¹³C₂]-2-Bromoacetic Acid (10): Trifluoroacetic anhydride (67.3 mL,

484 mmol, 3.0 equiv.) was slowly added to [1,2-¹³C₂]-acetic acid (9;

10 g, 161 mmol, 1.0 equiv.) while stirring. Bromine (8.30 mL,

161 mmol, 1.0 equiv.) was added, and the reaction mixture was

stirred at room temperature for 20 h. The mixture was then cooled

to 0 °C, and water (10.2 mL, 564 mmol, 3.5 equiv.) was added. The

excess bromine was removed by a flow of argon. The crude mixture

was then dissolved in toluene (200 mL), and the solution was con-

centrated in vacuo. This procedure was repeated twice to give

[¹³C₂]-2-bromoacetic acid (10; 23.2 g, 142 mmol, 88%) as an off-

white solid, which was used without further purification.^[8]

[1-¹³C₁]-Decanitrile (14): [¹³C₁]-Potassium cyanide (5.00 g,

76.0 mmol, 1.0 equiv.) was added to a solution of 1-bromononane

(13; 16.5 g, 79.0 mmol, 1.05 equiv.) in a mixture of ethanol and

water (9:1; 140 mL), and the reaction mixture was heated overnight

at 80 °C. The mixture was then cooled to room temperature, diluted

with Et₂O (500 mL), and washed with water (2ϫ 500 mL) and

brine (400 mL). The aqueous layers were extracted with Et₂O

(400 mL), and the combined organic extracts were dried (Na₂SO₄),

filtered, and concentrated in vacuo. Purification by column

chromatography (0–2% EtOAc in petroleum ether) gave compound

14 (11.1 g, 72.0 mmol, 95%) as a colourless oil. R_f= 0.23 (3%

[1,2-¹³C₂]-2-Bromo-N-methoxy-N-methylacetamide and [1,2-¹³C₂]-2-

Chloro-N-methoxy-N-methylacetamide (11): [¹³C₂]-2-Bromoacetic

acid (10; 8.46 g, 60 mmol, 1.0 equiv.) was dissolved in anhydrous

CH₂Cl₂(100 mL), and the solution was put under an atmosphere

of argon, and cooled to 0 °C. Oxalyl chloride (10.5 mL, 120 mmol,

2.0 equiv.) was added, followed by DMF (one drop). The reaction

mixture was kept under a flow of argon and stirred at room tem-

perature. When the evolution of gas stopped (ca. 2 h), the mixture

was concentrated in vacuo (10–15 °C, 180 mbar).

1

EtOAc in petroleum ether). H NMR (400 MHz, CDCl₃): δ = 2.33

(dt, J = 9.6, 7.1 Hz, 2 H), 1.65 (m, 2 H), 1.44 (m, 2 H), 1.35–1.22

(m, 10 H), 0.88 (t, J = 6.9 Hz, 3 H) ppm. ¹³C NMR (101 MHz,

CDCl₃): δ = 119.8, 31.7, 29.2, 29.1, 28.7, 28.5 (d, J = 3.3 Hz), 25.3

(d, J = 0.4 Hz), 22.5, 17.0 (d, J = 55.8 Hz), 14.0 ppm. IR (neat): ν

˜

= 2925, 2856, 2194, 1467, 1425, 1378, 721 cm^–1. HRMS: calcd. for

[C₉¹³CH₁₉N + H]⁺155.2623; found 155.2624.

[1-¹³C₁]-Decanal (15): [1-¹³C₁]-Decanitrile (14; 11.1 g, 72.0 mmol,

1.0 equiv.) was dissolved in anhydrous THF (250 mL), and the

solution was cooled to 0 °C. Then DIBAL-H (1.5 m in hexanes;

52.9 mL, 79.0 mmol, 1.1 equiv.) was added, and the reaction mix-

ture was stirred at ambient temperature for 2.5 h. The mixture was

then transferred to an extraction funnel, diluted with Et₂O

The residue was dissolved in anhydrous CH₂Cl₂(40 mL), and the

solution was cooled to –70 °C. A solution of N,O-dimethylhydrox-

ylamine (12.3 mL, 168 mmol, 2.8 equiv.) in anhydrous CH₂Cl₂

(30 mL) was slowly added to the acyl chloride solution at –70 °C.

2666

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Eur. J. Org. Chem. 2015, 2661–2677

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

(200 mL), and washed with HCl (1 m aq.; 2ϫ 400 mL), and satd.

aq. NaHCO₃(400 mL). The aqueous layers were extracted with

Et₂O (2ϫ 400 mL), and the combined organic extracts were dried

(MgSO₄), filtered through Celite, and concentrated in vacuo. Puri-

fication by column chromatography (0–10% CH₂Cl₂in petroleum

ether) gave compound 15 (10.4 g, 66.1 mmol, 92%) as a colourless

oil. R_f= 0.22 (20% CH₂Cl₂in petroleum ether). ¹H NMR

(400 MHz, CDCl₃): δ = 9.76 (dt, J = 169.8, 1.9 Hz, 1 H), 2.42 (dtd,

J = 7.4, 6.2, 1.8 Hz, 2 H), 1.62 (m, 2 H), 1.36–1.23 (m, 12 H), 0.88

(t, J = 6.9 Hz, 3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 203.0,

43.9 (d, J = 38.8 Hz), 31.8, 29.35, 29.32, 29.2, 29.1 (d, J = 3.4 Hz),

in petroleum ether) gave compound 17 (6.85 g, 27.8 mmol, 82%)

as a colourless oil. R_f= 0.38 (20% EtOAc in petroleum ether). H

1

NMR (400 MHz, CDCl₃): δ = 3.68 (s, 3 H), 3.18 (s, 3 H), 2.41 (dm,

J = 127.3 Hz, 2 H), 1.62 (dm, J = 127.9 Hz, 2 H), 1.35–1.23 (m,

16 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm. ¹³C NMR (101 MHz, CDCl₃):

δ = 174.6 (br. d, J = 51.5 Hz), 61.1, 31.9, 31.8 (dd, J = 51.5,

37.5 Hz), 29.7–29.1 (m), 24.6 (dd, J = 34.9, 1.3 Hz), 22.6, 14.1 ppm.

IR (neat): ν = 2923, 2854, 1627, 1464, 1369, 1174, 1119, 998, 722,

˜

436 cm^–1. HRMS: calcd. for [C₁₁¹³C₃H₂₉NO₂+ H]⁺247.2372;

found 247.2373.

[1,2,3,4,5-¹³C₅]-(E/Z)-N-Methoxy-N-methyltetradec-2-enamide

(18): [1,2,3-¹³C₃]-N-Methoxy-N-methyldodecanamide (17; 3.91 g,

15.9 mmol, 1.0 equiv.) was dissolved in anhydrous THF (120 mL),

and the solution was cooled to 0 °C. Then lithium aluminium

hydride (4.0 m in THF; 2.38 mL, 9.52 mmol, 0.6 equiv.) was added.

The reaction mixture was stirred for 45 min, and then it was cooled

to –15 °C. Sat. aq. KHSO₄(100 mL) and Et₂O (300 mL) were

added. The two-phase system was stirred vigorously for 30 min,

then the phases were separated, and the organic phase was then

dried with MgSO₄, followed by Na₂SO₄. The solids were filtered

and washed with Et₂O (200 mL). The filtrate was concentrated in

vacuo to give crude [1,2,3-¹³C₃]-dodecanal (2.96 g, 15.8 mmol) as a

colourless oil, which was used without further purification.

22.6, 22.0 (d, J = 1.6 Hz), 14.0 ppm. IR (neat): ν = 2922, 2855,

˜

1728, 1466, 719 cm^–1.

[1,2,3-¹³C₃]-(E/Z)-N-Methoxy-N-methyldodec-2-enamide (16): Di-

ethyl ([1,2-¹³C₂]-N-methoxy-N-methylcarbamoylmethyl)phosphon-

ate (12; 10.4 g, 43.1 mmol, 1.1 equiv.) was dissolved in dry THF

(200 mL), and the solution was cooled to 0 °C. Then n-butyllithium

(1.6 m in hexanes; 26.5 mL, 42.3 mmol, 1.08 equiv.) was added, and

the reaction mixture was stirred for 10 min at 0 °C. A solution of

[1-¹³C₁]-decanal (15; 6.16 g, 39.2 mmol, 1.0 equiv.) in anhydrous

THF (40 mL) was then added to the phosphonate carbanion solu-

tion, and the reaction mixture was stirred at room temperature

overnight. The mixture was then transferred to an extraction funnel

with Et₂O (50 mL), and washed with water (250 mL) and brine

(200 mL). The aqueous layers were extracted with Et₂O (2ϫ

250 mL), and the combined organic extracts were dried (Na₂SO₄),

filtered, and concentrated in vacuo. Purification by column

chromatography (0–15% EtOAc in petroleum ether) gave [1,2,3-

Diethyl (N-methoxy-N-methylcarbamoylmethyl)phosphonate (12;

4.20 g, 17.4 mmol, 1.1 equiv.) was dissolved in anhydrous THF

(80 mL), and the solution was cooled to 0 °C. n-Butyllithium (1.6 m

in hexanes; 10.4 mL, 16.6 mmol, 1.05 equiv.) was added, and the

reaction mixture was stirred for 10 min at 0 °C. The crude [1,2,3-

¹³C₃]-dodecanal was dissolved in anhydrous THF (20 mL), and the

resulting solution was added to the Horner–Wadsworth–Emmons

reagent at 0 °C. The reaction mixture was then stirred at room tem-

perature overnight. The mixture was transferred to an extraction

funnel with Et₂O (50 mL), and washed with water (100 mL) and

brine (100 mL). The aqueous layers were extracted with Et₂O (2ϫ

100 mL) and the combined organic extracts were dried (Na₂SO₄),

filtered, and concentrated in vacuo. Purification by column

chromatography (5–15% EtOAc in petroleum ether) gave [1,2,3,4,5-

¹³C₅]-(E)-N-methoxy-N-methyl-tetradec-2-enamide (18E; 3.05 g,

11.1 mmol, 70 %) and [1,2,3,4,5-¹³C₅]-(Z)-N-methoxy-N-methyl-

tetradec-2-enamide (18Z; 310 mg, 1.13 mmol, 7%) as colourless

oils (combined yield 77%).

¹³C₃]-(E)-N-methoxy-N-methyldodec-2-enamide (16E;

7.52 g,

30.8 mmol, 79%) and [1,2,3-¹³C₃]-(Z)-N-methoxy-N-methyldodec-

2-enamide (16Z; 0.75 mg, 3.07 mmol, 8%) as a colourless oil (com-

bined yield 87%).

Data for E isomer 16E: R_f= 0.42 (20% EtOAc in petroleum ether).

¹H NMR (400 MHz, CDCl₃): δ = 6.98 (dm, J = 153.8 Hz, 1 H),

6.38 (ddd, J = 160.8, 15.4, 4.1 Hz, 1 H), 3.70 (s, 3 H), 3.24 (s, 3

H), 2.23 (m, 2 H), 1.46 (m, 2 H), 1.35–1.23 (m, 12 H), 0.88 (t, J =

6.8 Hz, 3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 167.1 (d, J

= 67.1 Hz), 148.0 (d, J = 71.6 Hz), 118.5 (dd, J = 71.6, 67.1 Hz),

61.6, 32.5 (m), 32.3 (m), 31.9, 29.5, 29.4, 29.3, 29.2 (d, J = 3.6 Hz),

28.3 (m), 22.7, 14.1 ppm. IR (neat): ν = 2926, 5856, 1622, 1584,

˜

1462, 1368, 1175, 993 cm^–1. HRMS: calcd for [C₁₁¹³C₃H₂₇NO₂H]⁺

245.2215; found 245.2216.

Data for E isomer 18E: R_f= 0.39 (15% EtOAc in petroleum ether).

¹H NMR (600 MHz, CDCl₃): δ = 6.98 (dm, J = 153.8 Hz, 1 H),

6.39 (ddm, J = 161.1, 15.4 Hz, 1 H), 3.70 (s, 3 H), 3.24 (s, 3 H),

2.23 (ddt, J = 126.2, 7.0, 6.1 Hz, 2 H), 1.60–1.20 (m, 18 H), 0.88

(t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 167.1

(dd, J = 67.1, 6.1 Hz), 148.0 (ddd, J = 71.6, 41.8, 2.1 Hz), 118.6

(dddd, J = 71.6, 67.1, 3.6, 1.5 Hz), 61.6, 32.5 (dddd, J = 41.8, 33.7,

6.1, 1.5 Hz), 32.3, 31.9, 29.6–29.0 (m), 28.3 (ddd, J = 33.7, 3.6,

Data for Z isomer 16Z: R_f= 0.64 (20% EtOAc in petroleum ether).

¹H NMR (400 MHz, CDCl₃): δ = 6.22 (dd, J = 161.8, 11.5 Hz, 1

H), 6.11 (dm, J = 152.0 Hz, 1 H), 3.68 (s, 3 H), 3.21 (s, 3 H), 2.61

(m, 2 H), 1.43 (m, 2 H), 1.35–1.22 (m, 12 H), 0.88 (t, J = 6.9 Hz,

3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 167.6 (d, J =

63.6 Hz), 147.8 (d, J = 67.1 Hz), 117.9 (dd, J = 67.1, 63.6 Hz), 61.5,

31.9, 31.6, 29.6, 29.5, 29.38 (d, J = 4.0 Hz), 29.35–29.29 (m), 29.1

(m), 22.7, 14.1 ppm. IR (neat): ν = 2925, 2855, 1618, 1459, 1334,

˜

1178, 996, 776 cm^–1. HRMS: calcd. for [C₁₁¹³C₃H₂₇NO₂+ H]⁺

245.2215; found 245.2216.

2.1 Hz), 22.7, 14.1 ppm. IR (neat): ν = 2924, 2854, 1618, 1583,

˜

1464, 1368, 991 cm^–1. HRMS: calcd for [C₁₁¹³C₅H₃₁NO₂+ H]⁺

275.2595; found 275.2595.

[1,2,3-¹³C₃]-N-Methoxy-N-methyldodecanamide (17): [1,2,3-¹³C₃]-

(E/Z)-N-Methoxy-N-methyldodec-2-enamide

(16E/Z;

8.25 g,

Data for Z isomer 18Z: R_f= 0.58 (15% EtOAc in petroleum ether).

¹H NMR (600 MHz, CDCl₃): δ = 6.23 (dm, J = 160.7 Hz, 1 H),

6.12 (dm, J = 152.0 Hz, 1 H), 3.68 (s, 3 H), 3.21 (s, 3 H), 2.62 (dm,

J = 125.3 Hz, 2 H), 1.59–1.20 (m, 18 H), 0.88 (t, J = 7.1 Hz, 3 H)

ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 167.6 (dm, J = 67.1 Hz),

147.8 (dd, J = 69.9, 35.2 Hz), 117.9 (dd, J = 69.9, 67.1 Hz), 61.4,

33.8 mmol, 1.0 equiv.) was dissolved in EtOAc (200 mL). The solu-

tion was bubbled with argon while stirring, and palladium (10% on

charcoal; 0.72 g, 0.67 mmol, 0.02 equiv.) was added. The reaction

mixture was then stirred under a flow of hydrogen gas for 30 min,

and left overnight under a hydrogen atmosphere. The palladium

was removed by filtration through a Whatmann paper, and rinsed

with EtOAc (100 mL). The solvent was removed from the filtrate

in vacuo. Purification by column chromatography (5–20% EtOAc

32.0, 31.9, 30.2–28.4 (m), 22.7, 14.1 ppm. IR (neat): ν = 2923, 2854,

˜

1618, 1464, 1331, 1176, 1086, 999, 775 cm^–1. HRMS: calcd. for

[C₁₁¹³C₅H₃₁NO₂+ H]⁺275.2595; found 275.2595.

Eur. J. Org. Chem. 2015, 2661–2677

www.eurjoc.org

2667

J. D. C. Codée, H. S. Overkleeft et al.

FULL PAPER

[1,2,3,4,5-¹³C₅]-N-Methoxy-N-methyltetradecanamide (19):

[1,2,3,4,5-¹³C₅]-(E/Z)-N-Methoxy-N-methyl-tetradec-2-enamide

(18E/Z; 3.20 g, 11.66 mmol, 1.0 equiv.) was dissolved in EtOAc

(100 mL). The solution was bubbled with argon while stirring, and

then palladium (10% on charcoal; 0.62 g, 0.58 mmol, 0.05 equiv.)

was added. The reaction mixture was then stirred under a flow of

argon for 36 h. The reaction mixture was concentrated in vacuo,

and the residue was purified by column chromatography (0–10%

EtOAc in petroleum ether) to give compound 22a (1.30 g,

2.96 mmol, 74%) as a viscous oil. R_f= 0.19 (10% EtOAc in petro-

leum ether). [α]²_D²= –26 (c = 0.25, CHCl₃). ¹H NMR (400 MHz,

[D₆]DMSO, 363 K): δ = 5.56 (dt, J = 15.8, 6.5 Hz, 1 H), 5.45 (ddd,

hydrogen gas for 30 min, and was then left overnight under a J = 15.8, 6.6, 1.1 Hz, 1 H), 4.61 (br. s, 1 H), 4.03 (m, 1 H), 3.93

hydrogen atmosphere. The palladium residue was removed by fil-

tration through a Whatmann paper, and rinsed with EtOAc

(100 mL). The solvent was removed from the filtrate in vacuo. Puri-

fication by column chromatography (5–15% EtOAc in petroleum

ether) gave compound 19 (3.00 g, 10.85 mmol, 93%) as a colourless

oil. R_f= 0.38 (15 % EtOAc in petroleum ether). ¹H NMR

(br. d, J = 8.5 Hz, 1 H), 3.83 (br. t, J = 7.3 Hz, 1 H), 3.75 (m, 1

H), 1.98 (m, 2 H), 1.48 (s, 3 H), 1.43 (m, 12 H), 1.39–1.20 (m, 22

H), 0.87 (t, J = 6.6 Hz, 3 H) ppm. ¹³C NMR (100 MHz, [D₆]-

DMSO, 363 K): δ = 151.3, 130.8, 130.4, 92.8, 78.7, 71.4, 63.7, 61.0,

31.2, 30.8, 28.5, 28.4, 28.2, 28.12, 28.06, 27.7, 26.2, 21.5, 13.2 ppm.

IR (neat): ν = 3436, 2924, 2854, 1702, 1381, 1365, 1255, 1173, 1097,

˜

(600 MHz, CDCl₃): δ = 3.68 (s, 3 H), 3.18 (s, 3 H), 2.41 (dm, J = 848, 766 cm^–1. HRMS: calcd. for [C₂₆H₄₉NO₄+ H]⁺440.3734;

128.4 Hz, 2 H), 1.62 (dm, J = 127.1 Hz, 2 H), 1.46–1.12 (m, 20 H),

0.88 (t, J = 7.1 Hz, 3 H) ppm. ¹³C NMR (151 MHz, CDCl₃): δ =

174.8 (dm, J = 51.5 Hz), 61.1, 32.1, 31.9 (dd, J = 51.5, 35.6 Hz),

found 440.3733.

(E)-[5,6,7,8,9-¹³C₅]-1,2-O,N-Isopropylidene-N-(tert-butoxycarbon-

yl)-D-erythro-sphingosine (22b): (2S,3R)-2-Amino-N-(tert-butyloxy-

29.7–29.1 (m), 24.6 (m), 22.6, 14.1 ppm. IR (neat): ν = 2922, 2853,

˜

carbonyl)-1,3-dihydroxy-1,2-O,N-isopropylidene-4-pentene (21;

3.58 g, 13.9 mmol, 3.0 equiv.) and [2,3,4,5,6-¹³C₅]-pentadec-1-ene

(20; 1.00 g, 4.64 mmol, 1.0 equiv.) were dissolved in anhydrous

CH₂Cl₂(4 mL), and the flask was flushed with argon. Grubbs 2^nd

generation catalyst (79 mg, 93 μmol, 0.02 equiv.) and acetic acid

(53 μL, 0.93 mmol, 0.2 equiv.) were added. The reaction mixture

was heated at reflux under a flow of argon for 36 h. The reaction

mixture was concentrated in vacuo, and the residue was purified

by column chromatography (0–10% EtOAc in petroleum ether) to

give compound 22b (1.68 g, 3.29 mmol, 81%) as a viscous oil. R_f

= 0.19 (10% EtOAc in petroleum ether). [α]²_D²= –19 (c = 0.5,

1628, 1458, 1370, 1175, 996, 721 cm^–1. HRMS: calcd. for

[C₁₁¹³C₅H₃₃NO₂+ H]⁺277.2751; found 277.2752.

[2,3,4,5,6-¹³C₅]-Pentadec-1-ene (20): [1,2,3,4,5-¹³C₅]-N-Methoxy-N-

methyltetradecanamide (19; 1.57 g, 5.72 mmol, 1.0 equiv.) was dis-

solved in anhydrous THF (55 mL), and LiAlH₄(4 m in THF;

0.86 mL, 3.43 mmol, 0.6 equiv.) was added at 0 °C. The reaction

mixture was stirred for 45 min, and then it was cooled to ca.

–15 °C, and satd. aq. KHSO₄(40 mL) and Et₂O (100 mL) were

added. The resulting two-phase mixture was stirred vigorously for

30 min, then the phases were separated, and the organic phase

dried with MgSO₄, and then Na₂SO₄. The solids were removed by

filtration, and washed with Et₂O (100 mL). The filtrate was concen-

trated in vacuo to give crude [1,2,3,4,5-¹³C₅]-tetradecanal (1.24 g,

5.72 mmol) as a colourless oil, which was used without further pu-

rification.

1

CHCl₃). H NMR (400 MHz, [D₆]DMSO, 363 K): δ = 5.55 (dm, J

= 152.0 Hz, 1 H), 5.44 (m, 1 H), 4.60 (br. d, J = 5.4 Hz, 1 H), 4.05

(m, 1 H), 3.94 (dd, J = 8.6, 2.0 Hz, 1 H), 3.82 (dd, J = 8.6, 6.1 Hz,

1 H), 3.75 (td, J = 6.1, 2.0 Hz, 1 H), 1.98 (dm, J = 124.2 Hz, 2 H),

1.56–1.06 (m, 37 H), 0.87 (t, J = 6.9 Hz, 3 H) ppm. ¹H NMR

(400 MHz, CDCl₃): δ = 5.74 (dm, J = 149.4 Hz, 1 H), 5.45 (dd, J

= 15.4, 6.0 Hz, 1 H), 4.39–3.74 (m, 5 H), 2.04 (dm, J = 125.2 Hz,

2 H), 1.72–1.01 (m, 37 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm. ¹H NMR

(400 MHz, CDCl₃, ¹³C-decoupled): δ = 5.74 (dt, J = 15.4, 6.6 Hz,

1 H), 5.45 (dd, J = 15.4, 6.4 Hz, 1 H), 4.39–3.74 (m, 5 H), 2.04 (q,

J = 7.0 Hz, 2 H), 1.71–1.16 (m, 37 H), 0.88 (t, J = 6.8 Hz, 3 H)

ppm. ¹³C NMR (100 MHz, [D₆]DMSO, 363 K): δ = 151.3, 130.8

(d, J = 42.3 Hz), 130.4 (d, J = 73.4 Hz), 92.8, 78.4, 71.4 (d, J =

5.2 Hz), 63.7, 61.0 (d, J = 2.7 Hz), 31.9–30.5 (m), 29.7–26.1 (m),

Methyltriphenylphosphonium bromide (3.06 g, 8.58 mmol,

1.5 equiv.) was suspended in anhydrous THF (150 mL), and n-but-

yllithium (1.6 m in hexanes; 4.65 mL, 7.44 mmol, 1.3 equiv.) was

added at 0 °C. The reaction mixture was then stirred for 10 min at

0 °C. The crude [1,2,3,4,5-¹³C₅]-tetradecanal was dissolved in anhy-

drous THF (20 mL), and this solution was then added to the phos-

phorylide at 0 °C. The reaction mixture was stirred overnight at

room temperature, and then transferred to an extraction funnel

using Et₂O (100 mL). The reaction mixture was washed with water

(2ϫ 200 mL) and brine (200 mL). The aqueous phases were ex-

tracted with Et₂O (200 mL), and the combined organic extracts

were dried (Na₂SO₄), filtered, and concentrated in vacuo. Purifica-

tion by column chromatography (100% petroleum ether) gave com-

pound 20 (1.15 g, 5.34 mmol, 93%) as a colourless oil. R_f= 0.98

(100% petroleum ether). ¹H NMR (400 MHz, CDCl₃): δ = 5.81

(dm, J = 150.3 Hz, 1 H), 4.99 (dd, J = 17.1, 6.5 Hz, 1 H), 4.92 (t,

J = 10.8 Hz, 1 H), 2.03 (dm, J = 125.4 Hz, 2 H), 1.57–1.11 (m, 22

H), 0.88 (t, J = 6.8 Hz, 3 H) ppm. ¹³C NMR (101 MHz, CDCl₃):

δ = 139.2 (dm, J = 42.1 Hz), 114.0 (dd, J = 69.1, 3.1 Hz), 33.9 (m),

21.5, 13.2 ppm. IR (neat): ν = 3436, 2922, 2853, 1698, 1458, 1386,

˜

1365, 1256, 1173, 1098, 965, 848, 766 cm^–1. HRMS: calcd. for

[C₂₁¹³C₅H₄₉NO₄+ H]⁺445.3902; found 445.3902.

3-O-Benzoyl-N-(tert-butoxycarbonyl)-D-erythro-sphingosine (23a):

(E)-1,2-O,N-Isopropylidene-N-(tert-butoxycarbonyl)-d-erythro-

sphingosine (22a; 0.59 g, 1.3 mmol, 1.0 equiv.) was dissolved in a

mixture of pyridine and CH₂Cl₂(2:1; 10 mL). DMAP (16 mg,

0.13 mmol, 0.1 equiv.) was added, followed by benzoyl chloride

(0.23 mL, 2.0 mmol, 1.5 equiv.). The reaction mixture was stirred

overnight, and was then quenched with methanol (0.5 mL). The

mixture was concentrated in vacuo, and the residue was dissolved

in EtOAc (50 mL). The organic phase was washed with HCl (1 m

aq.; 50 mL), satd. aq. NaHCO₃(50 mL), and brine (50 mL). The

32.0, 29.9–28.6 (m), 22.7, 14.1 ppm. IR (neat): ν = 2922, 2853,

˜

1628, 1458, 1370, 1175, 1117, 996, 721 cm^–1.

(E)-1,2-O,N-Isopropylidene-N-(tert-butoxycarbonyl)-

D-erythro- aqueous layers were extracted with EtOAc (50 mL), and the com-

sphingosine (22a): (2S,3R)-2-Amino-N-(tert-butyloxycarbonyl)-1,3- bined organic layers were dried (Na₂SO₄), filtered, and concen-

dihydroxy-1,2-O,N-isopropylidene-4-pentene (21; 1 g, 4.0 mmol,

1.0 equiv.) and pentadec-1-ene (1.70 g, 8.0 mmol, 2.0 equiv.) were

dissolved in anhydrous CH₂Cl₂(4 mL), and the flask was flushed

trated in vacuo. Purification by column chromatography (1.5%

EtOAc in petroleum ether) gave 1,2-O,N-isopropylidene-3-O-

benzoyl-N-(tert-butyloxycarbonyl)-d-erythro-sphingosine (0.61 g,

with argon. Grubbs 2^ndgeneration catalyst (67 mg, 79 μmol, 1.1 mmol, 84%) as a colourless oil. R_f= 0.82 (10% EtOAc in petro-

0.02 equiv.) and acetic acid (45 μL, 0.79 mmol, 0.2 equiv.) were leum ether). [α]²_D²= –29 (c = 0.66, CHCl₃). ¹H NMR (400 MHz,

added. The reaction mixture was heated at reflux under a flow of

[D₆]DMSO, 363 K): δ = 8.00 (dm, J = 7.9 Hz, 1 H), 7.63 (m, 1 H),

2668

www.eurjoc.org

Eur. J. Org. Chem. 2015, 2661–2677

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

7.55–7.47 (m, 2 H), 5.82 (br. s, 1 H), 5.75 (dt, J = 15.4, 6.5 Hz, 1 δ = 8.10 (d, J = 7.4 Hz, 2 H), 7.55 (t, J = 7.4 Hz, 1 H), 7.44 (t, J

H), 5.53 (ddd, J = 15.4, 6.2, 1.4 Hz, 1 H), 4.09 (m, 1 H), 4.06–3.97 = 7.6 Hz, 2 H), 5.93–5.82 (m, 1 H), 5.82 (dm, J = 149.8 Hz, 1 H),

(m, 2 H), 2.01 (m, 2 H), 1.43 (s, 9 H), 1.40 (s, 3 H), 1.36–1.17 (m,

5.46 (m, 1 H), 4.25–4.10 (m, 1.5 H), 4.07–3.96 (m, 1.5 H), 2.03

25 H), 0.86 (t, J = 6.3 Hz, 3 H) ppm. ¹³C NMR (100 MHz, [D₆] (dm, J = 125.7 Hz, 2 H), 1.58–1.00 (m, 37 H), 0.88 (t, J = 6.9 Hz,

DMSO, 363 K): δ = 164.5, 134.4, 132.7, 129.7, 128.9, 128.1, 125.4,

93.2, 79.1, 73.4, 62.9, 59.1, 31.1, 30.8, 28.5 (ϫ2), 28.4 (ϫ2), 28.4,

3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 165.5, 165.4, 152.5,

151.7, 135.8 (d, J = 42.6 Hz) 135.7 (d, J = 42.6 Hz), 132.9, 132.8,

28.2, 27.8 (ϫ2), 27.6 (ϫ2), 21.5 (m), 13.3 ppm. IR (neat): ν = 2924, 130.5, 130.3, 129.8, 128.3, 125.0 (d, J = 72.8 Hz), 94.6, 94.0, 80.4,

˜

2854, 1724, 1701, 1365, 1268, 1097, 1070, 855, 709 cm^–1. HRMS:

calcd. for [C₃₃H₅₃NO₅+ Na]⁺566.3816; found 566.3814.

80.2, 74.4 (d, J = 5.6 Hz), 74.2 (d, J = 5.6 Hz), 63.70, 63.66, 60.00,

59.97, 32.8–31.7 (m), 29.8–28.2 (m), 22.7 (CH₂), 14.1 ppm. HRMS:

calcd. for [C₂₈¹³C₅H₅₃NO₅+ Na]⁺571.3984; found 571.3982.

1,2-O,N-Isopropylidene-3-O-benzoyl-N-(tert-butyloxycarbonyl)-d-

erythro-sphingosine (0.5 g, 0.92 mmol, 1.0 equiv.) was dissolved in

methanol/ethanol (1:1; 15 mL), and p-toluenesulfonic acid (mono-

hydrate; 87 mg, 0.46 mmol, 0.5 equiv.) was added. The reaction

mixture was stirred at ambient temperature overnight, and then

the reaction was quenched with triethylamine (0.32 mL, 2.3 mmol,

2.5 equiv.). The mixture was diluted with toluene (10 mL), and then

concentrated in vacuo. The residue was dissolved in EtOAc

(60 mL), and this solution was washed with satd. aq. Na₂HCO₃

(60 mL) and brine (50 mL). The aqueous layers were back-ex-

tracted with EtOAc (60 mL). The combined organic extracts were

dried (Na₂SO₄), filtered, and concentrated in vacuo. Purification

by column chromatography (10% EtOAc in petroleum ether) gave

compound 23a (0.25 g, 0.50 mmol, 54%; 88% based on recovered

starting material) as a colourless waxy solid. R_f= 0.07 (10% EtOAc

in petroleum ether). [α]²_D²= +15 (c = 1.0, CHCl₃). ¹H NMR

(400 MHz, CDCl₃): δ = 8.04 (dm, J = 7.5 Hz, 2 H), 7.57 (t, J =

7.4 Hz, 1 H), 7.44 (t, J = 7.7 Hz, 2 H), 5.87 (dt, J = 14.9, 6.6 Hz,

1 H), 5.60 (dd, J = 14.9, 7.7 Hz, 1 H), 5.53 (t, J = 7.3 Hz, 1 H),

5.12 (d, J = 8.9 Hz, 1 H), 3.95 (m, 1 H), 3.76–3.67 (m, 2 H), 2.82

(br. s, 1 H), 2.05 (m, 2 H), 1.43 (s, 9 H), 1.40–1.20 (m, 22 H), 0.88

(t, J = 6.8 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 166.2,

155.8, 137.3, 133.2, 129.8, 129.7, 128.4 (ϫ2), 124.6, 79.6, 74.8, 61.7,

54.5, 32.2, 31.9, 29.62 (ϫ3), 29.60, 29.5, 29.4, 29.3, 29.2, 28.9, 28.3,

[5,6,7,8,9-¹³C₅]-1,2-O,N-Isopropylidene-3-O-benzoyl-N-(tert-but-

oxycarbonyl)-d-erythro-sphingosine (120 mg, 0.22 mmol,

1.0 equiv.) was dissolved in methanol/ethanol (1:1; 10 mL), and p-

toluenesulphonic acid (monohydrate; 8.3 mg, 44 μmol, 0.2 equiv.)

was added. The reaction mixture was stirred overnight at room

temperature. The mixture was then transferred to an extraction

funnel using EtOAc (60 mL), and washed with satd. aq. NaHCO₃/

water (2:1; 60 mL), and brine (50 mL). The aqueous layer was ex-

tracted with EtOAc (60 mL). The combined organic extracts were

dried (Na₂SO₄), filtered, and concentrated in vacuo. Purification by

column chromatography (5–10% EtOAc in petroleum ether) gave

compound 23b (70 mg, 0.14 mmol, 63%; 83% based on recovered

starting material) as an amorphous solid. R_f= 0.07 (10% EtOAc

in petroleum ether). [α]²_D²= +16 (c = 0.5, CHCl₃). ¹H NMR

(400 MHz, CDCl₃): δ = 8.03 (dm, J = 7.8 Hz, 2 H), 7.57 (tt, J =

7.0, 1.5 Hz, 1 H), 7.45 (t, J = 7.8 Hz, 2 H), 5.88 (dm, J = 149.8 Hz,

1 H), 5.60 (m, 1 H), 5.52 (m, 1 H), 5.08 (d, J = 8.9 Hz, 1 H), 3.93

(m, 1 H), 3.76–3.67 (m, 2 H), 2.66 (br. s, 1 H), 2.08 (dm, J =

125.5 Hz, 2 H), 1.58–1.01 (m, 31 H), 0.88 (t, J = 6.8 Hz, 3 H)

ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 166.3, 155.8, 137.4 (d, J

= 42.5 Hz), 133.3, 129.80, 129.75, 128.4, 124.6 (d, J = 71.5 Hz),

79.7, 74.9 (d, J = 5.4 Hz), 61.9, 54.6, 33.0–31.6 (m), 29.8–28.1 (m),

22.7, 14.1 ppm. IR (neat): ν = 3372, 2922, 2853, 1696, 1505, 1452,

˜

1267, 1169, 1111, 1070, 1026, 966, 710 cm^–1. HRMS: calcd. for

[C₂₅¹³C₅H₄₉NO₅+ Na]⁺531.3671; found 531.3667.

22.6, 14.1 ppm. IR (neat): ν = 3372, 2924, 2854, 1715, 1268, 1171,

˜

1111, 1070, 969, 710 cm^–1. HRMS: calcd. for [C₃₀H₄₉NO₅+ Na]⁺

526.3503; found 526.3500.

[1,2,3-¹³C₃]-N-Methoxy-N-(methyl)-tetradecanamide (25): [1,2,3-

¹³C₃]-Myristic acid (24; 3.00 g, 13.0 mmol, 1.0 equiv.) was dissolved

in anhydrous CH₂Cl₂(26 mL), and the solution was put under an

atmosphere of argon and cooled to 0 °C. Oxalyl chloride (2.28 mL,

26.0 mmol, 2.0 equiv.) was added, followed by a drop of DMF. The

reaction mixture was then stirred under a flow of argon at room

temperature. When the evolution of gas stopped (ca. 2 h), the mix-

ture was concentrated in vacuo.

[5,6,7,8,9-¹³C₅]-3-O-Benzoyl-N-(tert-butoxycarbonyl)-

D-erythro-

sphingosine (23b): [5,6,7,8,9-¹³C₅]-1,2-O,N-Isopropylidene-N-(tert-

butyloxycarbonyl)-d-erythro-sphingosine (22b; 1.14 g, 2.56 mmol,

1.0 equiv.) was dissolved in a mixture of pyridine and CH₂Cl₂(2:1;

20 mL). DMAP (16 mg, 0.13 mmol, 0.05 equiv.) was added, fol-

lowed by benzoyl chloride (0.45 mL, 3.85 mmol, 1.5 equiv.). The

reaction mixture was stirred overnight, and then the reaction was

quenched with methanol (0.5 mL). The solvent was removed in

vacuo, and the resulting residue was dissolved in EtOAc (50 mL).

The residue was dissolved in anhydrous CH₂Cl₂(13 mL), and the

This solution was washed with HCl (1 m; 50 mL), satd. aq. solution was cooled to –78 °C. A solution of N,O-dimethylhydrox-

NaHCO₃(50 mL), and brine (40 mL). The aqueous layers were

extracted with EtOAc (50 mL), and the combined organic extracts

were dried (Na₂SO₄), filtered, and concentrated in vacuo. Purifica-

tion by column chromatography (1.5% EtOAc in petroleum ether)

gave [5,6,7,8,9-¹³C₅]-1,2-O,N-isopropylidene-3-O-benzoyl-N-(tert-

butoxycarbonyl)-d-erythro-sphingosine (1.13 g, 2.37 mmol, 92%)

as a colourless oil. R_f= 0.29 (5 % EtOAc in petroleum ether).

[α]²_D²= –30 (c = 0.5, CHCl₃). ¹H NMR (400 MHz, [D₆]DMSO,

363 K): δ = 8.00 (d, J = 7.6 Hz, 2 H), 7.64 (t, J = 7.4 Hz, 1 H),

7.52 (t, J = 7.6 Hz, 2 H), 5.82 (br. s, 1 H), 5.75 (dm, J = 149.2 Hz,

ylamine (2.30 mL, 32.5 mmol, 2.5 equiv.) in anhydrous CH₂Cl₂

(13 mL) was slowly added to the myristoyl chloride solution at

–78 °C. Then the stirred reaction mixture was allowed to reach

room temperature over 2 h. The reaction mixture was stirred at

room temperature for 30 min. The solids were removed by filtration

through a Whatmann paper, and washed with CH₂Cl₂. The filtrate

was concentrated in vacuo, and the residue was purified by column

chromatography (5–20% EtOAc in pentane) to give compound 25

(3.45 g, 12.7 mmol, 98%) as a colourless oil. R_f= 0.42 (20% EtOAc

1

in pentane). H NMR (400 MHz, CDCl₃): δ = 3.68 (s, 3 H), 3.13

1 H), 5.53 (m, 1 H), 4.15–3.97 (m, 3 H, 2-H), 2.04 (dm, J = (d, J = 2.0 Hz, 3 H), 2.41 (dm, J = 127.2 Hz, 2 H), 1.62 (dm, J =

126.1 Hz, 2 H), 1.54–1.01 (m, 37 H), 0.86 (t, J = 6.3 Hz, 3 H) ppm.

¹³C NMR (100 MHz, [D₆]DMSO, 363 K): δ = 164.5, 151.1, 134.4

(d, J = 42.6 Hz), 132.8, 129.7, 128.9, 128.2 (ϫ2), 125.2 (d, J =

72.2 Hz), 93.2, 79.2, 73.4 (d, J = 5.6 Hz), 62.9, 59.1, 31.8–30.4 (m),

128.8 Hz, 2 H), 1.35–1.22 (m, 20 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm.

¹³C NMR (100 MHz, CDCl₃): δ = 175.0 (d, J = 51.0 Hz), 61.4,

32.04, 31.99 (dd, J = 51.0, 34.0 Hz), 29.8–29.3 (m), 24.77 (dd, J =

35.0, 2.0 Hz), 22.8, 14.2 ppm. IR (neat): ν = 2924, 2855, 1616, 1462,

˜

28.8–27.3 (m), 21.6, 13.4 ppm. The same sample in CDCl₃at room 1375, 1176, 908, 729 cm^–1. HRMS: calcd. for [C₁₃¹³C₃H₃₃NO₂

+

temperature showed two rotamers: ¹H NMR (400 MHz, CDCl₃):

H]⁺275.2612; found 275.2683.

Eur. J. Org. Chem. 2015, 2661–2677

www.eurjoc.org

2669

J. D. C. Codée, H. S. Overkleeft et al.

Ethyl (E)-[3,4,5-¹³C₃]-Hexadec-2-enoate (27): [1,2,3-¹³C₃]-N-(Meth- tracts were dried (Na₂SO₄), filtered, and concentrated in vacuo.

FULL PAPER

oxy)-N-methyl-tetradecanamide (25; 2.74 g, 10.0 mmol, 1.0 equiv.)

Purification by column chromatography (5% EtOAc, 1% AcOH in

was dissolved in dry THF (20 mL). The solution was cooled to

pentane) gave 29 (1.89 g, 6.94 mmol, 95%) as a white solid. R_f=

–78 °C, and then DIBAL-H (1.5 m in toluene; 8.0 mL, 12.0 mmol, 0.6 (10 % EtOAc, 1 % AcOH in pentane). ¹H NMR (400 MHz,

1.2 equiv.) was added. The reaction mixture was stirred for 30 min, CDCl₃): δ = 11.40 (br. s, 1 H), 2.35 (m, 2 H), 1.61 (dm, J =

then it was quenched with satd. aq. Rochelle salt (12 mL). The

mixture was then transferred to an extraction funnel with EtOAc

(50 mL), and washed with water (40 mL) and brine (40 mL). The

130.0 Hz, 2 H), 1.52–1.06 (m, 24 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm.

¹³C NMR (100 MHz, CDCl₃): δ = 180.1, 32.1, 29.9–28.7 (m), 25.2–

24.4 (m), 22.8, 14.3 ppm. IR (neat): ν = 2912, 2847, 1694, 1470,

˜

aqueous layers were extracted with EtOAc (50 mL). The combined 1430, 1308, 1288, 941, 718, 679 cm^–1.

organic extracts were dried (Na₂SO₂), filtered, and concentrated in

[3,4,5-¹³C₃]-Palmitoyl Chloride (30): [3,4,5-¹³C₃]-Palmitic acid

vacuo to give crude [1,2,3-¹³C₃]-tetradecanal (2.15 g, 10.0 mmol) as

a colourless oil, which was used without further purification.

(1.80 g, 6.93 mmol, 1.0 equiv.) was dissolved in dry CH₂Cl₂

(72 mL), and the solution was put under an atmosphere of argon,

and cooled to 0 °C. Oxalyl chloride (1.2 mL, 14 mmol, 2 equiv.)

was added, followed by DMF (one drop). The reaction mixture

was then kept under a flow of argon at room temperature. When

the evolution of gas stopped (ca. 2 h), the mixture was concentrated

in vacuo to give compound 30 (1.90 g, 6.93 mmol, 100 %). ¹H

NMR (400 MHz, CDCl₃): δ = 2.88 (m, 2 H), 1.70 (dm, J =

130.5 Hz, 2 H), 1.56–1.05 (m, 24 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm.

¹³C NMR (100 MHz, CDCl₃): δ = 166.4, 32.0, 29.8–28.0 (m),

Triethyl phosphonoacetate (26; 3.14 g, 14.0 mmol, 1.4 equiv.) was

dissolved in dry THF (50 mL), and the solution was cooled to 0 °C.

n-Butyllithium (1.6 m in hexanes; 7.8 mL, 12.5 mL, 1.25 equiv.) was

added, and the reaction mixture was stirred for 10 min at 0 °C. The

crude [1,2,3-¹³C₃]-tetradecanal was dissolved in anhydrous in THF

(10 mL), and this solution was added to the Horner–Wadsworth–

Emmons reagent at 0 °C. The mixture was then stirred overnight

at room temperature. The mixture was transferred to an extraction

funnel with Et₂O (50 mL), and washed with water (50 mL) and

brine (50 mL). The aqueous layers were extracted with Et₂O

(50 mL), and the combined organic extracts were dried with

(Na₂SO₄), filtered, and concentrated in vacuo. Purification by col-

umn chromatography (0–2% EtOAc in pentane) gave compound

27 (2.3 g, 8.1 mmol, 81%) as a colourless oil. R_f= 0.58 (2% EtOAc

in pentane). ¹H NMR (400 MHz, CDCl₃): δ = 6.96 (dm, J =

152.0 Hz, 1 H), 5.81 (dd, J = 15.6, 5.2 Hz, 1 H), 4.18 (q, J = 7.2 Hz,

2 H), 2.19 (dm, J = 126.0 Hz, 2 H), 1.62–1.22 (m, 25 H), 0.88 (t, J

= 6.8 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 166.9 (d,

J = 6.0 Hz), 150.84 (dt, J = 39.0, 17.0 Hz), 149.65 (dd, J = 41.0,

2.0 Hz), 60.24, 32.33 (dd, J = 41.0, 34.0 Hz), 29.8–29.0 (m), 28.1

25.15, 24.14 (dd, J = 32.0, 1.5 Hz), 22.8, 14.2 ppm. IR (neat): ν =

˜

; 2918, 2848, 2747, 1800, 1660, 1384, 1305, 1161, 1033, 908,

802 cm^–1.

D

-erythro-Sphingosine (31a): 3-O-Benzoyl-N-(tert-butyloxycarb-

onyl)-d-erythro-sphingosine (23a; 90 mg, 0.30 mmol, 1.0 equiv.)

was dissolved in methanol (10 mL), and sodium methoxide (30%

in methanol; 20 μL, 0.15 mmol, 0.5 equiv.) was added. The reaction

mixture was stirred at room temperature until TLC showed full

conversion into a lower-running spot. Potassium hydroxide (0.5 m

in water; 1.2 mL, 0.60 mmol, 2.0 equiv.) was added, and the reac-

tion mixture was stirred overnight at ambient temperature. The re-

action was quenched with acetic acid (0.09 mL, 1.5 mmol, 5 equiv.),

and then the mixture was concentrated in vacuo.

(dd, J = 34.0, 2.0 Hz), 22.8, 14.4, 14.3 ppm. IR (neat): ν = 2922,

˜

2852, 1720, 1626, 1466, 1365, 1301, 1263, 1175, 1034, 977,

721 cm^–1. HRMS: calcd. for [C₁₅¹³C₃H₃₄O₂+ H]⁺286.2738; found

286.2733.

The residue was cooled to 0 °C, and then water (1.0 mL) and TFA

(3 mL) were added. The reaction mixture was stirred for 2 min at

0 °C, then it was diluted with toluene (40 mL), and concentrated

in vacuo. Purification by HPLC–MS (52–62% B, following the ge-

neral procedure for HPLC–MS purifications) gave compound 31a

(41 mg, 0.1 mmol, 54%) as a TFA adduct. [α]²_D²= –2.0 (c = 0.5,

MeOH). ¹H NMR (600 MHz, [D₄]methanol): δ = 5.85 (m, 1 H),

5.47 (m, 1 H), 4.28 (m, 1 H), 3.79 (dd, J = 11.6, 4.0 Hz, 1 H), 3.66

(dd, J = 11.6, 8.4 Hz, 1 H), 3.19 (dt, J = 8.6, 4.4 Hz, 1 H), 2.10 (q,

J = 7.1 Hz, 2 H), 1.45–1.32 (m, 2 H), 1.35–1.26 (m, 20 H), 0.90 (t,

J = 7.0 Hz, 3 H) ppm. ¹³C NMR (150 MHz, [D₄]methanol): δ =

136.6, 128.5, 71.0, 59.4, 58.5, 33.4, 33.1, 30.81, 30.80 (2ϫ), 30.77,

Ethyl [3,4,5-¹³C₃]-hexadecanoate (28): Ethyl (E)-[3,4,5-¹³C₃]-hexa-

dec-2-enoate (27; 2.20 g, 7.71 mmol, 1.0 equiv.) was dissolved in

EtOAc (40 mL). The solution was purged with argon while stirring,

and then palladium (10 % on charcoal; 0.41 g, 0.38 mmol,

0.05 equiv.) was added. The reaction mixture was then stirred under

a flow of hydrogen gas for 30 min, and then it was left under a

hydrogen atmosphere overnight. The palladium residue was re-

moved by filtration through a Whatmann paper, and rinsed with

EtOAc (50 mL). The solvent was removed from the filtrate in

vacuo. Purification by column chromatography (1 % EtOAc in

pentane) gave compound 28 (2.21 g, 7.32 mmol, 95%) as a colour-

less oil. R_f= 0.58 (2% EtOAc in pentane). ¹H NMR (400 MHz,

CDCl₃): δ = 4.12 (q, J = 7.2 Hz, 2 H), 2.28 (m, 2 H), 1.61 (dm, J

= 130.8 Hz, 2 H), 1.46–1.08 (m, 27 H), 0.88 (t, J = 6.8 Hz, 3 H)

ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 174.0, 62.0, 32.1, 29.8–

30.75, 30.65, 30.5, 30.4, 30.2, 23.8, 14.4 ppm. IR (neat): ν = 3289,

˜

2918, 2850, 1668, 1520, 1470, 1192, 1134, 986, 720 cm^–1. HRMS:

calcd. for [C₁₈H₃₇NO₂+ H]⁺300.2897; found 300.2899.

[5,6,7,8,9-¹³C₅]- -erythro-Sphingosine (31b): [5,6,7,8,9-¹³C₅]-3-O-

D

Benzoyl-N-(tert-butoxycarbonyl)-d-erythro-sphingosine 23b

(90 mg, 0.29 mmol, 1 equiv.) was dissolved in methanol (10 mL),

and sodium methoxide (30 % in methanol; 19 μL, 0.14 mmol,

0.5 equiv.) was added. The reaction mixture was stirred at room

temperature until TLC showed full conversion into a lower-running

spot. Potassium hydroxide (0.5 m in water; 1.2 mL, 0.59 mmol,

2 equiv.) was added, and the reaction mixture was stirred overnight

at ambient temperature. The reaction was quenched with acetic

acid (0.08 mL, 1.45 mmol, 5 equiv.), and then the mixture was con-

centrated in vacuo.

28.8 (m), 25.4–24.7 (m), 22.8, 14.3, 14.2 ppm. IR (neat): ν = 2920,

˜

2851, 1738, 1463, 1238, 1174, 1035, 733 cm^–1. HRMS: calcd. for

[C₁₅¹³C₃H₃₆O₂+ H]⁺288.2894; found 288.2889.

[3,4,5-¹³C₃]-Palmitic Acid (29): Ethyl [3,4,5-¹³C₃]-hexadecanoate

(28; 2.10 g, 7.30 mmol, 1.0 equiv.) was dissolved in THF/EtOH/

H₂O (1:1:1; 35 mL), and lithium hydroxide (0.52 g, 21.9 mmol,

3.0 equiv.) was added. The reaction mixture was stirred at room

temperature overnight. The mixture was then transferred to an ex-

traction funnel with EtOAc (50 mL), and washed with HCl (1 m

aq.; 50 mL), water (50 mL), and brine (50 mL). The aqueous layers

were extracted with EtOAc (50 mL), and the combined organic ex-

The residue was cooled to 0 °C, then water (1 mL) and TFA (3 mL)

were added. The reaction mixture was stirred for 2 min at 0 °C,

then it was diluted with toluene (40 mL) and concentrated in vacuo.

2670

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Eur. J. Org. Chem. 2015, 2661–2677

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

Purification by HPLC–MS (52–62% B, following the general pro-

cedure for HPLC–MS purifications) gave compound 31b (52 mg,

0.17 mmol, 59%) as a TFA adduct. [α]²_D²= –2.0 (c = 0.5, MeOH).

(600 MHz, CDCl₃): δ = 6.26 (d, J = 7.8 Hz, 1 H), 5.76 (dm, J =

150.0 Hz, 1 H), 5.53 (m, 1 H), 4.31 (m, 1 H), 3.95 (dd, J = 11.2,

4.0 Hz, 1 H), 3.90 (m, 2 H), 3.70 (dd, J = 11.4, 3.2 Hz, 1 H), 3.00–

¹H NMR (600 MHz, [D₄]methanol): δ = 5.85 (dm, J = 150 Hz, 1 2.60 (br. s, 2 H), 2.23 (m, 2 H), 2.05 (dm, J = 124.0 Hz, 2 H), 1.63

H), 5.47 (dt, J = 15.6, 6.0 Hz, 1 H), 4.28 (dd, J = 11.4, 4.8 Hz, 1 (dm, J = 130 Hz, 2 H), 1.50–1.15 (m, 46 H), 0.88 (t, J = 7.0 Hz, 6

H), 3.79 (dd, J = 11.6, 4.0 Hz, 1 H), 3.66 (dd, J = 11.6, 8.3 Hz, 1

H), 3.19 (dt, J = 8.5, 4.3 Hz, 1 H), 2.1 (dm, J = 126.0 Hz, 2 H),

H) ppm. ¹³C NMR (151 MHz, CDCl₃): δ = 174.1, 134.4 (d, J =

43.0 Hz), 128.8 (d, J = 72.0 Hz), 74.5 (d, J = 20.8 Hz), 62.6, 54.6

1.56–1.20 (m, 22 H), 0.90 (t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR (d, J = 2.6 Hz), 37.0 (d, J = 35.0 Hz), 32.8–32.0 (m), 29.9–29.0 (m),

(151 MHz, [D₄]methanol): δ = 135.6 (dd, J = 42.0, 3.0 Hz), 128.5 28.2–27.7 (m), 26.2–25.6 (m), 21.7, 14.3 (2ϫ) ppm. IR (neat): ν =

˜

(dd, J = 72.0, 3.0 Hz), 71.0 (dd, J = 5.5, 1.3 Hz), 59.5, 58.5 (d, J = 3294, 2914, 2847, 1699, 1636, 1547, 1464, 1040, 970, 719 cm^–1

.

3.0 Hz), 33.8–32.9 (m), 30.9–29.8 (m), 23.7, 14.5 ppm. IR (neat): ν HRMS: calcd. for [C₂₆¹³C₈H₆₇NO₃+ H]⁺546.5121; found

˜

= 3287, 2914, 2847, 1661, 1526, 1470, 1198, 1136, 966, 721 cm^–1. 546.5461.

HRMS: calcd. for [C₁₃¹³C₅H₃₇NO₂+ H]⁺305.2897; found

305.3065.

Sphinganine (33a): 3-O-Benzoyl-N-(tert-butyloxycarbonyl)-d-

erythro-sphingosine (23a; 36.8 mg, 0.07 mmol, 1.0 equiv.) was dis-

solved in methanol (2.4 mL), and sodium methoxide (30% in meth-

anol; 4.6 μL, 0.035 mmol, 0.5 equiv.) was added. The reaction mix-

ture was stirred at room temperature until TLC showed full conver-

sion into a lower-running spot. Potassium hydroxide (0.5 m in

water; 0.28 mL, 0.14 mmol, 2.0 equiv.)was added, and the reaction

mixture was stirred overnight at ambient temperature. The reaction

was quenched with acetic acid (0.019 mL, 0.35 mmol, 5.0 equiv.),

and then the mixture was concentrated in vacuo. The residue was

coevaporated once with toluene (4.0 mL).

Ceramide (32a): See the general procedure for the synthesis of

ceramides from sphingosine, yield (20 mg, 37 μmol, 79 %). R_f=

0.48 (EtOAc/pentane, 1:1). [α]²_D²= –7.6 (c = 1.0, MeO/CHCl₃, 1:1).

¹H NMR (600 MHz, CDCl₃): δ = 6.26 (d, J = 7.8 Hz, 1 H), 5.78

(dt, J = 15.4, 7.0 Hz, 1 H), 5.53 (dd, J = 15.4, 6.5 Hz, 1 H), 4.31

(t, J = 4.7 Hz, 1 H), 3.95 (dd, J = 11.2, 3.8 Hz, 1 H), 3.90 (m, 1

H), 3.70 (dd, J = 11.4, 3.6 Hz, 1 H), 3.00–2.60 (br. s, 2 H), 2.23 (t,

J = 7.7 Hz, 2 H), 2.05 (q, J = 7.2 Hz, 2 H), 1.63 (m, 2 H), 140–

1.21 (m, 46 H), 0.88 (t, J = 7.0 Hz, 6 H) ppm. ¹³C NMR (150 MHz,

CDCl₃): δ = 174.1, 134.4, 128.9, 74.5, 62.4, 54.7, 37.0, 32.4, 32.08,

29.86 (4 ϫ), 29.85 (4 ϫ), 29.82 (3 ϫ), 29.80, 29.78, 29.67, 29.65,

29.53, 29.52 ϫ2, 29.45, 29.39, 29.28, 25.92, 22.84, 14.3 (2ϫ) ppm.

The residue was dissolved in EtOAc (1 mL). The solution was

purged with argon, and then platinum dioxide (1.5 mg,

0.007 mmol, 0.1 equiv.) was added. The reaction mixture was

stirred under a flow of hydrogen gas for 30 min, and then it was

left under a hydrogen atmosphere overnight. The platinum dioxide

residue was removed by filtration through a plug of Celite, and

then rinsed with EtOAc. The filtrate was concentrated in vacuo.

IR (neat): ν = 3308, 2914, 2865, 1645, 1548, 1464, 1049, 959,

˜

719 cm^–1. HRMS: calcd. for [C₃₄H₆₇NO₃+ H]⁺538.5121; found

538.5192.

2-N-([3,4,5-¹³C₃]-Hexadecanoyl)-sphingosine (32b): See the general

procedure for the synthesis of ceramides from sphingosine, yield

The resulting residue was cooled to 0 °C, and then water (1 mL)

and TFA (2 mL) were added. The reaction mixture was stirred for

2 min at 0 °C, then it was diluted with toluene (4 mL), and concen-

trated in vacuo. Purification by HPLC–MS (52–62% B, following

the general procedure for HPLC–MS purifications) gave compound

33a (10 mg, 33 μmol, 47%) as a TFA adduct. [α]²_D²= –7.0 (c = 0.1,

MeOH). ¹H NMR (600 MHz, [D₄]methanol): δ = 3.83 (dd, J =

11.6, 4.0 Hz, 1 H), 3.77 (dt, J = 8.4, 4.2 Hz, 1 H), 3.70 (dd, J =

11.5, 8.7 Hz, 1 H), 3.19 (dt, J = 8.3, 3.9 Hz, 1 H), 1.55–1.22 (m, H

28), 0.90 (t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR (151 MHz, [D₄]meth-

anol): δ = 70.3, 58.8, 58.4, 34.2, 33.1, 30.84 (4ϫ), 30.78, 30.77,

(14 mg, 25 μmmol, 71%). R_f= 0.48 (EtOAc/pentane, 1:1). [α]²_D²

=

–8.0 (c = 0.1, MeOH/CHCl₃, 1:1). ¹H NMR (600 MHz, CDCl₃/

[D₄]methanol): δ = 6.26 (d, J = 7.8 Hz, 1 H), 5.78 (dt, J = 15.4,

7.0 Hz, 1 H), 5.53 (dd, J = 15.4, 6.5 Hz, 1 H), 4.31 (t, J = 4.7 Hz,

1 H), 3.95 (dd, J = 11.2, 3.8 Hz, 1 H), 3.90 (m, 1 H), 3.70 (dd, J =

11.4, 3.6 Hz, 1 H), 3.00–2.60 (br. s, 2 H), 2.23 (m, 2 H), 2.05 (q, J

= 7.2 Hz, 2 H), 1.63 (dm, J = 130 Hz, 2 H), 1.45–1.15 (m, 46 H),

0.88 (t, J = 7.0 Hz, 6 H) ppm. ¹³C NMR (151 MHz, CDCl₃/[D₄]

methanol): δ = 174.1, 134.4, 128.9, 74.8, 62.6, 54.7, 37.0 (d, J =

35.0 Hz), 32.4, 32.0, 29.91, 29.85–29.38 (m), 26.15–25.79 (m),

22.84, 14.3 (2ϫ) ppm. IR (neat): ν = 3293, 2914, 2847, 1636, 1547,

˜

30.74, 30.70, 30.57, 30.49, 27.0, 23.8, 14.5 ppm. IR (neat): ν = 3150,

˜

1465, 1038, 972, 721 cm^–1. HRMS: calcd. for [C₃₁¹³C₃H₆₇NO₃

H]⁺541.5121; found 541.5293.

+

2914, 2849, 1676, 1207, 1186, 1153, 1126, 1053, 840, 800, 721 cm^–1.

HRMS: calcd. for [C₁₈H₃₉NO₂+ H]⁺302.2981; found 302.3054.

2-N-(Hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphingosine (32c): See the ge-

neral procedure for the synthesis of ceramides from sphingosine,

yield (12 mg, 22 μmol, 73 %). R_f= 0.48 (EtOAc/pentane, 1:1).

[α]²_D²= –7.2 (c = 0.25, MeOH/CHCl₃, 1:1). ¹H NMR (600 MHz,

CDCl₃): δ = 6.31 (d, J = 7.8 Hz, 1 H), 5.76 (dm, J = 150.0 Hz, 1

H), 5.53 (m, 1 H), 4.30 (m, 1 H), 3.95 (m, 1 H), 3.90 (m, 1 H), 3.70

(m, 1 H), 3.00–2.60 (br. s, 2 H), 2.22 (t, J = 7.9 Hz, 2 H), 2.05 (dm,

J = 124.0 Hz, 2 H), 1.63 (m, 2 H), 1.52–1.13 (m, 46 H), 0.88 (t, J

= 7.0 Hz, 6 H) ppm. ¹³C NMR (151 Hz, CDCl₃): δ = 174.2, 133.0

(d, J = 43.0 Hz), 129.1 (d, J = 72.0 Hz), 74.7 (d, J = 20.8 Hz), 62.6,

54.6 (d, J = 2.6 Hz), 37.0, 32.8–32.0 (m), 29.9–29.0 (m), 28.25–

[5,6,7,8,9-¹³C₅]-Sphinganine (33b): [5,6,7,8,9-¹³C₅]-3-O-Benzoyl-N-

(tert-butoxycarbonyl)-d-erythro-sphingosine (23b; 81.4 mg,

0.16 mmol, 1.0 equiv.) was dissolved in methanol (5.5 mL), and so-

dium methoxide (30% in methanol; 10 μL, 0.08 mmol, 0.5 equiv.)

was added. The reaction mixture was stirred at room temperature

until TLC showed full conversion into a lower-running spot. Potas-

sium hydroxide (0.5 m in water; 0.64 mL, 0.32 mmol, 2.0 equiv.)was

added, and the reaction mixture was stirred overnight at ambient

temperature. The reaction was quenched with acetic acid (4.4 μL,

0.8 mmol, 5.0 equiv.), then the mixture was concentrated in vacuo.

The residue was coevaporated once with toluene (10 mL).

27.74 (m), 25.92 (m), 22.84, 14.3 (2ϫ) ppm. IR (neat): ν = 3300,

˜

The residue was dissolved in EtOAc (2 mL). The solution was

purged with argon, then platinum dioxide (3.6 mg, 0.016 mmol,

0.1 equiv.) was added. The reaction mixture was stirred under a

2914, 2847, 1701, 1635, 1547, 1464, 1124, 970, 719 cm^–1. HRMS:

calcd. for [C₂₉¹³C₅H₆₇NO₃+ H]⁺543.5121; found 543.5358.

2-N-([3,4,5-¹³C₃]-Hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphingosine (32d): flow of hydrogen gas for 30 min, and then it was left under a

See the general procedure for the synthesis of ceramides from

sphingosine, yield (18 mg, 33 μmol, 81%). R_f= 0.48 (EtOAc/pent-

ane, 1:1). [α]²_D²= –7.0 (c = 0.33, MeOH/CHCl₃, 1:1). ¹H NMR

hydrogen atmosphere overnight. The platinum dioxide residue was

removed by filtration through a plug of Celite, and then rinsed with

EtOAc. The filtrate was concentrated in vacuo.

Eur. J. Org. Chem. 2015, 2661–2677

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2671

J. D. C. Codée, H. S. Overkleeft et al.

FULL PAPER

The residue was cooled to 0 °C, then water (0.5 mL) and TFA

(1.5 mL) were added. The reaction mixture was stirred for 2 min at

0 °C, then it was diluted with toluene (10 mL) and concentrated in

vacuo. Purification by HPLC–MS (52–62% B, following the gene-

ral procedure for HPLC–MS purifications) gave compound 33b

(25 mg, 83 μmol, 52%) as a TFA adduct. [α]²_D²= –7.5 (c = 0.1,

MeOH). ¹H NMR (600 MHz, [D₄]methanol): δ = 3.83 (dd, J =

11.5, 4.0 Hz, 1 H), 3.76 (dt, J = 8.3, 4.3 Hz, 1 H), 3.69 (dd, J =

11.5, 8.8 Hz, 1 H), 3.18 (dt, J = 8.9, 4.0 Hz, 1 H), 1.65–1.15 (m, 28

H), 0.90 (t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR (151 MHz, [D₄]meth-

anol): δ = 70.3, 58.6, 58.4, 34.1 (d, J = 34.7 Hz), 33.1, 31.1–30.2

1726, 1630, 1572, 1470, 1047, 716 cm^–1. HRMS: calcd. for

[C₂₆¹³C₈H₆₇NO₃+ H]⁺548.5121; found 548.5611.

Phenyl 4,6-O-(Di-tert-butylsilanediyl)-1-thio-β-D-glucosylpyranoside

(35b): Phenyl 1-thio-β-d-glucoside (35a; Scheme 5; 1.85 g,

6.8 mmol, 1.05 equiv.) was dissolved in dry DMF (27 mmol) under

an argon atmosphere. This solution was cooled down to –40 °C,

and then di-tert-butylsilylbis(trifluoromethanesulfonate) (2.1 mL,

6.5 mmol, 1 equiv.) was added dropwise. The resulting reaction

mixture was stirred at –40 °C for 30 min, and then pyridine

(1.58 mL, 19.5 mmol, 3 equiv.) was added. The reaction mixture

was stirred for a further 15 min, and then it was transferred to an

extraction funnel with Et₂O (50 mL). The organic phase was

washed with water (2ϫ100 mL) and brine (100 mL). The aqueous

layers were extracted with Et₂O (50 mL), and the combined organic

extracts were dried (Na₂SO₄), filtered, and concentrated in vacuo.

Purification by silica column chromatography (10% Et₂O in petro-

(m), 27.3–26.7 (m), 23.8, 14.5 (C-18) ppm. IR (neat): ν = 3120,

˜

2914, 2847, 1676, 1206, 1186, 1153, 1130, 840, 800, 723 cm^–1

HRMS: calcd. for [C₁₈H₃₉NO₂+ H]⁺307.2981; found 307.3222.

.

Dihydroceramide (34a): See the general procedure for the synthesis

of ceramides from sphingosine, yield (12 mg, 22 μmol, 68%). R_f=

0.50 (EtOAc/pentane, 1:1). [α]²_D²= +4.5 (c = 0.15, MeOH/CHCl₃,

leum ether) gave compound 35b (2.16 g, 5.2 mmol, 77%). R_f= 0.8

1

1:1). H NMR (600 MHz, CDCl₃, 318 K): δ = 6.36 (d, J = 7.6 Hz, (50% EtOAc in petroleum ether). [α]²_D²= –37 (c = 1.0, CHCl₃). H

1 H), 4.01 (d, J = 11.3 Hz, 1 H), 3.83 (m, 1 H), 3.80–3.72 (m, 2 NMR (400 MHz, CDCl₃): δ = 7.53–7.50 (m, 2 H), 7.33–7.28 (m, 3

H), 2.90–2.50 (br. s, 2 H), 2.23 (t, J = 7.4 Hz, 2 H), 1.68–1.59 (m, H), 4.61 (d, J = 10.0 Hz, 1 H), 4.21 (dd, J = 10.3, 5.2 Hz, 1 H),

4 H), 1.59–1.45 (m, 2 H), 1.38–1.19 (m, 46 H), 0.88 (t, J = 7.2 Hz,

6 H) ppm. ¹³C NMR (151 MHz, CDCl₃, 318 K): δ = 173.7, 74.4,

62.7, 54.2, 37.1, 34.7, 32.10, 29.86 (4ϫ), 29.84 (3ϫ), 29.82 (3ϫ),

29.79 (2ϫ), 29.75 (3ϫ), 29.72, 29.71, 29.67, 29.66, 29.52, 29.51

3.90 (t, J = 10.2 Hz, 1 H), 3.69 (t, J = 9.2 Hz, 1 H), 3.61 (t, J =

8.7 Hz, 1 H), 3.49 (m, 2 H), 3.25 (br. s, 2 H), 1.05 (s, 3 H), 0.98 (s,

3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 132.9, 131.9, 129.1,

128.3, 88.6, 77.9, 76.5, 74.6, 71.9, 66.2, 27.5, 27.1, 22.8, 20.0 ppm.

(2ϫ), 29.48, 22.84, 14.2 (2ϫ) ppm. IR (neat): ν = 3395, 2914, 2849,

IR (neat): ν = 3380, 2931, 2858, 1472, 1055, 823, 731, 651 cm^–1.

˜

1738, 1630, 1570, 1470, 1047, 719 cm^–1. HRMS: calcd. for

[C₃₄H₆₇NO₃+ H]⁺540.5121; found 540.5347.

HRMS: calcd. for [C₂₀H₃₂O₅SSi + H]⁺413.1740; found 413.1801.

2-N-([3,4,5-¹³C₃]-Hexadecanoyl)-sphinganine (34b): See the general

procedure for the synthesis of ceramides from sphingosine (15 mg,

27 μmol, 74%). R_f= 0.50 (EtOAc/pentane, 1:1). [α]²_D²= +4.8 (c =

1

0.1, MeOH/CHCl₃, 1:1). H NMR (600 MHz, CDCl₃, 318 K): δ =

6.26 (d, J = 7.6 Hz, 1 H), 4.01 (d, J = 11.3 Hz, 1 H), 3.83 (m, 1

H), 3.80–3.72 (m, 2 H), 2.80–2.40 (br. s, 2 H), 2.23 (m, 2 H), 1.80–

1.10 (m, 54 H), 0.88 (t, J = 7.2 Hz, 6 H) ppm. ¹³C NMR (151 MHz,

CDCl₃, 318 K): δ = 173.7, 74.5, 62.7, 54.1, 37.1 (d, J = 34.0 Hz),

34.8, 32.09, 29.9–29.3 (m), 29.18, 26.2–25.8 (m), 25.67, 22.83, 14.2

(2ϫ) ppm. IR (neat): ν = 3394, 2914, 2849, 1738, 1630, 1570, 1470,

˜

1049, 719 cm^–1. HRMS: calcd. for [C₃₁¹³C₃H₆₇NO₃+ H]⁺

543.5121; found 543.5442.

Scheme 5. Reagents and conditions: (a) tBu₂SiOTf₂, pyridine,

DMF, –40 °C, 30 min, 77%; (b) BzCl, pyridine, room temp., 3 h,

98%; (c) NIS (N-iodosuccinimide), TFA, CH₂Cl₂, 0 °C, 3 h, 98%;

(d) ClC(NPh)CF₃, CsCO₃, acetone, 0 °C, 2 h, 80%.

2-N-(Hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphinganine (34c): See the ge-

neral procedure for the synthesis of ceramides from sphingosine,

yield (14 mg, 25 μmol, 65 %). R_f= 0.50 (EtOAc/pentane, 1:1).

[α]²_D²= +5.2 (c = 0.25, MeOH/CHCl₃, 1:1). ¹H NMR (600 MHz,

CDCl₃, 318 K): δ = 6.26 (d, J = 7.6 Hz, 1 H), 4.01 (d, J = 11.3 Hz,

1 H), 3.83 (m, 1 H), 3.80–3.72 (m, 2 H), 2.70–2.40 (br. s, 2 H) 2.22

(t, J = 7.9 Hz, 2 H), 1.65–1.15 (m, 54 H), 0.89 (t, J = 7.2 Hz, 6 H)

ppm. ¹³C NMR (151 MHz, CDCl₃, 318 K): δ = 173.7, 74.4, 62.7,

54.1 (d, J = 2.6 Hz), 37.1, 34.8 (d, J = 35.0 Hz), 34.0, 32.10 29.9–

Phenyl 2,3-Di-O-benzoyl-4,6-O-(di-tert-butylsilanediyl)-1-thio-β-D-

glucopyranoside (35c): Phenyl 4,6-O-(di-tert-butylsilanediyl)-1-thio-

β-d-glucosylpyranoside (35b; 2.16 g, 5.2 mmol, 1.0 equiv.) was dis-

solved in dry pyridine (13 mL), and benzoyl chloride (3.25 mL,

28.0 mmol, 2.4 equiv.) was added. The reaction mixture was stirred

until TLC showed full conversion into a higher-running product.

Then the reaction was quenched with methanol (1 mL), and the

mixture was concentrated in vacuo. The residue was dissolved in

29.5 (m), 26.4–25.8 (m), 22.8, 14.2 (2ϫ) ppm. IR (neat): ν = 3399,

˜

2914, 2849, 1630, 1568, 1470, 1047, 717 cm^–1. HRMS: calcd. for

[C₂₉¹³C₅H₆₇NO₃+ H]⁺545.5121; found 545.5515.

2-N-([3,4,5-¹³C₃]-Hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphinganine (34d): EtOAc (50 mL), and this solution was washed with HCl (1 n aq.;

See the general procedure for the synthesis of ceramides from

sphingosine, yield (18 mg, 33 μmol, 66%). R_f= 0.50 (EtOAc/pent-

ane, 1:1). [α]²_D²= +5.0 (c = 0.25, MeOH/CHCl₃, 1:1). ¹H NMR

(600 MHz, CDCl₃, 318 K): δ = 6.26 (d, J = 7.6 Hz, 1 H), 4.01 (d,

J = 11.3 Hz, 1 H), 3.83 (m, 1 H), 3.80–3.72 (m, 2 H), 2.55 (br. s, 1

50 mL), satd. aq. NaHCO₃(50 mL), and brine (50 mL). The aque-

ous layers were extracted with EtOAc (50 mL), and the combined

organic extracts were dried (Na₂SO₄), filtered, and concentrated in

vacuo. Purification by silica gel column chromatography (10%

Et₂O in petroleum ether) gave compound 35c (3.18 g, 5.12 mmol,

H), 2.45 (br. s, 1 H), 2.23 (m, 2 H), 1.72–1.12 (m, 54 H), 0.88 (t, J 98%). [α]²_D²= +47.2 (c = 1.0, CHCl₃); R_f= 0.7 (15% EtOAc in

= 7.2 Hz, 6 H) ppm. ¹³C NMR (151 MHz, CDCl₃, 318 K): δ = δ

= 173.7, 74.4, 62.7, 54.1 (d, J = 2.6 Hz), 37.1 (d, J = 35.0 Hz), 34.8

petroleum ether). H NMR (400 MHz, CDCl₃): δ = 8.00–7.92 (m,

4 H), 7.55–7.26 (m, 11 H), 5.59 (t, J = 9.5 Hz, 1 H), 5.39 (t, J =

1

(d, J = 35.0 Hz), 34.0 (m), 32.1, 30.0–29.3 (m), 29.18, 26.4–25.7 9.8 Hz, 1 H), 4.99 (d, J = 10.0 Hz, 1 H), 4.31 (dd, J = 10.0, 5.1 Hz,

(m), 25.67, 22.8, 14.2 (2ϫ) ppm. IR (neat): ν = 3390, 2914, 2849, 1 H), 4.10 (t, J = 9.2 Hz, 1 H), 4.01 (t, J = 10.0 Hz, 1 H), 3.69 (td,

˜

2672

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Eur. J. Org. Chem. 2015, 2661–2677

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

J = 10.0, 5.2 Hz, 1 H), 0.973 (s, 9 H), 0.967 (s, 9 H) ppm. ¹³C

NMR (101 MHz, CDCl₃): δ = 165.8, 165.1, 134.5, 133.2, 132.9,

132.1, 130.5, 129.8, 129.7, 129.6, 128.3, 129.0, 128.8, 128.3, 128.2,

(180 mg, 0.357 mmol, 1.0 equiv.) were coevaporated twice with tol-

uene (10 mL), and then dissolved in anhydrous CH₂Cl₂(4 mL).

Activated molecular sieves (3 Å) were added, and the mixture was

87.0, 76.2, 75.1, 74.9 70.6, 66.1, 27.3, 26.9, 22.5, 19.9 ppm. IR stirred for 1 h at ambient temperature. The mixture was then cooled

(neat): ν = 2959, 2932, 2883, 2858, 1732, 1271, 1177, 1126, 827, to 0 °C, and BF₃·OEt₂(44 μL, 0.36 mmol, 1.0 equiv.) was added.

˜

708 cm^–1. HRMS: calcd. for [C₃₄H₄₀O₇SSi + H]⁺621.2344; found

621.2337.

The reaction mixture was stirred until TLC showed a lower-run-

ning spot (removal of the Boc group from the sphingosine ac-

ceptor) (ca. 1 h). The reaction mixture was transferred to an extrac-

tion funnel with EtOAc (50 mL), and washed with satd. aq.

NaHCO₃(50 mL) and brine (50 mL). The aqueous layers were ex-

tracted with EtOAc (50 mL), and the combined organic extracts

were dried (Na₂SO₄), filtered, and concentrated in vacuo. Purifica-

tion by column chromatography (2–5% Et₂O, 20% CH₂Cl₂in pe-

troleum ether) gave compound 36a (177 mg, 0.17 mmol, 49%) as

an amorphous solid. R_f= 0.45 (10% Et₂O, 20% CH₂Cl₂in petro-

leum ether). [α]²_D²= +6.8 (c = 0.1, CHCl₃). ¹H NMR (400 MHz,

CDCl₃): δ = 8.03–7.95 (m, 6 H), 7.56–7.48 (m, 3 H) 7.45–7.35 (m,

6 H), 5.79 (m, 1 H), 5.56 (t, J = 9.4 Hz, 1 H), 5.50–5.41 (m, 2 H),

5.35 (dd, J = 10.4, 7.8 Hz, 1 H), 4.80 (d, J = 9.5 Hz, 1 H), 4.68 (d,

J = 7.8 Hz, 1 H), 4.11–4.02 (m, 3 H), 3.97 (dd, J = 10.2, 4.5 Hz, 1

H), 3.74 (t, J = 10.2 Hz, 1 H), 3.62 (m, 1 H), 3.55 (m, 1 H), 1.96

(q, J = 6.8 Hz, 2 H), 1.34 (s, 9 H), 1.32–1.18 (m, 22 H), 0.95 (s, 18

H), 0.88 (t, J = 6.8 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ = 165.9, 165.4, 165.1, 155.4, 137.6, 133.3, 133.1, 133.0, 130.6,

129.9, 129.8, 129.7, 129.5, 128.5, 128.4, 124.7, 101.4, 79.5, 75.1,

74.8, 74.4, 72.1, 70.8, 67.9, 66.0, 52.4, 32.4, 32.0, 29.80 (4ϫ), 29.71,

29.59, 29.48, 29.35, 28.92, 28.4, 27.4, 26.9 22.8, 22.6, 20.0,

2,3-Di-O-benzoyl-4,6-O-(di-tert-butylsilanediyl)-α/β-D-glucopyran-

ose (35d): Phenyl 2,3-di-O-benzoyl-4,6-O-(di-tert-butylsilanediyl)-

1-thio-β-d-glucopyranoside (35c; Scheme 5; 6.27 g, 10.7 mmol,

1.0 equiv.) was dissolved in CH₂Cl₂(100 mL), and the solution was

cooled to 0 °C. N-Iodosuccinimide (4.81 g, 21.4 mmol, 2.0 equiv.)

was added, followed by trifluoroacetic acid (0.82 mL, 10.7 mmol,

1.0 equiv.). The reaction mixture was stirred under air until TLC

showed full conversion. The mixture was transferred to an extrac-

tion funnel with EtOAc (200 mL), and it was washed with sodium

thiosulfate (20% aq.; 200 mL), satd. aq. NaHCO₃(200 mL), and

brine (200 mL). The aqueous layers were extracted with EtOAc

(100 mL), and the combined organic extracts were dried (Na₂SO₄),

filtered, and concentrated in vacuo. Purification by silica gel col-

umn chromatography (5% EtOAc in petroleum ether) gave com-

pound 35d (6.63 g, 10.5 mmol, 98%, 5:3 α/β). R_f= 0.1 (10% EtOAc

1

in petroleum ether). H NMR (400 MHz, CDCl₃): δ = 8.10 (m, 1

H), 8.03–7.94 (m, 6 H), 7.60 (m, 0.6 H), 7.55–7.43 (m, 4.5 H), 7.41–

7.32 (m, 6.3 H), 5.92 (t, J = 9.1 Hz, 1 H), 5.66–5.61 (m, 1.6 H),

5.22 (dd, J = 9.5, 7.8 Hz, 0.6 H), 5.18 (dd, J = 10.3, 3.9 Hz, 1 H),

4.97 (d, J = 8.1 Hz, 0.6 H), 4.31–4.13 (m, 3.2 H), 4.12–4.06 (m, 2

H), 4.03–3.91 (m, 2 H), 3.67 (td, J = 10.4, 5.2 Hz, 0.6 H), 1.00–

0.97 (m, 25.2 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 167.1,

166.2, 166.15, 166.0, 133.73, 133.69, 133.50, 133.20, 133.00, 130.27,

130.11, 130.09, 130.02, 129.72, 129.69, 129.16, 128.95, 128.54,

128.46, 128.40, 96.4, 90.9, 75.6, 75.3, 74.51, 74.47, 72.4, 74.3, 71.2,

66.73, 66.52, 66.32, 27.44, 27.41, 27.00, 26.96, 22.75, 22.72, 20.08,

14.3 ppm. IR (neat): ν = 3070, 2958, 2924, 2854, 1728, 1271, 1174,

˜

1103, 1070, 709 cm^–1. HRMS: calcd. for [C₅₈H₈₃NO₁₂Si + Na]⁺

1036.5685; found 1036.5584.

5,6,7,8,9-¹³C₅]-Glucosylsphingosine (36b): 2,3-di-O-Benzoyl-4,6-O-

(di-tert-butylsilanediyl)-1-O-(N-[phenyl]-trifluoracetimidoyl)-α/β-

d-glucopyranose (35; 0.27 g, 0.4 mmol, 1.5 equiv.) and sphingosine

acceptor 23b (137 mg, 0.27 mmol, 1.0 equiv.) were coevaporated

twice with toluene (10 mL), and then dissolved in anhydrous

CH₂Cl₂(3 mL). Activated molecular sieves (3 Å) were added, and

the mixture was stirred for 1 h at ambient temperature. Then the

mixture was cooled to 0 °C, and BF₃·OEt₂(35 μL, 0.27 mmol,

1.0 equiv.) was added. The reaction mixture was stirred until TLC

showed a lower-running spot (removal of the Boc group from the

sphingosine acceptor) (ca. 1 h). The mixture was transferred to an

extraction funnel with EtOAc (40 mL), and it was washed with

satd. aq. NaHCO₃(40 mL) and brine (40 mL). The aqueous layers

were extracted with EtOAc (40 mL), and the combined organic ex-

tracts were dried (Na₂SO₄), filtered, and concentrated in vacuo.

Purification by column chromatography (2–5% Et₂O, 20% CH₂Cl₂

in petroleum ether) gave compound 36b (147 mg, 0.145 mmol,

54%) as an amorphous solid. R_f= 0.45 (10% Et₂O, 20% CH₂Cl₂

in petroleum ether). [α]²_D²= +6.0 (c = 0.1, CHCl₃). ¹H NMR

(400 MHz, CDCl₃): δ = 8.03–7.95 (m, 6 H), 7.57–7.48 (m, 3 H)

7.45–7.34 (m, 6 H), 5.79 (dm, J = 151.2 Hz, 1 H), 5.55 (t, J =

9.4 Hz, 1 H), 5.50–5.41 (m, 2 H), 5.35 (dd, J = 10.4, 7.8 Hz, 1 H),

4.79 (d, J = 8.9 Hz, 1 H), 4.67 (d, J = 7.8 Hz, 1 H), 4.11–4.02 (m,

3 H, 4-H), 3.98 (dd, J = 10.2, 4.5 Hz, 1 H), 3.74 (t, J = 10.2 Hz, 1

H), 3.62 (m, 1 H), 3.55 (m, 1 H), 1.96 (dm, J = 126.2 Hz, 2 H),

1.34–1.10 (m, 31 H), 0.95 (s, 18 H), 0.88 (t, J = 6.8 Hz, 3 H) ppm.

¹³C NMR (100 MHz, CDCl₃): δ = 166.0, 165.4, 165.1, 155.4, 137.6

(d, J = 42.6 Hz), 133.3, 133.1, 133.0, 130.6, 129.9, 129.8, 129.7,

129.5, 128.5, 128.4, 124.7 (d, J = 71.2 Hz), 101.4, 79.5, 75.1, 74.8,

74.4 (d, J = 5.2 Hz), 72.1, 70.8, 67.9, 66.0, 52.4, 32.4 (m), 32.0,

20.05 ppm. IR (neat): ν = 3431, 2934, 2859, 1728, 1277, 1177, 1070,

˜

827, 708 cm^–1. HRMS: calcd. for [C₂₆H₃₈O₈Si + H]⁺529.2259;

found 529.2256.

2,3-Di-O-benzoyl-4,6-O-(di-tert-butylsilanediyl)-1-O-(N-[phenyl]-tri-

fluoroacetimidoyl)-α/β-D-glucopyranose (35): 2,3-Di-O-benzoyl-4,6-

O-(di-tert-butylsilanediyl)-α/β-d-glucopyranose (35d; 1.71 g,

3.45 mmol, 1.0 equiv.) was dissolved in acetone (20 mL), and the

solution was cooled to 0 °C. Cesium carbonate (1.69 g, 5.18 mmol,

1.5 equiv.) was added, followed by chloro-N-phenyl-trifluoroimidi-

ate (0.78 mL, 5.18 mmol, 1.5 equiv.), and the reaction mixture was

stirred at 0 °C for 2 h. The mixture was filtered, and the filtrate

was concentrated in vacuo. Purifiaction by silica gel column

chromatography (using silica gel that was neutralised by running

an eluent of 3% Et₃N in petroleum ether (100 mL) through the

column; 0–5% EtOAc, 20% CH₂Cl₂in petroleum ether) gave com-

pound 35 (1.93 g, 2.76 mmol, 80%). R_f= 0.1 (10% EtOAc in petro-

1

leum ether). H NMR (400 MHz, CDCl₃): δ = 8.05–8.00 (m, 4 H),

7.54–7.44 (m, 2 H), 7.41–7.31 (m, 4 H), 7.28 (m, 1 H), 7.11 (t, J =

8.0 Hz, 2 H), 7.00 (t, J = 8.0 Hz, 1 H), 6.74 (m, 1 H), 6.45 (m, 1

H), 5.98 (m, 1 H), 5.50 (m, 1 H), 4.32–4.20 (m, 2 H), 4.00 (m, 1

H), 1.05–0.97 (m, 18 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ =

165.55, 165.49, 142.96, 133.68, 133.10, 130.00, 129.86, 129.70,

129.65, 128.82, 128.67, 128.63, 128.49, 128.40, 119.21, 75.00, 72.08,

70.53, 69.27, 66.29, 27.31, 26.83, 22.65, 20.00 ppm. IR (neat): ν =

˜

2959, 2936, 2860, 1728, 1273, 1211, 995, 766, 710 cm^–1. HRMS:

calcd. for [C₃₆H₄₀F₃NO₈Si + H]⁺700.2555; found 700.2549.

Glucosylsphingosine (36a): 2,3-di-O-Benzoyl-4,6-O-(di-tert-butylsil-

anediyl)-1-O-(N-[phenyl]-trifluoracetimidoyl)-α/β-d-glucopyranose

35 (0.325 g, 0.465 mmol, 1.3 equiv.) and sphingosine acceptor 23a

29.80–28.4 (m), 27.4, 26.9, 22.8, 22.6, 20.0, 14.3 ppm. IR (neat): ν

˜

= 3070, 2922, 2854, 1724, 1267, 1172, 1069, 827, 708 cm^–1. HRMS:

calcd. for [C₅₃¹³C₅H₈₃NO₁₂Si + H]⁺1041.5748; found 1041.5748.

Eur. J. Org. Chem. 2015, 2661–2677

www.eurjoc.org

2673

J. D. C. Codée, H. S. Overkleeft et al.

FULL PAPER

Glucosylsphingosine (37a): Protected glucosyl sphingosine 36a

(130 mg, 0.128 mmol, 1.0 equiv.) was dissolved in THF/pyridine

(4:1) (15 mL), and hydrogen fluoride (70 % in pyridine; 53 μL,

at 0 °C, and then it was diluted with toluene (20 mL), and concen-

trated in vacuo. Purification by HPLC–MS (52–62% B, following

the general procedure for HPLC–MS purifications) gave compound

0.256 mmol, 2.0 equiv.) was added. The reaction mixture was 37b (10.7 mg, 23 μmol, 49%) as a TFA adduct. [α]²_D²= –5.1 (c =

1

stirred at room temperature until TLC showed full conversion [ca.

0.1, MeOH). H NMR (600 MHz, [D₄]methanol): δ = 5.85 (dm, J

2 h; product R_f= 0.75 (40% EtOAc in CH₂Cl₂]. The mixture was = 150.2 Hz, 1 H), 5.48 (dt, J = 15.8, 6.4 Hz, 1 H), 4.34–4.29 (m, 2

concentrated in vacuo, the residue was redissolved in EtOAc H), 3.97–3.88 (m, 3 H), 3.66 (dd, J = 11.7, 6.1 Hz, 1 H), 3.40–3.31

(30 mL), and this solution was washed with HCl (1 m aq.; 30 mL), (m, 2 H), 3.29–2.21 (m 3 H), 2.10 (dm, J = 126.9 Hz, 2 H), 1.56–

satd. aq. NaHCO₃(30 mL), and brine (30 mL). The aqueous layers

were extracted with EtOAc (30 mL), and the combined organic ex-

tracts were dried (Na₂SO₄), filtered, and concentrated in vacuo.

1.15 (m, 22 H), 0.90 (t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR (151 MHz,

[D₄]methanol): δ = 136.8 (d, J = 43.0 Hz), 128.2 (dd, J = 72.5,

3.5 Hz), 104.1, 78.1, 77.9, 74.9, 71.5, 70.9 (m), 67.3, 62.5, 56.8 (d,

J = 3.4 Hz), 33.6–32.9 (m), 30.9–29.6 (m), 23.4, 14.5 ppm. IR

The crude mixture was dissolved in MeOH (13 mL), and sodium

methoxide (30% in methanol; 18 μL, 0.128 mmol, 1.0 equiv.) was

added. The reaction mixture was stirred overnight at room tem-

perature, and the progress of the reaction was monitored by

HPLC–MS. Aqueous potassium hydroxide (0.5 m; 3.8 mL,

1.9 mmol, 15 equiv.) was added, and the reaction mixture was

stirred overnight at ambient temperature. The reaction was then

quenched with AcOH (0.73 mL, 13 mmol, 100 equiv.), and the mix-

ture was concentrated in vacuo. The crude reaction product was

coevaporated in toluene.

(neat): ν = 3300, 2918, 2851, 1670, 1433, 1200, 1134, 1074, 1024,

˜

800, 721 cm^–1. HRMS: calcd. for [C₁₉¹³C₅H₄₇NO₇+ H]⁺467.3598;

found 467.3591.

Glucosylceramide (38a): See the general procedure for the synthesis

of ceramides from sphingosine, yield (3.1 mg, 4.4 μmol, 57%). R_f

= 0.25 (CHCl₃/MeOH, 9:1). [α]²_D²= +6.0 (c = 0.1, MeOH/CHCl₃,

1

1:1). H NMR (600 MHz, CDCl₃/[D₄]methanol): δ = 5.68 (dt, J =

13.8, 6.9 Hz, 1 H), 5.44 (dd, J = 15.3, 7.8 Hz, 1 H), 4.26 (d, J =

7.9 Hz, 1 H), 4.16 (dd, J = 10.3, 4.8 Hz, 1 H), 4.06 (t, J = 8.4 Hz,

1 H), 3.97 (dt, J = 8.4, 4.0 Hz, 1 H), 3.86 (dd, J = 11.9, 1.8 Hz, 1

H), 3.66 (m 1 H), 3.59 (dd, J = 10.1, 3.3 Hz, 1 H), 3.36 (m, 1 H),

3.29–3.26 (m, 2 H), 3.21 (dd, J = 9.4, 7.8 Hz, 1 H), 2.17 (t, J =

7.2 Hz, 2 H), 2.02 (m, 2 H), 1.58 (m, 2 H), 1.42–1.23 (m, 46 H),

0.90 (t, J = 7.0 Hz, 6 H) ppm. ¹³C NMR (151 MHz, CDCl₃/[D₄]-

methanol): δ = 173.9, 133.0, 129.2, 102.5, 75.8, 75.7, 73.0, 70.9,

69.5, 67.8, 60.5, 52.6, 35.3, 31.4, 31.0, 28.80, 28.77 (3ϫ), 28.76

(4ϫ), 28.74 (2ϫ), 28.73, 28.71, 28.70, 28.64, 28.57, 28.50, 28.43,

28.42, 28.38, 28.37, 28.34, 25.10, 21.67, 12.4 (2ϫ) ppm. IR (neat):

The residue was cooled in an ice-bath, and then water (1 mL) and

TFA (3 mL) were added. The reaction mixture was stirred for 2 min

at 0 °C, then it was diluted with toluene (20 mL), and concentrated

in vacuo. Purification by HPLC–MS (52–62% B, following the ge-

neral procedure for HPLC–MS purifications) gave compound 37a

(31 mg, 0.067 mmol, 53%) as a TFA adduct. [α]²_D²= –5.0 (c = 0.1,

MeOH). ¹H NMR (600 MHz, [D₄]methanol): δ = 5.87 (dtd, J =

15.0, 6.8, 1.2 Hz, 1 H), 5.48 (ddt, J = 15.4, 6.9, 1.5 Hz, 1 H), 4.33–

4.29 (m, 2 H), 3.97–3.88 (m, 3 H), 3.66 (m, 1 H), 3.40–3.32 (m, 2

H), 3.29–2.21 (m 3 H), 2.1 (q, J = 7.2 Hz, 2 H), 1.42 (m, 2 H),

1.36–1.22 (m, 20 H), 0.9 (t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR:

(151 MHz, [D₄]methanol): δ = 136.8, 128.4, 104.1, 78.1, 77.9, 74.8,

71.5, 70.9, 67.3, 62.5, 56.8, 33.4, 33.1, 30.82, 30.81 (2ϫ), 30.78,

ν = 3300, 2916, 2848, 1670, 1540, 1467, 1200, 1134, 1074, 1028,

˜

721 cm^–1. HRMS: calcd. for [C₄₀H₇₇NO₈+ H]⁺700.5727; found

700.5720.

Glucosyl-2-N-([3,4,5-¹³C₃]-hexadecanoyl)-sphingosine (38b): See the

general procedure for the synthesis of ceramides from sphingosine,

yield (3.2 mg, 4.5 μmol, 59 %). R_f= 0.25 (CHCl₃/MeOH, 9:1).

[α]²_D²= +4.8 (c = 0.2, MeOH/CHCl₃, 1:1). ¹H NMR (600 MHz,

[D₄]methanol): δ = 5.68 (dt, J = 13.8, 6.9 Hz, 1 H), 5.44 (dd, J =

15.3, 7.8 Hz, 1 H), 4.26 (d, J = 7.9 Hz, 1 H), 4.16 (dd, J = 10.3,

4.8 Hz, 1 H), 4.06 (t, J = 8.4 Hz, 1 H), 3.97 (dt, J = 8.4, 4.0 Hz, 1

H), 3.86 (dd, J = 11.9, 1.8 Hz, 1 H), 3.66 (m, 1 H), 3.59 (dd, J =

10.1, 3.3 Hz, 1 H), 3.36 (m, 1 H), 3.29–3.26 (m, 2 H), 3.21 (dd, J

= 9.4, 7.8 Hz, 1 H), 2.17 (m, 2 H), 2.02 (m, 2 H), 1.58 (dm, J =

130 Hz, 2 H), 1.42–1.16 (m, 46 H), 0.90 (t, J = 7.0 Hz, 6 H) ppm.

¹³C NMR (151 MHz, [D₄]methanol): δ = 176.0, 135.1, 131.3, 104.7,

78.0, 77.9, 75.2, 73.0, 71.6, 69.9, 62.6, 54.7, 34.8 (d, J = 35.0 Hz),

33.1, 31.0–30.0 (m), 27.51, 27.4–27.0 (m), 26.87, 23.77, 14.4 (2ϫ)

30.77, 30.66, 30.50, 30.41, 30.18, 23.6, 14.2 ppm. IR (neat): ν =

˜

3300, 2918, 2850, 1668, 1435, 1202, 1134, 1074, 1026, 800,

721 cm^–1. HRMS: calcd. for [C₂₄H₄₇NO₇+ H]⁺462.3431; found

462.3424.

Glucosyl-[5,6,7,8,9-¹³C₅]-sphingosine (37b): Protected glucosyl-

[5,6,7,8,9-¹³C₅]-sphingosine 36a (48 mg, 47 μmol, 1.0 equiv.) was

dissolved in THF/pyridine (4:1) (10 mL), and hydrogen fluoride

(70% in pyridine; 20 μL, 94 μmol, 2.0 equiv.) was added. The reac-

tion mixture was stirred at room temperature until TLC showed

full conversion (ca. 2 h) [product R_f= 0.75 (40 % EtOAc in

CH₂Cl₂)]. The mixture was concentrated in vacuo, the residue was

redissolved in EtOAc (20 mL), and this solution was washed with

HCl (1 m aq.; 20 mL), satd. aq. NaHCO₃(20 mL), and brine

(20 mL). The aqueous layers were extracted with EtOAc (20 mL),

and the combined organic extracts were dried (Na₂SO₄), filtered,

and concentrated in vacuo.

ppm. IR (neat): ν = 3260, 2914, 2847, 1643, 1541, 1468, 1205, 1134,

˜

1076, 1030, 717 cm^–1. HRMS: calcd. for [C₃₇¹³C₃H₇₇NO₈+ H]⁺

703.5828; found 703.5821.

The crude mixture was dissolved in MeOH (8 mL), and sodium

methoxide (30 % in methanol; 6.5 μL, 47 μmol, 1.0 equiv.) was

added. The reaction mixture was stirred overnight at room tem-

perature, and the progress of the reaction was monitored by

HPLC–MS. Aqueous potassium hydroxide (0.5 m; 1.4 mL,

0.7 mmol, 15 equiv.) was added, and the reaction mixture was

stirred overnight at ambient temperature. The reaction was then

quenched with AcOH (0.3 mL, 4.7 mmol, 100 equiv.), and the mix-

ture was concentrated in vacuo. The crude product mixture was

coevaporated with toluene.

Glucosyl-2-N-(hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphingosine (38c): See

the general procedure for the synthesis of ceramides from sphingo-

sine, yield (2.8 mg, 3.9 μmol, 52%). R_f= 0.25 (CHCl₃/MeOH, 9:1).

[α]²_D²= +5.4 (c = 0.1, MeOH/CHCl₃). ¹H NMR (600 MHz, [D₄]-

methanol): δ = 5.68 (dm, J = 154.0 Hz, 1 H), 5.44 (m, 1 H), 4.26

(d, J = 7.9 Hz, 1 H), 4.16 (dd, J = 10.3, 4.8 Hz, 1 H), 4.06 (m, 1

H), 3.97 (m, 1 H), 3.86 (dd, J = 11.9, 1.8 Hz, 1 H), 3.66 (m 1 H),

3.59 (dd, J = 10.1, 3.3 Hz, 1 H), 3.36 (m, 1 H), 3.29–3.26 (m, 2 H),

3.21 (dd, J = 9.4, 7.8 Hz, 1 H), 2.17 (m, 2 H), 2.02 (dm, J =

128.0 Hz, 2 H), 1.58 (dm, J = 130.0 Hz, 2 H), 1.42–1.14 (m, 46 H),

0.90 (t, J = 7.0 Hz, 6 H) ppm. ¹³C NMR (151 MHz, [D₄]methanol):

δ = 176.0, 135.1 (d, J = 44.0 Hz), 131.3 (d, J = 72.5 Hz), 104.6,

The residue was cooled in an ice-bath, and then water (0.3 mL) and

TFA (1 mL) were added. The reaction mixture was stirred for 2 min

2674

www.eurjoc.org

Eur. J. Org. Chem. 2015, 2661–2677

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

78.0, 77.9, 75.2, 73.0, 71.6, 69.9, 62.6, 54.7, 37.4, 33.9–33.0 (m), acceptor 23b (40.7 mg, 80 μmol, 1.0 equiv.) were coevaporated

31.1–30.1 (m), 27.20, 23.78, 14.5 ppm. IR (neat): ν = 3300, 2913, twice with toluene (5 mL), and then dissolved in anhydrous CH₂Cl₂

˜

2847, 1643, 1544, 1468, 1260, 1085, 1030, 718 cm^–1. HRMS: calcd.

(2 mL). Activated molecular sieves (3 Å) were added, and the mix-

ture was stirred at ambient temperature for 1 h. The mixture was

then cooled to 0 °C, and then BF₃·OEt₂(48 % in Et₂O; 23 μL,

88 μmol, 1.1 equiv.) was added. The reaction mixture was stirred

until TLC showed complete conversion of the [¹³C₅]-sphingosine

acceptor (ca. 2 h). The mixture was then transferred to an extrac-

tion funnel with EtOAc (40 mL), and it was washed with satd. aq.

NaHCO₃(40 mL) and brine (30 mL). The aqueous layers were then

extracted with EtOAc (40 mL), and the combined organic extracts

were dried (Na₂SO₄), filtered, and concentrated in vacuo. Purifica-

tion by column chromatography (12% Et₂O, 10% CH₂Cl₂in petro-

leum ether) gave compound 40b (87 mg, 44 μmol, 55 %) as an

amorphous solid. R_f= 0.54 (30% Et₂O, 20% CH₂Cl₂in petroleum

for [C₃₅¹³C₅H₇₇NO₈+ H]⁺705.5895; found 705.5884.

Glucosyl-2-N-([3,4,5-¹³C₃]-hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphingo-

sine (38d): See the general procedure for the synthesis of ceramides

from sphingosine, yield (4.9 mg, 6.9 μmol, 61%). R_f= 0.25 (CHCl₃/

MeOH, 9:1). [α]²_D²= +5.0 (c = 0.2, MeOH/CHCl₃, 1:1). H NMR

1

(600 MHz, [D₄]methanol): δ = 5.68 (dm, J = 154.0 Hz, 1 H), 5.44

(m, 1 H), 4.26 (d, J = 7.9 Hz, 1 H), 4.16 (dd, J = 10.3, 4.8 Hz, 1

H), 4.06 (m, 1 H), 3.97 (m, 1 H), 3.86 (dd, J = 11.9, 1.8 Hz, 1 H),

3.66 (m 1 H), 3.59 (dd, J = 10.1, 3.3 Hz, 1 H), 3.36 (m, 1 H), 3.29–

3.26 (m, 2 H), 3.21 (dd, J = 9.4, 7.8 Hz, 1 H), 2.17 (m, 2 H), 2.02

(dm, J = 128.0 Hz, 2 H), 1.58 (dm, J = 130.0 Hz, 2 H), 1.42–1.14

(m, 46 H), 0.90 (t, J = 7.0 Hz, 6 H) ppm. ¹³C NMR (151 MHz,

[D₄]methanol): δ = 176.0, 135.1 (d, J = 44.0 Hz), 131.3 (d, J =

72.5 Hz), 104.6, 78.0, 77.9, 75.2, 73.0, 71.6, 69.9, 62.6, 54.7, 37.4

(d, J = 35.0 Hz), 33.9–33.0 (m), 31.1–30.0 (m), 27.50, 27.4–27.0

1

ether). [α]²_D²= +30 (c = 1.0, CHCl₃). H NMR (400 MHz, CDCl₃):

δ = 8.17 (m, 2 H), 8.06–8.00 (m, 4 H), 7.95 (m, 2 H), 7.92–7.84 (m,

6), 7.67 (m, 2 H), 7.55 (m, 2 H), 7.53–7.42 (m, 7 H), 7.40–7.27 (m,

16 H), 7.21 (m, 2 H), 7.11 (m, 2 H), 5.77 (t, J = 9.3 Hz, 1 H), 5.71

(dd, J = 10.7, 3.7 Hz, 1 H), 5.67 (dm, J = 151.2 Hz, 1 H), 5.59 (dd,

J = 10.8, 7.8 Hz, 1 H), 5.49 (dd, J = 10.7, 3.0 Hz, 1 H), 5.47–5.31

(m, 4 H), 5.24 (dd, J = 10.9, 2.1 Hz, 1 H), 5.10 (d, J = 3.0 Hz, 1

H), 4.80–4.73 (m, 2 H), 4.65 (d, J = 7.8 Hz, 1 H), 4.54 (d, J =

12.0 Hz, 1 H), 4.44 (d, J = 12.0 Hz, 1 H), 4.38–4.30 (m, 3 H), 4.12

(t, J = 9.4 Hz, 1 H), 4.07 (d, J = 1.5 Hz, 1 H), 4.06–3.99 (m, 2 H),

3.97 (dd, J = 10.9, 5.4 Hz, 1 H), 3.81–3.71 (m, 2 H), 3.58 (m, 1 H),

3.51 (dd, J = 13.9, 6.9 Hz, 1 H), 1.87 (dm, J = 124.6 Hz, 2 H),

1.40–1.14 (m, 31 H), 1.05 (s, 9 H), 1.00 (s, 9 H), 0.87 (t, J = 6.8 Hz,

3 H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 166.2, 166.0, 165.7,

165.6, 165.2, 165.00, 164.98, 164.79, 164.75, 155.2, 137.2 (d, J =

42.4 Hz), 133.4, 133.2, 133.1, 133.00, 132.99, 132.96, 132.89, 132.7,

130.2, 130.1, 130.00, 129.95, 129.8, 129.67, 129.62, 129.60, 129.50,

129.48, 129.4, 129.2, 129.0, 128.6, 128.53, 128.50, 128.48, 128.41,

128.37, 128.32, 128.30, 128.18, 128.14, 128.09, 124.4 (d, J =

71.2 Hz), 101.3, 100.9, 98.7, 79.4, 76.6, 76.3, 74.3 (d, J = 5.4 Hz),

73.04, 72.97, 72.8, 72.7, 71.9, 71.2, 71.0, 69.7, 69.6, 68.3, 67.8, 66.9,

62.3, 60.5, 52.3 (d, J = 2.4 Hz), 32.2 (m), 31.9, 29.8–28.1 (m), 27.5,

(m), 26.87, 23.78, 14.4 (2ϫ) ppm. IR (neat): ν = 3295, 2913, 2847,

˜

1643, 1545, 1468, 1260, 1086, 1032, 718 cm^–1. HRMS: calcd. for

[C₃₂¹³C₈H₇₇NO₈+ H]⁺708.5995; found 708.5989.

Globotriaosylsphingosine (40a): Globotriaosyl imidate donor 39

(0.54 g, 0.33 mmol, 1.2 equiv.) and sphingosine acceptor 23a

(0.14 g, 0.27 mmol, 1.0 equiv.) were coevaporated twice with tolu-

ene (5 mL), and then dissolved in anhydrous CH₂Cl₂(3 mL). Acti-

vated molecular sieves (3 Å) were added, and the mixture was

stirred for 1 h at ambient temperature. The mixture was then cooled

to 0 °C, and BF₃·OEt₂(48% in Et₂O; 38 μL, 0.3 mmol, 1.1 equiv.)

was added. The reaction mixture was stirred until TLC showed

complete conversion of the sphingosine acceptor (ca. 2 h). The mix-

ture was then transferred to an extraction funnel with EtOAc

(40 mL), and it was washed with satd. aq. NaHCO₃(40 mL) and

brine (40 mL). The aqueous layers were extracted with EtOAc

(40 mL), and the combined organic extracts were dried (Na₂SO₄),

filtered, and concentrated in vacuo. Purification by column

chromatography (12% Et₂O, 10% CH₂Cl₂in petroleum ether) gave

compound 40a (0.32 g, 0.16 mmol, 60%) as an amorphous solid.

R_f= 0.54 (30% Et₂O, 20% CH₂Cl₂in petroleum ether). [α]²_D²= +31

27.2, 23.2, 22.7, 20.7, 14.1 ppm. IR (neat): ν = 3070, 2925, 2853,

˜

1718, 1452, 1266, 1094, 1069, 706 cm^–1. HRMS: calcd. for

[C₁₀₇¹³C₅H₁₂₇O₂₈Si + Na]⁺1989.8374; found 1989.8370.

1

(c = 1.0, CHCl₃). H NMR (400 MHz, CDCl₃): δ = 8.19 (m, 2 H),

7.92 (m, 2 H), 7.92–7.85 (m, 6 H), 7.68 (dm, J = 7.2 Hz, 2 H),

7.58–7.41 (m, 9 H), 7.40–7.18 (m, 18 H), 7.11 (m, 2 H), 5.78 (t, J

= 9.3 Hz, 1 H), 5.72 (dd, J = 10.7, 3.7 Hz, 1 H), 5.66 (m, 1 H),

5.60 (dd, J = 10.8, 7.8 Hz, 1 H), 5.50 (dd, J = 10.7, 3.0 ,Hz, 1 H),

5.47–5.32 (m, 4 H), 5.25 (dd, J = 10.8, 2.1 Hz, 1 H), 5.10 (d, J =

2.9 Hz, 1 H), 4.81–4.74 (m, 2 H), 4.66 (d, J = 7.8 Hz, 1 H), 4.55

(d, J = 11.9 Hz, 1 H), 4.46 (d, J = 11.9 Hz, 1 H), 4.39–4.32 (m, 3

H), 4.12 (t, J = 9.3 Hz, 1 H), 4.08 (br. s, 1 H), 4.07–4.00 (m, 2 H),

3.97 (dd, J = 10.9, 5.3 Hz, 1 H), 3.81–3.72 (m, 2 H), 3.59 (m, 1 H),

3.53 (m, 1 H), 1.88 (m, 2 H), 1.33 (s, 9 H), 1.30–1.11 (m, 22 H),

1.06 (s, 9 H), 1.00 (s, 9 H), 0.87 (t, J = 6.8 Hz, 3 H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 166.2, 165.9, 165.7, 165.6, 165.2,

Globotriaosylsphingosine (41a): Protected globotriaosylsphingosine

40a (200 mg, 0.10 mmol, 1.0 equiv.) was dissolved in THF/pyridine

(4:1; 20 mL), and hydrogen fluoride (70 % in pyridine; 53 μL,

0.26 mmol, ca. 20 equiv.) was added. The reaction mixture was

stirred at room temperature until TLC showed full conversion into

a lower-running spot (ca. 4 h). The mixture was then concentrated

in vacuo, the residue was redissolved in EtOAc (50 mL), and this

solution was washed with HCl (1 m aq.; 50 mL), satd. aq. NaHCO₃

(50 mL), and brine (50 mL). The aqueous phases were extracted

with EtOAc (50 mL), and the combined organic extracts were dried

(Na₂SO₄), filtered, and concentrated in vacuo.

164.98, 164.95, 164.8, 164.7, 155.2, 137.2, 133.4, 133.13, 133.10, The crude mixture was then dissolved in methanol (20 mL), and

132.97, 132.94, 132.87, 132.7, 130.2, 130.1, 130.0, 129.9, 129.8,

129.64, 129.59, 129.58, 129.5, 129.4, 129.3, 129.2, 129.0, 128.6,

sodium methoxide (30% in methanol; 14 μL, 0.10 mmol, 1.0 equiv.)

was added. The reaction mixture was stirred overnight at ambient

128.50, 128.47, 128.45, 128.39, 128.35, 128.29, 128.28, 128.16, temperature, and the progress of the reaction was monitored by

128.12, 128.07, 124.4, 101.3, 100.9, 98.68, 79.3, 76.6, 76.3, 74.3, HPLC–MS. Aqueous potassium hydroxide (0.5 m; 4.1 mL,

73.02, 72.94, 72.8, 72.6, 71.9, 71.2, 71.0, 69.7, 69.5, 68.3, 67.8, 66.9, 2.0 mmol, 20 equiv.) was added, and the reaction mixture was

62.3, 60.5, 52.3, 32.2, 31.9, 29.62 (3ϫ), 29.61, 29.5, 29.3, 29.2, 28.7, stirred overnight at ambient temperature. The reaction was then

28.2, 27.5, 27.2, 23.2, 22.6, 20.7, 14.1 ppm. IR (neat): ν = 3070, quenched with AcOH (0.58 mL, 100 equiv.), and the mixture was

˜

2926, 2856, 1722, 1451, 1267, 1095, 1070, 1028, 708 cm^–1. HRMS:

concentrated in vacuo. The crude product mixture was coevapo-

rated with toluene.

calcd. for [C₁₁₂H₁₂₇NO₂₈Si + Na]⁺1984.8206; found 1984.8204.

[5,6,7,8,9-¹³C₅]-Globotriaosylsphingosine (40b): Globotriaosyl imid-

ate donor 39; 158 mg, 96 μmol, 1.2 equiv.) and [¹³C₅]-sphingosine

The residue was cooled in an ice-bath, and then trifluoracetic acid

(5 mL) was added. After 1 min, the residue had completely dis-

Eur. J. Org. Chem. 2015, 2661–2677

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2675

J. D. C. Codée, H. S. Overkleeft et al.

solved, and the reaction mixture was then stirred for a further as a TFA adduct. [α]²_D²= +33.0 (c = 0.20, MeOH). ¹H NMR

FULL PAPER

1 min at 0 °C. The solution was then transferred to a round-bot-

tomed flask containing toluene (50 mL), and the mixture was con-

centrated to ca. 10 mL in vacuo. The coevaporation was repeated

twice with toluene (40 mL), then the mixture was concentrated to

dryness. The completion of the reaction was confirmed by HPLC–

MS. The residue was then purified over a short silica column

(MeOH/CH₂Cl₂, 1:9, followed by H₂O/MeOH/CH₂Cl₂, 3:27:70;

TLC visualised with ninhydrin spray). Further purification by

HPLC–MS (40–48 % B, following the general procedure for

HPLC–MS purifications) gave globotriaosylsphingosine 41a

(43 mg, 54 μmol, 53%) as a TFA adduct. [α]²_D²= +34.0 (c = 0.5,

MeOH). ¹H NMR (600 MHz, [D₄]methanol): δ = 5.87 (m, 1 H),

5.49 (m, 1 H), 4.94 (d, J = 3.9 Hz, 1 H), 4.40 (d, J = 6.9 Hz, 1 H),

4.37 (d, J = 7.8 Hz, 1 H), 4.32 (ddd, J = 6.8, 4.7, 1.3 Hz, 1 H), 4.25

(ddd, J = 7.1, 5.2, 1.3 Hz, 1 H), 4.01–3.96 (m, 2 H), 3.94 (dd, J =

11.9, 2.6 Hz, 1 H), 3.93–3.91 (m, 2 H), 3.89 (dd, J = 7.7, 4.1 Hz, 1

H), 3.88–3.81 (m, 3 H), 3.77 (dd, J = 10.2, 3.2 Hz, 1 H), 3.74 (dd,

J = 11.1, 7.1 Hz, 1 H), 3.40 (ddd, J = 8.5, 4.7, 3.6 Hz, 1 H), 3.30

(t, J = 7.7 Hz, 1 H), 2.10 (q, J = 7.0 Hz, 2 H), 1.42 (m, 2 H), 1.36–

1.22 (m, 20 H), 0.90 (t, J = 7.0 Hz, 3 H) ppm. ¹³C NMR (151 MHz,

[D₄]methanol): δ = 136.8, 128.3, 105.4, 103.7, 102.7, 80.8, 79.8,

76.6, 76.3, 74.7, 74.6, 72.8, 72.6, 71.3, 71.0, 70.8, 70.5, 67.1, 62.7,

61.6, 61.5, 56.7, 33.4, 33.1, 30.79 (3ϫ), 30.76, 30.74, 30.6, 30.5,

(600 MHz, [D₄]methanol): δ = 5.85 (dm, J = 150.2 Hz, 1 H), 5.47

(m, 1 H), 4.94 (d, J = 3.8 Hz, 1 H), 4.39 (d, J = 7.1 Hz, 1 H), 4.36

(d, J = 7.8 Hz, 1 H), 4.31 (ddd, J = 6.4, 4.8 Hz, 1 H), 4.25 (ddd, J

= 6.8, 5.2, 1.3 Hz, 1 H), 4.01–3.96 (m, 2 H), 3.94 (dd, J = 12.0,

2.4 Hz, 1 H), 3.92–3.90 (m, 2 H), 3.89 (dd, J = 7.7, 4.0 Hz, 1 H),

3.88–3.79 (m, 3 H), 3.77 (dd, J = 10.1, 3.1 Hz, 1 H), 3.74 (dd, J =

11.2, 7.3 Hz, 1 H), 3.70–3.65 (m, 2 H), 3.58–3.50 (m, 4 H, 4-H),

3.46 (m, 1 H), 3.40 (ddd, J = 8.5, 4.7, 3.6 Hz, 1 H), 3.30 (m, 1 H),

2.10 (dm, J = 126.9 Hz, 2 H), 1.56–1.14 (m, 22 H), 0.89 (t, J =

7.0 Hz, 3 H) ppm. ¹³C NMR (151 MHz, [D₄]methanol): δ = 136.8

(d, J = 42.8 Hz), 128.3 (d, J = 72.3 Hz), 105.4, 103.7, 102.7, 80.8,

79.8, 76.6 (2ϫ), 76.3, 74.65, 74.2, 72.8, 72.6, 71.3, 71.0, 70.8 (d, J

= 5.1 Hz), 70.5, 67.1, 62.7, 61.6, 61.5, 56.7 (d, J = 2.2 Hz), 33.8–

32.9 (m), 30.9–29.8 (m), 23.7, 14.4 ppm. IR (neat): ν = 3344 br. s,

˜

2925, 2855, 1674, 1202, 1134, 1067, 1027, 974, 801, 721 cm^–1

HRMS: calcd. for [C₃₁¹³C₅H₆₇NO₁₇H]⁺791.4650; found 791.4654.

Globotriaosylceramide (42a): See the general procedure for the syn-

thesis of ceramides from sphingosine, yield (6 mg, 5.8 μg, 49%). R_f

= 0.35 (CHCl₃/MeOH/H₂O, 70:27:3). [α]²_D²= +24 (c = 0.75, MeOH/

1

CHCl₃, 1:1). H NMR (600 MHz, CDCl₃/[D₄]methanol): δ = 5.69

(dt, J = 14.7, 6.9 Hz, 1 H), 5.45 (dd, J = 15.3, 7.8 Hz, 1 H), 4.96

(d, J = 3.8 Hz, 1 H), 4.41 (d, J = 6.9 Hz, 1 H), 4.30 (d, J = 7.8 Hz,

1 H), 4.25 (ddd, J = 6.8, 4.7, 1.3 Hz, 1 H), 4.19 (dd, J = 10.1,

4.5 Hz, 1 H), 4.07 (t, J = 8.8 Hz, 1 H), 4.04–3.96 (m, 4 H), 3.92 (d,

J = 3.0 Hz, 1 H) 3.89 (d, J = 3.2 Hz, 1 H), 3.85–3.81 (m, 3 H),

3.79–3.73 (m, 2 H), 3.71–3.3.63 (m, 3 H), 3.60–3.51 (m, 4 H), 2.17

(t, J = 7.2 Hz, 2 H), 2.03 (m, 2 H), 1.58 (m, 1 H), 1.43–1.20 (m, 46

30.4, 30.2, 23.7, 14.4 ppm. IR (neat): ν = 3345 br. s, 2925, 2855,

˜

1674, 1202, 1134, 1067, 1027, 974, 801, 721 cm^–1. HRMS: calcd.

for [C₃₆H₆₇NO₁₇+ H]⁺786.4482; found 786.4485.

Globotriaosyl-[5,6,7,8,9-¹³C₅]-sphingosine (41b): Globally protected

globotriaosyl-[5,6,7,8,9-¹³C₅]-sphingosine 40b (87 mg, 0.45 μmol, H), 0.90 (t, J = 6.9 Hz, 6 H) ppm. ¹³C NMR (151 MHz, CDCl₃/

1.0 equiv.) was dissolved in THF/pyridine (4:1; 10 mL), and

[D₄]methanol): δ = 177.5, 133.1, 132.6, 103.3, 102.3, 100.6, 78.8,

hydrogen fluoride (70 % in pyridine; 24 μL, 0.11 mmol, ca. 77.7, 74.3, 74.1, 72.8, 72.5, 70.9, 70.7, 70.5, 69.2, 68.4, 67.8, 60.6,

20 equiv.) was added. The reaction mixture was stirred at room

temperature until TLC showed full conversion into a lower-running

spot (ca. 4 h). The mixture was then concentrated in vacuo, the

residue was redissolved in EtOAc (50 mL), and this solution was

58.8, 57.2, 52.5, 35.3, 31.4, 31.0 28.78 (2 ϫ), 28.77 (4 ϫ), 28.74

(2 ϫ), 28.72 (2 ϫ), 28.71 (2 ϫ), 28.64, 28.59, 28.54, 28.52, 28.44

(2ϫ), 28.41, 28.40, 28.38, 28.36, 21.7, 12.5 ppm. IR (neat): ν =

˜

3300, 2918, 2851, 1636, 1465, 1379, 1205, 144, 1070, 1016,

washed with HCl (1 n aq.; 50 mL), satd. aq. NaHCO₃(50 mL), 719 cm^–1. HRMS: calcd. for [C₅₂H₉₇NO₁₈+ H]⁺1024.6784; found

and brine (50 mL). The aqueous phases were extracted with EtOAc

(50 mL) and the combined organic extracts were dried (Na₂SO₄),

filtered, and concentrated in vacuo.

1024.6783.

Globotriaosyl-2-N-([3,4,5-¹³C₃]-hexadecanoyl)-sphingosine (42b):

See the general procedure for the synthesis of ceramides from

sphingosine, yield (9 mg, 8.7 μmol, 71 %). R_f= 0.35 (CHCl₃/

MeOH/H₂O, 70:27:3). [α]²_D²= +24 (c = 0.25, MeOH/CHCl₃, 1:1).

¹H NMR (600 MHz, CDCl₃/[D₄]methanol): δ = 5.69 (dt, J = 14.7,

6.9 Hz, 1 H), 5.45 (dd, J = 15.3, 7.8 Hz, 1 H), 4.96 (d, J = 3.8 Hz,

1 H), 4.41 (d, J = 6.9 Hz, 1 H), 4.30 (d, J = 7.8 Hz, 1 H), 4.25

(ddd, J = 6.8, 4.7, 1.3 Hz, 1 H), 4.19 (dd, J = 10.1, 4.5 Hz, 1 H),

4.07 (t, J = 8.8 Hz, 1 H), 4.04–3.96 (m, 4 H), 3.92 (d, J = 3.0 Hz,

The crude mixture was then dissolved in methanol (8 mL), and

sodium methoxide (30% in MeOH; 6.2 μL, 0.45 μmol, 1.0 equiv.)

was added. The reaction mixture was stirred overnight at room

temperature. The progress of the reaction was monitored by

HPLC–MS. Aqueous potassium hydroxide (0.5 m; 1.8 mL,

0.89 mmol, 20 equiv.) was added, and the reaction mixture was

stirred overnight at room temperature. The reaction was then

quenched with AcOH (0.25 mL, 100 equiv.), and the mixture was 1 H) 3.89 (d, J = 3.2 Hz, 1 H), 3.85–3.81 (m, 3 H), 3.79–3.73 (m,

concentrated in vacuo. The crude reaction mixture was coevapo-

rated with toluene.

2 H), 3.71–3.3.63 (m, 3 H), 3.60–3.51 (m, 4 H), 2.17 (m, 2 H), 2.03

(m, 2 H), 1.58 (dm, J = 130.0 Hz, 1 H), 1.43–1.14 (m, 46 H), 0.90

(t, J = 6.9 Hz, 6 H) ppm. ¹³C NMR (151 MHz, CDCl₃/[D₄]meth-

anol): δ = 133.0, 132.7, 100.7, 78.9, 74.8, 60.7, 52.6, 31.5, 31.3,

29.0–28.2 (m), 25.5–24.8 (m), 24.3–24.0 (m), 21.71, 21.60, 19.68,

The residue was cooled in an ice-bath, and then trifluoroacetic acid

(3 mL) was added. The product had completely dissolved after ca.

1 min, and the reaction mixture was stirred for a further 1 min at

0 °C. The solution was then transferred to a round-bottomed flask

containing toluene (50 mL), and the mixture was concentrated in

vacuo to ca. 10 mL. The coevaporation was repeated twice with

toluene (40 mL), and then the mixture was concentrated to dryness.

The completion of the reaction was monitored by HPLC–MS. The

reaction mixture was filtered through a small silica column

(MeOH/CH₂Cl₂, 1:9, then H₂O/MeOH/CH₂Cl₂, 3:27:70; TLC visu-

alised with ninhydrin spray). Further purification by HPLC–MS

(40–48% B, following the general procedure for HPLC–MS purifi-

cations) gave globotriaosylsphingosine 41b (17 mg, 21 μmol, 48%)

12.6 ppm. IR (neat): ν = 3300, 2914, 2849, 1632, 1551, 1470, 1370,

˜

1203, 1070, 1024, 716 cm^–1. HRMS: calcd. for [C₄₉¹³C₃H₉₇NO₁₈

H]⁺1027.6884; found 1027.6881.

+

Globotriaosyl-2-N-(hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphingosine

(42c): See the general procedure for the synthesis of ceramides from

sphingosine, yield (5.5 mg, 5.3 μmol, 51%). R_f= 0.35 (CHCl₃/

MeOH/H₂O, 70:27:3). [α]²_D²= +26 (c = 0.15, MeOH/CHCl₃, 1:1).

¹H NMR (850 MHz, CDCl₃/[D₄]methanol): δ = 5.69 (dm, J =

150.0 Hz, 1 H), 5.45 (m, 1 H), 4.96 (d, J = 3.8 Hz, 1 H), 4.41 (d, J

= 6.9 Hz, 1 H), 4.30 (d, J = 7.8 Hz, 1 H), 4.25 (ddd, J = 6.8, 4.7,

2676

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Eur. J. Org. Chem. 2015, 2661–2677

Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids

Groener, M. Maas, F. A. Wijburg, D. Speijer, A. Tylki-Szyman-

ska, P. K. Mistry, R. G. Boot, J. M. Aerts, Blood 2011, 118,

e118–127.

1.3 Hz), 4.19 (dd, J = 10.1, 4.5 Hz, 1 H), 4.07 (t, J = 8.8 Hz, 1 H,

1 H), 4.04–3.96 (m, 4 H), 3.92 (d, J = 3.0 Hz, 1 H) 3.89 (d, J =

3.2 Hz, 1 H), 3.85–3.81 (m, 3 H), 3.79–3.73 (m, 2 H), 3.71–3.63 (m,

3 H), 3.60–3.51 (m, 4 H), 2.17 (m, 2 H), 2.03 (m, 2 H), 1.58 (dm,

J = 130.0 Hz, 1 H), 1.43–1.14 (m, 46 H), 0.90 (t, J = 6.9 Hz, 6 H)

ppm. ¹³C NMR (213 MHz, CDCl₃/[D₄]methanol): δ = 174.0, 133.2

(d, J = 42.0 Hz), 103.4, 102.4, 100.7, 74.4, 72.9, 72.6, 70.9, 70.5,

69.0, 68.4, 67.6, 66.1, 60.8, 38.6 35.6, 31.8–31.0 (m), 29.1–28.1 (m),

[4] T. Wennekes, R. J. van den Berg, R. G. Boot, G. A.

van der Marel, H. S. Overkleeft, J. M. Aerts, Angew. Chem. Int.

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25.2, 25.1, 21.8, 21.6, 19.7, 12.6 ppm. IR (neat): ν = 3300, 2914,

˜

2849, 1633, 1549, 1468, 1204, 1069, 1026, 719 cm^–1. HRMS: calcd.

for [C₄₇¹³C₅H₉₇NO₁₈+ H]⁺1029.6951; found 1029.6949.

Globotriaosyl-2-N-([3,4,5-¹³C₃]-hexadecanoyl)-[5,6,7,8,9-¹³C₅]-sphin-

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1

1:1). H NMR (850 MHz, CDCl₃/[D₄]methanol): δ = 5.69 (dm, J

= 150.0 Hz, 1 H), 5.45 (m, 1 H), 4.95 (d, J = 3.8 Hz, 1 H), 4.41 (d,

J = 6.9 Hz, 1 H), 4.30 (d, J = 7.8 Hz, 1 H), 4.25 (m, 1 H), 4.19

(dd, J = 10.1, 4.5 Hz, 1 H), 4.07 (m, 1 H), 4.04–3.96 (m, 4 H), 3.92

(d, J = 3.0 Hz, 1 H) 3.89 (m, 1 H), 3.85–3.81 (m, 3 H), 3.79–3.73

(m, 2 H), 3.71–3.63 (m, 3 H), 3.60–3.51 (m, 4 H), 2.17 (m, 2 H),

2.02 (dm, J = 128.0 Hz, 2 H), 1.65–1.14 (m, 48 H), 0.90 (t, J =

6.9 Hz, 6 H) ppm. ¹³C NMR (213 MHz, CDCl₃/[D₄]methanol): δ

= 133.1 (d, J = 44.6 Hz), 31.8–30.8 (m), 29.8–28.0 (m), 25.95,

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25.27–24.95 (m), 21.67, 21.52, 19.56, 12.4 ppm. IR (neat): ν = 3300,

˜

2955, 2849, 1634, 1549, 1466, 1070, 1028, 719 cm^–1. HRMS: calcd.

for [C₄₄¹³C₈H₉₇NO₁₈+ H]⁺1032.7052; found 1032.7053.

Supporting Information (see footnote on the first page of this arti-

1

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Acknowledgments

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The Netherlands Organization for Scientific Research (NWO-CW)

, Top grant, to H. S. O. and J. M. A.) and the European Research

Council (ERC AdG, to H. S. O.) are acknowledged for financial

support.

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Received: July 8, 2014

Published Online: March 6, 2015

Eur. J. Org. Chem. 2015, 2661–2677

www.eurjoc.org

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Synthesis of a panel of carbon-13-labelled (glyco)sphingolipids

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