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. Author manuscript; available in PMC: 2011 Nov 1.
Published in final edited form as: Chem Phys Lipids. 2010 Sep 17;163(8):809–813. doi: 10.1016/j.chemphyslip.2010.09.001

Selective deuterium labeling of the sphingoid backbone: facile syntheses of 3,4, 5-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin

Hoe-Sup Byun 1, Robert Bittman 1,*
PMCID: PMC2970728  NIHMSID: NIHMS237476  PMID: 20836998

Abstract

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen–deuterium exchange reaction of a β-ketophosphonate intermediate catalyzed by ND4Cl in D2O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,β-enone in perdeuteromethanol was used.

Keywords: Lipid synthesis, Sphingolipid, 2H-labeled

1. Introduction

Stable isotope-labeled natural products are widely used in the elucidation of the structures of metabolites, toxicity studies, and investigations of metabolism in normal and diseased states in vivo (Mutlib, 2008; Magkos and Mittendorfer, 2009; Postle and Hunt, 2009). There has been increased interest in lipids labeled with stable isotopes (principally deuterium) because of the dramatic success of lipidomics in identifying and quantifying individual molecular species present in lipid extracts by tandem mass spectrometry (Han, 2010; Shevchenko and Simons, 2010). Therefore, the development of efficient and facile synthetic methods to incorporate deuterium regioselectively into lipids is needed.

Deuterated sphingolipids have been used in deuterium NMR studies to determine the C–D bond order in the N-acyl chain of sphingomyelin bilayers (Mehnert et al., 2006; Bartels et al., 2008). However, selective deuteration of the interfacial region of the long-chain base of sphingomyelin has not yet been reported. In this communication we report a convenient methodology for inserting deuterium labels into the vinyl group and/or the C3 position of the sphingoid backbone, thereby enabling analysis of the conformational and order parameters in the lipid–water interfacial region of sphingolipid bilayers by solid-state 2H NMR spectroscopy.

Many deuterium-labeled lipids can be prepared by employing the routes for the synthesis of their radiolabeled counterparts. For example, tritium was incorporated at the C3 positions of sphingosine, sphinganine, and ceramide by sodium [3H]-borohydride or lithium aluminum [3H]-hydride reduction of 3-keto analogues, which affords a mixture of the erythro and threo stereoisomers of the C3-labeled compound (Hoffman and Tao, 1998; reviewed by Bielawska et al., 2000). Regiospecific oxidation of the primary hydroxy group was achieved by using C2- and C3-protected sphingolipids, giving a protected aldehyde that on reduction provided a C1-tritiated sphingolipid. Alternatively, an enantioselective aldol condensation of (E)-2-hexadecenal with a chiral iminoglycinate provided a 1-carboxylic ester intermediate (Solladié-Cavallo and Koessler, 1994); reduction of the 1-carboxylic ester with [3H]-NaBH4 afforded [1,1-3H]-sphingosine (Li et al., 1999).

The structures of the deuterium-labeled sphingolipids we prepared are shown in Fig. 1. A key step in our synthesis of 3,4,5-trideuterio-d-erythro-sphingosine (1) is a Horner–Wadsworth–Emmons (HWE) olefination reaction of 1-deuteromyristaldehyde with a dideutero-β-ketophosphonate derived from N-Boc-l-serine methyl ester. 3-Deuterio-d-erythro-sphingomyelin (2) was prepared via a NaBD4-mediated reduction of an unlabeled enone, which was synthesized in a HWE reaction of myristaldehyde with a β-ketophosphonate; in a subsequent step, the phosphocholine head group was installed.

Fig. 1.

Fig. 1

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Structures of 3,4,5-trideuterio-d-erythro-sphingosine (1) and 3-deutero-d-erythro-N-palmitoylsphingosylphosphocholine (2).

2. Results and discussion

2.1. Synthesis of compound 1

1-Deuteriotetradecanal (5) was prepared from methyl myristate (3) via 2,2-dideuteromyristyl alcohol (4) in two steps, as shown in Scheme 1. Lithium aluminum deuteride reduction of 3 afforded α,α-dideuterated alcohol 4 in 91% yield after purification by column chromatography. Oxidation of alcohol 4 with PCC in CH2Cl2 gave deuterated aldehyde 5 in 82% yield, which was spectroscopically pure by 1H NMR without the need for chromatography. As expected, the 1H NMR spectrum of 5 showed no CHO peak at δ 10 ppm. The deuterium atom in 5 became incorporated into the 5 position of sphingosine (1) as discussed below.

Scheme 1.

Scheme 1

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Synthesis of 1-deuteriomyristaldehyde (5) from methyl myristate (3).

The synthesis of 3,4,5-trideuteriosphingosine (1) is depicted in Scheme 2. Condensation of l-Garner methyl ester 6 (prepared from N-Boc-l-serine methyl ester as described by Garner and Park, 1987) with the lithium salt of dimethyl methylphosphonate in THF is known to afford β-ketophosphonate 7 (Koskinen and Koskinen, 2000) via enolate intermediate 8. Since the H–D exchange reaction is a straightforward method to introduce C–D bonds vicinal to carbonyl and phosphonyl groups, we first attempted to carry out a hydrogen–deuterium exchange reaction with 7 by using D2O in THF or MeCN and catalysis by either K2CO3 or ND4Cl. This was unsuccessful. However, we succeeded in preparing the desired dideuterio-β-ketophosphonate by the following sequence of reactions. First, lithium enolate 8 was converted to mono-deuterated β-ketophosphonate 9 with a catalytic amount of ND4Cl in D2O/THF (1:1) at room temperature. The hydrogen–deuterium exchange reaction was repeated three times, affording α,α-dideuterio-β-ketophosphonate 10. The product of the exchange reactions was confirmed by 1H NMR (disappearance of the PCH2CO multiplet peaks around δ 3.15 ppm). A precedent for hydrogen–deuterium exchange with ND4Cl in D2O/THF is the synthesis of 5-epi-[6-2H]-valiolone after reductive desulfurization of a bis(methylthio) moiety alpha to a carbonyl group (Mahmud et al., 2001).

Scheme 2.

Scheme 2

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Synthesis of 3,4,5-trideuteriosphingosine (1).

In the next step, HWE reaction of α,α-dideuterio-β-ketophosphonate 10 with 1-deuterotetradecanal (5) in D2O/THF (1:1) containing 3 equiv. of K2CO3 at 0°C for 4 h and then at room temperature for 72 h provided α,β-enone 11, which bears deuterium atoms in the vinyl group, in 61% yield. To incorporate deuterium at C-3, Luche reduction (Gemal and Luche, 1981) of the carbonyl group of 11 with sodium borodeuteride was employed. Thus, reaction of enone 11 with NaBD4 in the presence of CeCl3·7H2O (to suppress 1,4-hydride addition) in THF/CD3OD (10:1) for 4 h at room temperature gave alcohol 12 together with traces of its threo isomer (Chun et al., 2002a,b, 2003). In order to reduce amount of perdeuterated methanol, we used THF as the co-solvent. Column chromatography on silica gel (elution with hexane/EtOAc 10:1) gave erythro-enriched alcohol 12 in 89% yield. High-resolution mass spectral (HRMS) analysis of the relative intensities in the isotope pattern of 12 indicated that the isotopic composition was approximately 16% dideuterio, 64% trideuterio, and 20% tetradeuterio. The tetradeuterio species may arise from 1,4-addition of deuteride ion to 11, followed by protonation of the enolate ion intermediate with the traces of water present in the hydrated CeCl3 rather than deuteration with CD3OD. Therefore, the amount of tetradeuterio species may be enhanced by conversion of CeCl3·7H2O to CeCl3·7D2O via multiple treatments of the salt with D2O.

Treatment of acetonide 12 with 1M HCl in THF at room temperature provided 3,4,5-trideuterio-N-Boc-sphingosine 13 in 78% yield. At this stage, the traces of the threo isomer that had remained were completely removed by column chromatography on silica gel (elution with hexane/EtOAc 1:1). Finally, removal of the N-Boc group with 3M HCl in THF at 70 °C furnished deuterium-labeled sphingosine 1 as its hydrochloride salt in quantitative yield.

2.2. Synthesis of compound 2

Scheme 3 outlines the preparation of 3-deuterio-N-palmitoyl-d-erythro-sphingosylphosphocholine (2). Enone 14 was prepared by HWE reaction of 7 with unlabeled myristaldehyde. Reduction of 14 with NaBD4 in the presence of CeCl3·7H2O in THF/CD3OD (10:1) for 4 h at room temperature gave alcohol 15 together with traces of its threo isomer. After removal of the isopropylidene group, the phosphocholine moiety was inserted into N-Boc-3-deuterio-sphingosine 16 via a one-pot reaction with ethylene chlorophosphite, oxidation with bromine, and quaternization with trimethylamine (Byun et al., 1994; Erukulla et al., 1994). After the N-Boc group was removed by heating with 3M HCl in methanol, the resulting protonated amine was treated with diisopropylethylamine. An N-acylation reaction of C3-deuteriosphingosylphosphocholine with p-nitrophenyl palmitate provided 3-deuterio-N-palmitoyl-d-erythro-sphingosylphosphocholine (2). The extent of deuteration wasestimated to be approximately 95%, based on the relative intensities of the m/z peaks at 703.5752 ([M+H+] ion of nondeuterated 2), 704.5812 ([M+H+] ion of mono-deuterated 2), and 705.5844 (13C isotope peak of the [M+H+] ion of mono-deuterated 2).

Scheme 3.

Scheme 3

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Synthesis of 3-deuterio-N-palmitoyl-d-erythro-sphingosylphosphocholine (2).

In summary, we developed simple procedures for the preparation of deuterium-labeled sphingosine (1) and sphingomyelin (2) from l-Garner methyl ester 6. The methodology described here can be extended to prepare a variety of other deuterated sphingolipids. For example, other deuterium-labeled derivatives of sphingosine are readily accessible by using multi-deuterated analogues of 5, deuterated glyco- or phosphosphingolipids can be prepared from intermediate 13, and deuterium-labeled ceramides can be conveniently obtained from 1. Compounds containing deuterium at selected sites in the interfacial region of the sphingoid backbone may be useful as probes for 2H NMR spectroscopic analysis and as internal standards for analysis of sphingosine and sphingomyelin in biological samples by electrospray ionization/tandem mass spectrometry.

3. Experimental procedures

3.1. General methods

HRMS were obtained by electrospray ionization (ESI) on an Agilent Technologies G1969A or G6520A Q-TOF mass spectrometer, which have mass accuracies of <3 and <2 ppm, respectively. Analyses were done using two reference ions. 1H, 13C, and 31P NMR spectra were recorded at 400, 100, and 161.9 MHz, respectively, on a Bruker Avance I spectrometer, and were referenced to the residual chloroform at δ 7.24 (1H) and δ 77.2 (13C) and to external 85% H3PO4 at δ 0.00ppm (31P). TLC was carried out on aluminum-backed silica gel GF plates (250-μm thickness), and the compounds were visualized by charring with 10% sulfuric acid in ethanol and/or short wavelength UV light. For flash chromatography, silica gel 60 (230–400 ASTM mesh) was used. THF was distilled from Na and benzophenone before use. CH2Cl2 and i-Pr2NEt were dried over CaH2.

3.2. 1,1-Dideuteriotetradecanol (4)

To a solution of methyl myristate (3, 2.50 g, 10.3 mmol) in THF was added LiAlD4 (600 mg, 14.3 mmol) at −78 °C. After 2 h, the reaction mixture was gradually warmed to room temperature and stirred overnight. After the reaction was quenched with solid Na2SO4·10H2O (2.0 g, 6.2 mmol), the mixture was passed through a pad of Celite, which was washed with 100mL of EtOAc. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (elution with hexane/EtOAc 4:1) to give 2.03 g (91%) of alcohol 4 (which is available commercially from several vendors): 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.4 Hz), 1.26 (m, 22H), 1.55 (t, 2H, J = 6.4 Hz); 13C NMR (CDCl3) δ 14.1, 22.7, 25.7, 29.4, 29.4, 29.59, 29.60, 29.7, 31.9, 32.6, 62.3 (pentet, J = 21 Hz).

3.3. 1-Deuteriotetradecanal (5)

To a mixture of PCC (2.16 g, 10.0 mmol) and silica gel (3 g) in 50mL of CH2Cl2 was added a solution of alcohol 4 (984 mg, 4.55 mmol) in 10mL of CH2Cl2 at room temperature. After the mixture was stirred for 2 h, it was diluted with 300mL of Et2O and passed through a pad of silica gel, which was washed 100mL of Et2O. The filtrate was concentrated under reduced pressure. The residuewasdissolved in aminimal volume of Et2O and passed again through a pad of silica gel, which was washed with Et2O. The filtrate was concentrated under reduced pressure to give 795mg (82%) of aldehyde 5: 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz), 1.25 (s, 20H), 1.62 (m, 2H), 2.41 (t, 2H, J = 7.2 Hz); 13C NMR (CDCl3) δ 14.1, 21.3, 22.5, 28.8, 28.9, 29.3, 29.60, 29.61, 29.7, 32.1, 43.73 (t, J = 4 Hz), 202.6 (t, J = 25 Hz).

3.4. N-tert-Butoxycarbonyl (4S)-[2′,2′-dideuterio-2′-dimethylphosphonoacetyl]-2,2-dimethyl-1,3-oxazolidine (10)

To a solution of dimethyl methylphosphonate (1.65 g, 13.3 mmol) in 50mL of THF was added dropwise n-BuLi (5.2mL of a 2.5M solution in hexane, 13.0 mmol) at −78 °C. After 2 h, a solution of l-Garner methyl ester 6 (1.65 g, 6.36 mmol) in 10mL of THF was added slowly. The mixture was stirred at −78 °C for 2 h, then allowed to warm to room temperature, and stirred overnight. After the reaction was quenched by addition of solid ND4Cl (770 mg, 13.4 mmol), it was concentrated under reduced pressure. The residue was dissolved in EtOAc (300 mL) and washed with brine. The organic layer was dried (MgSO4) and concentrated. Purification by chromatography on silica gel (elution with hexane/EtOAc 1:1 and then EtOAc) gave 1.50 g (67%) of β-ketophosphonate 9 as a colorless oil. To a solution of 9 in 5mL of THF and 5mL of D2O was added ND4Cl (2.0 mg, 34.6μmol) at room temperature. After the mixture was stirred overnight, it was concentrated under reduced pressure to give a residue. This exchange reaction was repeated three times. Dideuterated β-ketophosphonate 10 was used without further purification; 1H NMR (CDCl3) δ 1.45–1.75 (m, 15H), 3.65–3.95 (m, 6H), 4.04–4.17 (m, 2H), 4.49–4.60 (m, 1H).

3.5. N-tert-Butoxycarbonyl (4S)-[1′-oxo-(2′E)-2′,3′-dideuteriohexadecenyl]-2,2-dimethyl-1,3-oxazolidine (11)

To a mixture of β-ketophosphonate 10 (1.50 g, 4.25 mmol) and K2CO3 (1.80 g, 13.0 mmol) in 10mL of D2O was added a solution of aldehyde 5 (788 mg, 3.69 mmol) in 10mL of THF at 0 °C. After 4 h, the reaction mixture was gradually raised to room temperature and stirred for 72 h. After dilution with EtOAc (250 mL), the mixture was washed with brine, dried (Na2SO4), and concentrated under reduced pressure. Purification of the residue by column chromatography on silica gel (elution with a gradient of hexane, hexane/EtOAc 50:1 and 20:1) gave 991 mg (61%) of enone 11: 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 7.0 Hz), 1.09–1.31 (m, 20 H), 1.48 (s, 9H), 1.54 (s, 3H), 1.83 (s, 3H), 1.95 (q, 2H, J = 6.5 Hz), 3.82 (m, 2H), 5.85 (m, 1H), 5.98 (m, 1H), 6.23 (d, 1H, J = 15.5 Hz), 7.30 (m, 1H); 13C NMR (CDCl3) δ 14.1, 20.1, 28.4, 29.0, 29.1, 29.4, 29.5, 29.7, 32.2, 33.2, 33.3, (64.7), 65.0, (66.0), 66.3, (79.9), 80.0, (94.4), 95.3, 123.3, (124.4), (128.2), 129.2, (144.0), 144.2, (145.8), 146.4, 151.7, (152.2), (195.5), 196.0. The 13C NMR chemical shifts in parentheses indicate the small peaks that arise from minor rotamers in the oxazolidine system, which undergoes a slow dynamic equilibrium at room temperature.

3.6. N-tert-Butoxycarbonyl (4S)-[(1′S)-hydroxy-1′,2′,3′-trideuterio-(2′E)-hexadecenyl]-2,2-dimethyl-1,3-oxazolidine (12)

To a mixture of enone 11 (985 mg, 2.24 mmol) and CeCl3·7H2O (186 mg, 0.50 mmol) in 40mL of THF and 4mL of CD3OD was added NaBD4 (123 mg, 2.94 mmol) at −78 °C. The mixture was gradually raised to room temperature. After 4 h, the mixture was filtered through a pad of Celite, which was washed with 150mL of EtOAc. The filtrate washed with brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (elution with hexane/EtOAc 9:1) to give 882mg(89%) of alcohol 12: 1HNMR(CDCl3) δ 0.89 (t, 3H, J = 6.6 Hz), 1.27 (m, 20H), 1.48 (s, 9H), 1.53 (s, 3H), 1.57 (s, 3H), 1.98 (br s, 1H), 2.08 (q, 2H, J = 6.8 Hz), 4.03 (m, 1H), 4.08 (br s, 1H), 4.16 (br m, 1H), 4.31 (br s, 1H), 6.28 (m, 1H); 13C NMR (CDCl3) δ 14.1, 10.0, 22.9, 24.4, 26.8, 28.1, 28.4, 29.5, 29.67, 29.72, 29.89, 29.99, 30.0, 32.3, 32.9, 33.1. 62.0, 73.6, 80.2, 94.6, 128.4, 128.7, 133.8, 134.1, 161.1; HRMS [M+H]+ m/z calcd. for C26H47D3NO4, 443.3923, found, 443.3924. Additional [M+H]+ ions were also present: m/z calcd. for C26H46D4NO4, 444.3985, found, 443.3972; m/z calcd. for C26H48D2NO4, 442.3860, found, 442.3858. The relative intensities of these peaks, after correction for the natural abundance of 13C, are 0.64, 0.20, and 0.16, respectively.

3.7. (2S,3R)-2-(N-tert-Butoxycarbonylamido)-4(E)-3,4,5-trideuteriooctadecene-1,3-diol (13)

A solution of acetonide 12 (874 mg, 1.97 mmmol) in 1M HCl (10 mL) and THF (20 mL) was stirred at room temperature until the reaction was completed. The mixture was neutralized with saturated aqueous NaHCO3 solution, and the product was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (elution with hexane/EtOAc 2:1 then 1:1) to give 621 mg (78%) of N-Boc-sphingosine-d3 13 (containing minor amounts of the corresponding d2 and d4 species as described above): 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz), 1.26 (m, 20H), 1.37 (m, 2H), 1.45 (s, 9H), 2.04 (q, 2H, J = 7.1 Hz), 3.07 (br s, 2H), 3.68 (m, 1H), 3.77 (m, 1H), 3.91 (m, 1H), 5.32 (br s, 1H); 13C NMR (CDCl3) δ 14.1, 22.7, 28.4, 29.04, 29.07, 29.3, 29.47, 29.59, 29.62, 29.7, 31.9, 32.1, 55.4, 62.5, 64.1, 74.1 (t, J = 18 Hz), 79.8, 128.4 (t, J = 16 Hz), 133.6 (t, J = 16 Hz), 156.2; HRMS [M+Na]+ m/z calcd for C23H42D3NNaO4, 425.3429, found, 425.3424.

3.8. (2S,3R)-2-Amino-(4E)-3,4,5-trideuteriooctadecene-1,3-diol hydrochloride salt (1)

A solution of N-Boc-sphingosine-d3 (13) (417 mg, 1.03 mmol) in 2mL of 3M HCl and 10mL of THF was heated at 70 °C with stirring overnight under nitrogen. The reaction mixture was cooled to room temperature, concentrated, and lyophilized from C6H6 to give 351 mg (100%) of sphingosine-d3 HCl salt 1: 1H NMR (CDCl3) δ 0.85 (t, 3H, J = 6.8 Hz), 1.26 (m, 20H), 1.36 (m, 2H), 2.08 (q, 2H, J = 7.1 Hz), 3.07 (br s, 2H), 3.68 (m, 1H), 3.77 (m, 1H), 3.91 (m, 1H), 5.32 (br s, 1H); 13C NMR (CDCl3/CD3OD) δ 14.2, 22.6, 28.4, 29.06, 29.08, 29.3, 29.48, 29.58, 29.63, 29.7, 31.9, 32.1, 55.4, 62.5, 64.1, 74.2 (t, J = 18 Hz), 128.3 (t, J = 18 Hz), 133.6 (t, J = 18 Hz); HRMS [M+H]+ m/z calcd. for C18H35D3NO2, 303.3085, found, 303.3089; HRMS [M+Na]+ m/z calcd for C23H42D3NNaO2, 325.2905, found, 325.2905.

3.9. N-tert-Butoxycarbonyl (4S)-4-[1′-hydroxy-1′-deutero-(2′E)-hexadecanyl]-2,2-dimethyl-1,3-oxazolidine (15)

To the mixture of enone 14 (298 mg, 0.68 mmol) and CeCl3·7H2O (56 mg, 0.15 mmol) in 20mL of THF and 2mL of CD3OD was added NaBD4 (37 mg, 0.89 mmol) at −78°C. The mixture was gradually raised to room temperature. After 10 h, the mixture was filtered through a pad of Celite, which was washed with 150mL of EtOAc. The filtrate washed with brine, dried (Na2SO4), and concentrated under reduced pressure. A residue was purified by column chromatography on silica gel (elution with hexane/EtOAc 9:1) to give 240mg (80%) of 3-deuterio-alcohol 15 as a colorless oil: 1H NMR δ 0.88 (t, 3H, J = 6.6 Hz), 1.25 (m, 20H), 1.49 (s, 9H), 1.30–1.70 (m, 10H), 2.05 (m, 2H), 3.75–4.25 (m, 4H), 5.44 (d, 1H, J = 15.2 Hz), 5.73 (m, 1H); 13C NMR δ 14.1, 10.0, 22.9, 24.4, 26.8, 28.1, 28.4, 29.5, 29.67, 29.72, 29.89, 29.99, 30.0, 32.3, 32.9, 33.1, 59.4, 61.3, 62.3, 64.8, 65.2, 66.1, 66.4, 73.4, 81.0 (t, J = 24 Hz), 94.4, (95.0), 128.0, (129.5), 131.8, 133.4, (135.4), 171.3, (170.92).

3.10. (2S,3R)-2-(N-tert-Butoxycarbonylamido)-3-deutero-4(E)-octadecene-1,3-diol (16)

A solution of alcohol 15 (233 mg, 0.53 mmol) in 10mL of 1M HCl and 50mL of THF was stirred at room temperature until the reaction was completed. The mixture was neutralized with saturated aqueous NaHCO3 solution, and the product was extracted with EtOAc (100mL×3). The combined organic layers were washed with brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (elution with hexane/EtOAc 2:1 then 1:1) to give 180mg (85%) of 3-deuterio-N-Boc-sphingosine 16: 1H NMR δ 0.88 (t, 3H, J = 6.8 Hz), 1.25 (m, 20H), 1.45 (s, 9H), 2.05 (q, 2H, J = 7.1 Hz), 2.45 (br s, 2H), 3.61 (br s, 1H), 3.71 (m, 1H), 3.95 (m, 1H), 5.81 (br s, 1H), 5.53 (d, 1H, J = 15.3 Hz), 5.78 (m, 1H); 13C NMR δ 14.1, 22.6, 22.7, 25.8, 29.0, 29.2, 29.3, 29.59, 29.62, 29.65, 31.7, 31.9, 32.7, 36.8, 55.3, 62.6, 64.2, 74.1 (t, J = 18 Hz), 79.8, 134.0, 138.0, 154.6.

3.11. 3-Deuterio-N-palmitoyl-d-erythro-sphingosylphosphocholine (2)

To a solution of 2-chloro-1,3,2-dioxaphospholane (173 mg, 1.36 mmol) in 25mL of CH2Cl2 was added 70 μL (1.36 mmol) of Br2 at −78°C. After 10 min, a solution of 16 (175 mg, 0.437 mmol) and N,N-diisopropylethylamine (230 mg, 1.78 mmol) in 10mL of CH2Cl2 was added. After the reaction mixture was stirred at room temperature for 48 h, it was concentrated under reduced pressure to give a residue, which was dried under high vacuum for 2 h. To a stirred solution of the residue in 9mL of CHCl3 were added 39mL of aqueous NMe3 (40%) solution, 15mL of acetonitrile, and 15mL of 2-propanol. After the mixture was stirred at room temperature for 72 h, it was concentrated under reduced pressure. The product was purified by column chromatography on silica gel (elution with CHCl3/MeOH/H2O 65:25:4) to give 134 mg (54%) of 3-deuterio-N-Boc-sphingomyelin 17; HRMS [M+H+] m/z calcd. for C28H57DN2O7P, 566.4044, found 566.4039. A solution of 17 (102 mg, 0.18 mmol) in 5mL of 3MHCl and 50mL of methanol was heated at reflux overnight. The mixture was concentrated under reduced pressure to give a residue. To the residue were added 20mL of THF/H2O (9:1), N,N-diisopropylethylamine (100μL, 0.57 mmol), and 4-nitrophenyl palmitate (300 mg, 0.79 mmol). After the mixture was stirred for 72 h at room temperature, it was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (elution with CHCl3/MeOH/H2O, 65:25:4 and then 65:35:8) to afford 58mg (35%) of 2 after filtration of a chloroform solution through a 0.45-μm Teflon syringe filter to remove suspended silica gel: 1H NMR (CDCl3) δ 0.88 (t, 6H, J = 6.0 Hz), 1.26 (m, 32H), 1.53 (m, 2H), 2.07 (m, 2H), 2.26 (m, 2H), 3.33 (s, 9H), 3.93–3.66 (m, 4H), 4.29 (m, 4H), 5.50 (br s, 1H), 5.52 (d, 1H,J = 15.6 Hz), 5.77 (m, 1H); 13C NMR (CDCl3/CD3OD) δ 13.5, 25.1, 25.6, 28.91, 28.98, 29.0 29.06, 29.1, 29.20, 29.21, 29.28, 29.31, 31.5, 32.0, 36.1, 36.6, 53.7, 58.6 (d, J = 5.0 Hz), 64.2 (d, J = 5.0 Hz), 66.1, 66.5 (d, J = 5.0 Hz), 74.9 (t, J = 18 Hz), 128.7, 134.1, 175.7; 31P NMR (CDCl3/CD3OD) δ 0.29; HRMS [M+H+] m/z calcd. for C39H79DN2O6P, 704.5811, found 704.5812.

Acknowledgments

We are grateful to Dr. Cliff Soll for HRMS analyses and to the National Institutes of Health, Grant HL-083187, for financial support.

References

  1. Bartels T, Lankalapalli RS, Bittman R, Brown MF, Beyer K. Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy. J Am Chem Soc. 2008;130:14521–14532. doi: 10.1021/ja801789t. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bielawska A, Hannum YA, Szulc Z. Radiolabeling of the sphingolipid backbone. Methods Enzymol. 2000;311:480–498. doi: 10.1016/s0076-6879(00)11101-2. [DOI] [PubMed] [Google Scholar]
  3. Byun H-S, Erukulla RK, Bittman R. Synthesis of sphingomyelin and ceramide 1-phosphate from ceramide without protection of the allylic hydroxyl group. J Org Chem. 1994;59:6495–6498. [Google Scholar]
  4. Chun J, Li G, Byun H-S, Bittman R. Synthesis of new trans double-bond sphingolipid analogues: Δ4,6 and Δ6 ceramides. J Org Chem. 2002a;67:2600–2605. doi: 10.1021/jo0162639. [DOI] [PubMed] [Google Scholar]
  5. Chun J, Li G, Byun H-S, Bittman R. A concise route to d-erythro-sphingosine from N-Boc-l-serine derivatives via sulfoxide or sulfone intermediates. Tetrahedron Lett. 2002b;43:375–377. [Google Scholar]
  6. Chun J, Byun H-S, Arthur G, Bittman R. Synthesis and growth inhibitory activity of chiral 5-hydroxy-2-N-acyl-3E-sphingenines: ceramides with an unusual sphingoid backbone. J Org Chem. 2003;68:355–359. doi: 10.1021/jo026242u. [DOI] [PubMed] [Google Scholar]
  7. Erukulla RK, Byun H-S, Bittman R. Antitumor phospholipids: a one-pot introduction of a phosphocholine moiety into lipid hydroxy acceptors. Tetrahedron Lett. 1994;35:5783–5784. [Google Scholar]
  8. Garner P, Park JM. The synthesis and configurational stability of differentially protected β-hydroxy-α-amino aldehydes. J Org Chem. 1987;52:2361–2364. [Google Scholar]
  9. Gemal AL, Luche J-L. Lanthanoids in organic synthesis 6. The reduction of α-enones by sodium borohydride in the presence of lanthanoid chlorides: synthetic and mechanistic aspects. J Am Chem Soc. 1981;100:5454–5459. [Google Scholar]
  10. Han X. Multi-dimensional mass spectrometry-based shotgun lipidomics and the altered lipids at the mild cognitive impairment stage of Alzheimer’s disease. Biochim Biophys Acta. 2010;1801:774–783. doi: 10.1016/j.bbalip.2010.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoffman RV, Tao J. A synthesis of d-erythro- and l-threo-sphingosine and sphinganine diastereomers via the biomimetic precursor 3-ketosphinganine. J Org Chem. 1998;63:3979–3985. [Google Scholar]
  12. Koskinen PM, Koskinen AMP. Total synthesis of sphingosine and its analogs. Methods Enzymol. 2000;311:458–479. doi: 10.1016/s0076-6879(00)11100-0. [DOI] [PubMed] [Google Scholar]
  13. Li S, Pang J, Wilson WK, Schroepfer GJ., Jr Efficient synthesis of deuteriumor tritium-labeled d-erythro-sphingosine. J Labeled Cpd Radiopharm. 1999;42:815–826. [Google Scholar]
  14. Magkos F, Mittendorfer B. Stable isotope-labeled tracers for the investigation of fatty acid and triglyceride metabolism in humans in vivo. Clin Lipidol. 2009;4:215–230. doi: 10.2217/clp.09.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mahmud T, Xu J, Choi YU. Synthesis of 5-epi-[6-2H2]valiolone and stereo-specifcally monodeuterated 5-epi-valiolones: exploring the steric course of 5-epi-valiolone dehdratase in validamycin A biosynthesis. J Org Chem. 2001;66:5066–5073. doi: 10.1021/jo0101003. [DOI] [PubMed] [Google Scholar]
  16. Mehnert T, Bittman R, Jacob R, Beyer K. Structure and lipid interaction of N-palmitoyl-d-erythro-sphingosylphosphocholine in bilayer membranes as revealed by 2H NMR spectroscopy. Biophys J. 2006;90:939–946. doi: 10.1529/biophysj.105.063271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mutlib AE. Application of stable isotope-labeled compounds in metabolism and in metabolism-based toxicity studies. Chem Res Toxicol. 2008;21:1672–1689. doi: 10.1021/tx800139z. [DOI] [PubMed] [Google Scholar]
  18. Postle AD, Hunt AN. Dynamic lipidomics with stable isotope labeling. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:2716–2721. doi: 10.1016/j.jchromb.2009.03.046. [DOI] [PubMed] [Google Scholar]
  19. Shevchenko A, Simons K. Lipidomics: coming to grips with lipid diversity. Nat Rev Mol Cell Biol. 2010;11:593–598. doi: 10.1038/nrm2934. [DOI] [PubMed] [Google Scholar]
  20. Solladié-Cavallo A, Koessler JL. A four-step diastereoselective synthesis of d-erythro-sphingosine by an enantioselective aldol reaction using a titanium enolate derived from a chiral iminoglycinate. J Org Chem. 1994;59:3240–3242. [Google Scholar]

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