Facile Chemoselective Strategy toward Capturing Sphingoid Bases by a Unique Glutaraldehyde-Functionalized Resin.

Sphingoid bases, which have a 2-amino-1,3-diol common functional group, are the structural backbone units of all sphingolipids. Recently, much attention has been focused on sphingoid bases because of their potentially beneficial bioactivities toward various cancer cells as well as their dietary interest. However, low abundance and the handling complexity caused by their amphiphilic character led to very limited research on them. Glutaraldehyde has two aldehyde groups, and it reacts rapidly with the 2-amino-1,3-diol functional group of sphingosine to give a tricyclic product. Immobilization of glutaraldehyde on a resin was successfully performed by organic synthesis, starting from trans-p-coumaric acid via eight steps. This approach suppresses the self-polymerization of glutaraldehyde, and addition of water to the developed resin causes the formation of cyclic double hemiacetal function, which avoids oxidation like a reducing sugar in nature and makes it stable even for up to 1 year incubation. The resin was applied to the solid-phase extracting experiment of free sphingosine from human serum at a concentration of 280 nM. Another extraction study of edible golden oyster mushrooms showed that the sphingoid base was selectively captured from complex natural extracts. These results demonstrate that the developed glutaraldehyde resin method is a highly selective method, and hence, the combination of it with the o-phthaldialdehyde HPLC method was confirmed as an efficient and sensitive method for analysis of sphingoid bases in biological samples.

Introduction

Sphingolipids are found essentially in all animals, plants, and fungi, as well as in some prokaryotic organisms and viruses. They are the basic constituents of the plasma membranes, and more importantly, they and their metabolites play crucial roles in many physiological processes, including immune response, cell proliferation, differentiation, and apoptosis.[1−3] Sphingolipids are composed of a structurally related family of hydrocarbon backbones having 2-amino-1,3-diol-termed sphingoid bases. The most predominant sphingoid base found in mammalian cells is d-erythro-sphingosine, and its cellular levels were controlled by the action of ceramide synthase and sphingosine kinase.[4] However, because of their low abundance as a free base in nature compared with other primary sphingolipids and their handling complexity resulting from their amphiphilic character, they have not been well-studied.[5] In the last few years, increasing attention has been focused on sphingoid bases because of their potentially useful bioactivities, such as cytotoxicity to various cancer cells in vitro and in vivo.[6−9] Structurally modified sphingoid bases have attracted more and more attention because some of their analogues have demonstrated the capability of introducing morphological changes in neuronal cells and behaving as enzyme inhibitors.[10] Also in terms of increasing dietary interest, applicable findings, such as improvement of diet-induced glucose intolerance in mice by oral administration of phytosphingosine[11] and activation of ceramide production in a three-dimensional human skin model by some sphingoid bases originated from dietary konjac, have been reported.[12]

Regarding chemical synthesis of the sphingolipid family, a sphingoid base is an essential component, which certainly improves the variety of ceramide library by diversity-oriented acylation of its 2-amino group,[13,14] while glycosylation of its 1-hydroxyl group leads to more complex glycosphingolipid derivatives. Recently, we reported a highly efficient preparation method for these sphingoid bases from naturally occurring glucosylceramides by the chemoenzymatic method to expand the sphingolipid chemical library.[15] However, handling the complexity of the sphingoid bases is still a challenging problem to be solved. The enigmatic character of the sphingoid bases disturbs their rapid purification; therefore, conventional solvent extraction methods, such as Bligh–Dyer method, Folch method, and solid-phase method,[16,17] are the mostly used extraction methods. Although sphingoid bases can be extracted by these solvent extraction methods, they have disadvantages such as limited efficiency and low environment adaptability.

Here, we report a facile and effective solid-phase extraction method of the sphingoid base by utilizing the reaction between a sphingoid base and glutaraldehyde. Also, its application in sphingoid-base selective extraction from human serum and natural resources by the glutaraldehyde-immobilized resin is demonstrated (Scheme ).

Scheme 1

Selective Solid-Phase Extraction Methodology of Sphingoid Bases by the Glutaraldehyde-Immobilized Merrifield Resin (GARI)

Results and Discussion

Glutaraldehyde (1, 1,5-pentanedial) has two terminal aldehyde functional groups and is used biochemically as an amine-reactive homobifunctional cross-linker and a fixative. Recently, we have reported that glutaraldehyde reacts rapidly with the 2-amino-1,3-diol of sphingosine at ambient temperature to give a sole and stable tricyclic product with a moderate yield without the presence of any catalyst.[18] This specific protection of the amino-diol function in sphingosine allows chiral discrimination of four sphingosine stereoisomers, which is still a challenging work for other methods. As described in this instance, the glutaraldehyde possesses a great advantage regarding its reactivity toward sphingosine. However, an aldehyde function is easily oxidized, which leads to possible aldol condensation, affording a complex mixture of glutaraldehyde oligomers and polymers. To overcome these two disadvantages, an immobilization strategy has been employed. Namely, immobilization of the glutaraldehyde on the resin suppresses its polymerization, and addition of water to this immobilized glutaraldehyde causes the formation of cyclic double hemiacetal (1b),[19] which could avoid its oxidation and likely form a cyclic hemiacetal of a reducing sugar in nature (Scheme ).

Scheme 2

Two Forms (1a, 1b) of Glutaraldehyde and Its Reaction with d-erythro-Sphingosine (2)

Synthesis of the glutaraldehyde resin was performed as shown in Scheme . The esterification of the starting material, trans-p-coumaric acid, sequential LiAlH4 reduction, and further oxidation with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) afforded trans-p-coumaric aldehyde (4) in 65% yield.[20,21] After protection of the phenolic hydroxyl group with tert-butyldiphenyl silyl ether (5), a microwave-assisted inverse-electron-demand hetero Diels-Alder reaction of ethyl vinyl ether[22] was performed in the presence of a catalytic amount of hydroquinone to give 6 as a diastereomixture. Deprotection of the silyl ether with tetrabutylammonium fluoride (TBAF) produced a free alcohol 7 in 83% yield, which was immobilized on the Merrifield resin through the SN2 reaction to afford 8 in 88% yield. The amount of reacted chloride in the Merrifield resin was estimated by the ion chromatography method (Table S1).[23,27] The structure of 8 was confirmed by comparing its IR spectrum with that of the model compound (Figure S4).[27] The following hydrolysis of 8 with hot hydrochloric acid in dioxane successfully afforded the desired GARI (9). Its IR spectra revealed the coexistence of a dialdehyde form (9a) and a cyclic double hemiacetal form (9b) (Figure S5).[27] It is noteworthy that this resin is quite stable even in the presence of a labile dialdehyde. The IR spectrum of the resin has not changed at all even after 1 year incubation in the solid state at room temperature (Figure S7).[27] This amazing stability could be ascribed to its cyclic double hemiacetal structure as shown in carbohydrates as well as the immobilization effect hampering their self-condensation reactions.

Scheme 3

Synthesis of the GARI and Its Reaction with d-erythro-Sphingosine (2)

Reagents and conditions: (a) TBDPSCl (1.1 equiv), imidazole (1.5 equiv), DMAP (0.2 equiv), DCM, rt, 4 h (76%); (b) ethyl vinyl ether (24 equiv), hydroquinone (0.6 equiv), n-butanol, 220 °C, microwave, 6 h (81%); (c) TBAF (1.2 equiv), THF, rt, 3 h (83%); (d) Merrifield resin (1.1 equiv), K2CO3 (1.5 equiv), KI (0.1 equiv), DMF, 90 °C, overnight; (e) conc. HCl, dioxane, 60 °C, 4 h; (f) THF, rt, 1.5 h; and (g) 0.25 M aq TFA, rt, 1 h; TBDPSCl = tert-butyldiphenylchlorosilane, DMAP = N,N-dimethyl-4-aminopyridine, and DCM = dichloromethane.

Conditions were optimized by changing various organic solvents, times, and concentrations of sphingosine during capturing experiments. A pilot solid-phase extraction study was carried out using standard d-erythro sphingosine as shown in Scheme . The GARI was stirred with sphingosine in THF for 1 h, and its capturing efficiency was estimated as 86% via quantitative analysis of the uncaptured sphingosine in the solution by the o-phthaldialdehyde (OPA) analysis method.[24] After careful washing by appropriate solvents, release experiments of the captured sphingosine, which is immobilized on the resin as a tricyclic adduct, were performed by the acid catalytic hydrolysis reaction. Optimization studies revealed that 0.25 M aqueous (aq) trifluoroacetic acid (TFA) solution gave free sphingosine as the single product, checked as the OPA derivative by HPLC (Table S2),[27] whose structure and purity were confirmed by the direct HPLC comparison of authentic samples through the OPA derivatization analysis (Figure S2).[27] Quantitative analysis of released OPA sphingosine showed that the TFA catalytic releasing efficiency was 81%. The total extraction and releasing efficiency of the resin was calculated about 70% (Figure S1).[27] Also, it is noteworthy that the recovered resin can capture a sphingoid base at almost the same efficiency (Figure S3).[27] To the best of our knowledge, this is the first unique methodology for the rapid and efficient extraction of free sphingoid bases, which are known as the minor components of total lipids, by a chemoselective reaction.

The purpose of this work is to develop a technology for the efficient extraction of sphingoid bases. On the basis of the above strategy, the GARI was applied to extract free sphingosine from human serum. A phosphate derivative at 1-OH of sphingosine, which is called as sphingosine-1-phosphate, is a bioactive lipid mediator. Its concentration is known to be regulated by some diseases, such as atherosclerosis. Also, its half-life time is 15 min in serum to produce a hydrolyzed product, sphingosine. Therefore, rapid analysis of sphingosine in human serum by the GARI would be a significant application for the future diagnosis purposes. The effectiveness of the GARI method was confirmed by the following study: 400 μL of methanol was added to 100 μL of human pooled serum, and after centrifugation, the upper layer was added to the GARI as a human serum extract. After selective capture and acidic releasing, the released filtrate was analyzed for free sphingosine using reversed-phase fluorescent HPLC with the OPA derivatization technique. Comparison of the chromatogram of the methanolic extract of human serum (Figure A) with the chromatogram of the elution released from the resin (Figure B) clearly shows the expected selective capture of sphingosine. The structure extracted from serum was confirmed by comparing its retention time with that of a standard sphingosine and electrospray ionization mass spectroscopy (ESI-MS) spectra of their OPA derivatives (Figure S2).[27] The quantitative analysis by calibration curves for authentic sphingosine revealed that the concentration of sphingosine in human serum estimated by the glutaraldehyde resin method was 280 nM, which is in reasonable agreement with that of the previous report.[25] These results demonstrate that the developed glutaraldehyde resin method is a highly selective method, and hence, the combination of it with the OPA HPLC method was confirmed as an extremely efficient and sensitive method for the analysis of sphingoid bases in biological samples.

Figure 1

HPLC profiles of OPA derivatives of the methanolic extract of human serum. (A) Direct OPA derivatives of the crude methanolic extract. (B) OPA derivatives of the methanolic extract after treatment by the GARI. The observed sole peak eluted at 8.6 min was identified as the OPA derivative of sphingosine (d18:14)[27] by direct comparison with its standard sample. * denotes the relevant peaks resulting from OPA derivatization reagents. HPLC conditions: a YMC-Pack C8 column (0.46 cm φ × 15 cm); acetonitrile-phosphate buffer, pH 7.0 (85:15); flow rate, 1 mL/min; and a fluorescence detector, 340 nm (excitation) and 450 nm (emission).

To demonstrate the general versatility of the GARI, a solid-phase extraction study from a natural resource was carried out using a fungus called the golden oyster mushroom (Pleurotus citrinopileatus). It is a significant edible mushroom found in the northern areas of Japan and is known to contain enriched glucosylceramide.[15] The methanolic extract of the fungus containing sphingolipids was hydrolyzed to liberate free sphingoid bases using the modified Cahoon and Lynch method.[26] The HPLC chromatogram of the crude reaction mixture showed numerous peaks (Figure A) as their OPA derivatives. After treating with the resin, it showed a few major peaks corresponding to 9-methylsphingadienine (9-Me d18:24) and glucosyl 9-methylsphingadienine (glc-9-Me d18:24) (hydrolyzed products of glucosylceramide) (Figure B) except several irrelevant peaks coming from the OPA derivatization reagents. These peaks are produced by the hydrolysis of a major sphingolipid component named glucosyl 9-methylsphingadienine ceramide of fruiting bodies in the golden oyster mushroom.[15] Both structures of 9-methylsphingadienine and glucosyl 9-methylsphingadienine were confirmed by the comparison of their retention time and their ESI-MS spectra with those of the standard samples (Figure S2).[27] The reaction of glucosyl 9-methylsphingadienine with glutaraldehyde forming a plausible bicyclic adduct expands its capture capability.[27]

Figure 2

HPLC profiles of OPA derivatives of the hydrolyzed methanolic extract of a golden oyster mushroom detected by UV and fluorescence. (A) Direct OPA derivatives of the crude hydrolysis mixture of the methanolic extract of the golden oyster mushroom. (B) OPA derivatives of the methanolic extract after treatment by the GARI. The observed peaks that eluted at 12.6 and 16.3 min were identified as the OPA derivative of 9-methylsphingadienine (9-Me d18:24) and glucosyl 9-methylsphingadienine (glc-9-Me d18:24)[27] by direct comparison with its standard samples. * denotes the relevant peaks resulting from OPA derivatization reagents. HPLC conditions: a YMC-Pack C8 column (0.46 cm φ × 15 cm); acetonitrile-phosphate buffer, pH 7.0 (85:15); flow rate, 0.5 mL/min; a UV detector, 254 nm; and a fluorescence detector, 340 nm (excitation) and 450 nm (emission).

Conclusions

In summary, a unique glutaraldehyde resin method was developed for the first time, and it was applied to the efficient extraction studies of endogenous sphingoid bases from human serum as well as a golden oyster mushroom. Although, the modern sensitive MS-based lipidomic approaches have revolutionized the study of lipids over the past couple of decades, this method is more advantageous over conventional solvent extraction methods in terms of chemoselectivity for sphingoid bases, efficiency, rapidity, and versatility. This method establishes an extraction method for rapid analysis of the nanomolar scale of sphingoid bases with high purity. We believe that this highly efficient extraction method of sphingoid bases from biological samples and other natural resources is a promising technology for the discovery of new sphingoid base chemotherapeutics.

Experimental Section

General Methods

1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Varian Inova instrument at 25 °C in CDCl3 and CD3OD. Chemical shifts (δ) are reported in ppm and coupling constant values (J) are in hertz relative to CDCl3 (1H, δ 7.26; 13C, δ 77.00) or tetramethylsilane. The following abbreviations were used for signal multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; and m = multiplet. High-resolution mass spectrometry (ESI) [HRMS (ESI)] spectra were obtained on a Thermo Scientific Exactive at the Instrumental Analysis Division, Equipment Management Center, Creative Research institution, Hokkaido University. Low-resolution mass spectra (ESI-MS) were obtained by a JEOL JMS-T100LP spectrometer. UV–vis and IR spectra were measured on a JASCO V-630 spectrophotometer and JASCO FT/IR-470 plus instrument, respectively. Analytical thin-layer chromatography was performed on 0.2 mm silica gel plates (Merck 60 F-254). SiO2 gel column chromatography was carried out using silica gel (Kanto Silica Gel 60, spherical, 40–50 μm) with air flashing. The microwave reaction was performed by using the Biotage Initiator+. HPLC analyses were conducted on the JASCO PU-2086 Plus pump equipped with JASCO FP-2020 Fluorescence and UV-2075 UV spectrophotometric detectors and were performed on an YMC-Pack C8 column (0.46 cm φ × 15 cm). The sample fractions collected from HPLC were manually infused into ESI-MS to acquire their MS spectrum.

Pooled human serum was purchased from Cosmo Bio Co. Ltd. (Tokyo Japan); trans-p-coumaric acid, DDQ, ethyl vinyl ether, TBAF, lithium aluminum hydride, benzyl chloride, tert-butyl(chloro)diphenylsilane, and TFA were purchased from Tokyo Chemical Industries Co. Ltd. (Tokyo Japan); imidazole, 4-dimethyl aminopyridine, hydroquinone, potassium carbonate, potassium iodide, boric acid, OPA, d-erythro-sphingosine, and all other solvents (HPLC grade) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Amberlite IR 120 was purchased from MP Biomedicals (Santa Ana, USA), the Merrifield resin was purchased from Sigma-Aldrich (St. Louis, USA), and barium hydroxide was purchased from Nacalai Tesque (Kyoto, Japan). Dried fruiting bodies of golden oyster mushrooms were obtained from Nissei Bio Co. Ltd. (Eniwa, Japan) and CDCl3 and CD3OD were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, USA). Libra tubes with filters (2.5 mL, P/N RT3C100) were purchased from Hipep laboratories (Kyoto, Japan).

Synthesis of Compound 4(20,21)

To a stirred solution of trans-p-coumaric acid (5.00 g, 30.4 mmol) in methanol, a catalytic amount of Amberlite IR 120 (H+) was added and refluxed at 75 °C for 45 h. The catalyst was filtered off and the reaction mixture was concentrated under reduced pressure, and the crude mixture was purified by column chromatography [hexane/ethyl acetate (EtOAc): 4:1] to give methyl trans-p-coumarate as an amorphous white solid in 97% yield. 1H NMR (500 MHz, CDCl3): δ 7.63 (1H, d, J = 15.9 Hz), 7.43–7.45 (2H, m), 6.84–6.87 (2H, m), 6.30 (1H, d, J = 15.8 Hz), 5.19 (1H, s), 3.80 (3H, s); HRMS (ESI): calcd for C10H10NaO3 (M + Na+), 201.0528; found, 201.0523.

To a stirred solution of LiAlH4 (1.63 g, 42.9 mmol, 1.5 equiv) in dry THF under a N2 atmosphere, benzyl chloride (4.9 mL, 42.9 mmol, 1.5 equiv) was added dropwise. After 15 min, methyl trans-p-coumarate (5.10 g, 28.6 mmol) dissolved in dry THF was added slowly through a dropping funnel; the reaction was continued to stir for 90 min, and the reaction mixture was quenched with water, filtered over celite, washed with aqueous sodium potassium tartrate, extracted with EtOAc, dried over Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography (hexane/EtOAc: 7:3) to give trans-p-coumaryl alcohol as an amorphous white solid in 94% yield. 1H NMR (500 MHz, CD3OD): δ 7.24 (2H, d, J = 8.8 Hz), 6.73 (2H, d, J = 8.4 Hz), 6.50 (1H, d, J = 15.8 Hz), 6.16 (1H, td, J = 5.9, 15.8 Hz), 4.18 (2H, dd, J = 1.1, 5.9 Hz); HRMS (ESI): calcd for C9H10NaO2 (M + Na+), 173.0578; found, 173.0574.

To a stirred solution of trans-p-coumaryl alcohol (3.50 g, 23.0 mmol) in 400 mL of dioxane, DDQ (5.82 g, 25.3 mmol, 1.10 equiv) was added; the reaction mixture was stirred at room temperature for 30 min, filtered over celite, and concentrated. The solid residue was purified by flash column chromatography (hexane/EtOAc: 9:1) to give trans-p-coumaraldehyde (4) as an amorphous light yellow solid in 71% yield. 1H NMR (500 MHz, CD3OD): δ 9.55 (1H, d, J = 7.8 Hz), 7.57 (1H, d, J = 15.8 Hz), 7.53–7.56 (2H, m), 6.83–6.86 (2H, m), 6.59 (1H, dd, J = 8.05, 15.9 Hz); HRMS (ESI): calcd for C9H8NaO2 (M + Na+), 171.0422; found, 171.0416.

Synthesis of Compound 5

To a stirred solution of trans-p-coumaraldehyde (4) (2.10 g, 14.0 mmol) in dry DCM (60 mL) were added imidazole (1.90 g, 28.0 mmol, 2.0 equiv) and tert-butyl(chloro)diphenylsilane (4.3 mL, 15.4 mmol, 1.1 equiv) at room temperature. The reaction mixture was stirred for 4 h, and then, DCM was evaporated; after addition of water, it was extracted with EtOAc (3 × 150 mL). The combined organic extracts were dried over MgSO4 and filtered, and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography (hexane/EtOAc = 9.5:0.5) to give 5 as an amorphous white solid in 76% yield. 1H NMR (500 MHz, CDCl3): δ 9.62 (1H, d, J = 7.8 Hz), 7.72–7.70 (4H, m), 7.45–7.32 (9H, m), 6.81 (2H, d, J = 8.7 Hz), 6.56–6.51 (1H, dd, J = 7.6, 15.6 Hz), 1.11 (9H, s). 13C NMR (125 MHz, CDCl3): δ 193.6, 158.5, 152.7, 135.2, 132.1, 130.0, 127.9, 127.1, 126.6, 120.4, 77.2, 77.0, 76.7, 26.3, 19.4. IR (KBr disk, cm–1): 1674, 1600, 1507, 1427, 1267, 1172, 1129. MS (ESI): 409 (M + Na+). HRMS (ESI): calcd for C25H26NaO2Si (M + Na+), 409.1594; found, 409.1590. UV (EtOH): λmax = 315 nm, ε = 38 200 M–1·cm–1.

Microwave-Assisted Synthesis of Compound 6

To a microwave reaction vial (2–5 mL) of compound 5 (500 mg, 1.29 mmol) in ethyl vinyl ether (1.5 mL, 15.5 mmol, 12 equiv) were added hydroquinone (80 mg, 0.774 mmol, 0.6 equiv) and n-butanol (1 mL) as a co-solvent to reduce vapor pressure. The vial is tightly sealed and placed inside the microwave, and the reaction temperature was kept at 220 °C, for 6 h. The resulting reaction mixture was concentrated and purified by flash chromatography (hexane/EtOAc = 9.7:0.3) to give 6, as a diastereomeric mixture (ca. 1:1), as a colorless oil in 81% yield. 1H NMR (500 MHz, CDCl3): δ 7.78–7.76 (4H, m), 7.47–7.39 (6H, m), 7.04 (0.5H, d, J = 8.3 Hz), 6.99 (0.5H, d, J = 8.5 Hz), 6.77–6.74 (2H, m), 6.42–6.36 (1H, m), 5.04–5.01 (1H, m), 4.84 (0.5H, d, J = 6.3 Hz), 4.71 (0.5 H, d, J = 6.1 Hz), 3.99–3.89 (1H, m), 3.65–3.52 (1H, m), 2.24–2.08 (1H, m), 1.87–1.77 (1H, m), 1.29–1.25 (3H, m), 1.15 (9H). 13C NMR (125 MHz, CDCl3): δ 154.1, 154.0, 141.9, 140.6, 137.5, 136.8, 135.5, 133.1, 133.0, 129.8, 128.2, 127.7, 119.6, 119.5, 105.5, 105.3, 99.5, 96.2, 77.3, 77.0, 76.8, 64.3, 63.8, 38.0, 37.0, 36.3, 32.4, 31.6, 26.6, 26.5, 22.7, 19.5, 15.2, 14.1. IR (KBr disk, cm–1): 1644, 1606, 1508, 1428, 1256, 1113, 1036, 919. MS (ESI): 481 (M + Na+). HRMS (ESI): calcd for C29H34NaO3Si (M + Na+), 481.2169; found, 481.2167. UV (cyclohexane): λmax = 269 nm, ε = 3760 M–1·cm–1.

Synthesis of Compound 7

To a stirred solution of compound 6 (3.39 g, 7.38 mmol) in 40 mL of THF, TBAF (8.85 mL, 8.86 mmol, 1.2 equiv) was added; the reaction mixture was stirred at room temperature for 3 h, Then, THF was evaporated, and after addition of water, it was extracted with Et2O. The combined organic extracts were dried over MgSO4 and filtered, and the filtrate was evaporated in vacuo. The residue was purified by flash column chromatography (hexane/EtOAc: 9.5:0.5) to give phenol (7), as a diastereomeric mixture (ca. 3:2), as colorless oil in 83% yield. 1H NMR (500 MHz, CDCl3): δ 7.15–7.09 (2H, m), 6.80–6.75 (2H, m), 6.43 (0.4H, dd, J = 2.2, 6.1 Hz), 6.37 (0.6H, dd, J = 2.4, 6.3 Hz), 5.05–5.01 (1H, m), 4.85–4.71 (1H, m), 3.99–3.86 (1H, m), 3.64–3.55 (2H, m), 2.25–2.09 (1H, m), 1.88–1.78 (1H, m), 1.28–1.23 (3H, m) 13C NMR (125 MHz, CDCl3): δ 154.0, 141.9, 140.6, 137.2, 136.4, 128.6, 128.2, 127.8, 115.4, 115.2, 105.5, 105.4, 99.5, 96.22, 77.27, 76.74, 64.4, 63.9, 37.9, 37.0, 36.3, 32.3, 15.1. IR (KBr disk, cm–1): 3443, 1643, 1511, 1444, 1382, 1231, 1092, 961. MS (ESI): 243 (M + Na+). HRMS (ESI): calcd for C13H16NaO3 (M + Na+), 243.0997; found, 243.0987. UV (cyclohexane): λmax = 277 nm, ε = 968 M–1·cm–1.

Synthesis of Compound 8

To a stirred solution of compound 7 (1.29 g, 5.89 mmol) in 40 mL of DMF, K2CO3 (0.814 g, 8.84 mmol, 1.5 equiv), KI (97.8 mg, 0.589 mmol, 0.10 equiv), and the Merrifield resin (1.43 g, 6.48 mmol, 1.1 equiv) were added, and the mixture was stirred at 90 °C for 30 min. It was filtrated through a sintered glass filter; the resulting resin was washed repeatedly with water, acetone, hexane, EtOAc, and MeOH. The structure of 8 was confirmed from its IR spectrum by comparing it with that of the model compound 8m(17) (for the structure of 8m, see Figure S4). Agreement of important IR absorption signals between 8 and 8m indicates their structural similarity.

Synthesis of Compound 9

The resin 8 (1.42 g, 5.89 mmol) in 40 mL of dioxane was swelled for an hour at room temperature, followed by the addition of conc. HCl (11.5 mL), and refluxed at 60 °C for 4 h. The hydrolyzed resin was filtered and washed repeatedly with water, acetone, hexane, EtOAc, and MeOH. The structure of 9 was confirmed by its IR spectrum and indicates that it is a mixture of 9a and 9b. Efficiency of the immobilization reaction was calculated to be 88% based on the elemental analysis of its chloride atom ratio in the Merrifield resin (Table S1).

Extraction of Spiked Standard Sphingosine by GARI

The GARI (5 mg) was swollen in THF (200 μL) for 1 h in a 2.5 mL Libra tube prior to its usage. To this resin suspension, 5 μL of authentic sphingosine in methanol (1 mg/mL) was added and it was mixed vigorously by a vortex mixer for 1.5 h at room temperature for its capturing reaction (the IR spectrum of GARI after capturing the reaction was shown in Figure S6). The resin was filtered off and washed well with 200 μL of water, methanol, and chloroform, sequentially. The resin was swollen with 200 μL of THF for 1 h again, and then, 0.25 M TFA (200 μL) was added for its releasing reaction. After 1 h of vortex stirring, the solution was filtered. The filtrate was analyzed as the sphingosine OPA derivative by the HPLC system, as described below in the extraction of sphingosine from the human serum section.

Extraction of Free Sphingosine from Human Serum

To 100 μL of serum, 400 μL of MeOH was added and the mixture was centrifuged at 20 °C, 3000 rpm, for 15 min. After centrifugation, the upper layer (350 μL) was removed and this solution was used as a human serum extract. The GARI (5 mg) was swollen in THF (200 μL) for 1 h in a 2.5 mL Libra tube prior to its usage. To this resin suspension, the human serum extract was added and it was mixed vigorously by a vortex mixer for 1.5 h at room temperature for its capturing reaction. The resin was filtered off and washed well with 200 μL of water, methanol, and chloroform, sequentially. The resin was swollen with 200 μL of THF for 1 h again, and then, 0.25 M TFA (200 μL) was added for its releasing reaction. After 1 h of vortex stirring, the solution was filtered. The filtrate was analyzed as the sphingosine OPA derivative by the HPLC system. The pH of the filtrate was adjusted to around 5 with the boric acid buffer to make sphingosine react with the OPA reagent effectively. To 50 μL of the filtrate, the same amount of freshly prepared OPA reagent [5 mg of OPA + 100 μL ethanol + 5 μL 2-mercaptoethanol + 9.9 mL 3% boric acid in water (pH adjusted to 10.5 with KOH)] was added, and the mixture was mixed vigorously by a vortex mixture for 15 min at room temperature. The mixture was added to 100 μL of the mobile phase (85:15 acetonitrile/phosphate buffer pH 7.0); 20 μL of the resulting solution was injected to the HPLC on a YMC-Pack C8 column (0.46 cm φ × 15 cm) using acetonitrile-phosphate buffer pH 7.0 (85:15). The fluorescence detector was set at 340 and 450 nm as an excitation and emission wavelengths, respectively. The flow rate was 1.0 mL/min.

Extraction of Fungal Sphingoids from the Golden Oyster Mushroom

About 1 g of the dried fruiting body of the golden oyster mushroom (P. citrinopileatus) was extracted with methanol for several times. Combined methanolic extracts were concentrated, and 20 mL of dioxane and 20 mL of 10% (w/v) Ba(OH)2 were added and refluxed at 110 °C for 24 h. After hydrolysis, 40 mL of 2% (w/v) (NH4)2SO4 was added to precipitate barium ions. After centrifugation, the centrifugate was extracted with diethyl ether (50 mL × 3). Combined ether extract was concentrated, dried, and redissolved in MeOH (3 mL). Capturing and releasing reactions of the free sphingoid base were carried out, as described above, by adding 200 μL of the hydrolyzed mushroom extract as a sample. OPA HPLC analyses were performed under the same condition as described above, except that its flow rate was 0.5 mL/min.