Synthesis of potentially caged sphingolipids, possible precursors of cellular modulators and second messengers1

Chemistry and Physics of Lipids 90 (1997) 55 – 61

Synthesis of potentially caged sphingolipids, possible precursors of cellular modulators and second messengers1 Uri Zehavi 2,* Institute of Biochemistry, Food Sciences and Nutrition, Faculty of Agricultural, Food and En6ironmental Sciences, The Hebrew Uni6ersity of Jerusalem, P.O. Box 12, Reho6ot 76100, Israel Received 14 July 1997; received in revised form 11 September 1997; accepted 11 September 1997

Abstract An increasing number of sphingolipids, glycosphingolipids and some of their degradation products have been recognized in recent years as second messengers involved in signal transduction and as modulators of numerous cellular functions. These can be converted into inert, caged compounds, introduced into cells and tissues and subsequently photolysed to active compounds thus enabling the study of fast biological processes. The novel, potentially caged compounds synthesized here are substituted 2-nitrobenzyl urethans and 2-nitrobenzyl amines derived from sphingosine, dihydrosphingosine, N-methylsphingosine, N-methyldihydrosphingosine, psychosine and glucosylsphingosine. Upon irradiation of the afore mentioned compounds they release, or are expected to release, the free biologically active amines. © 1997 Elsevier Science Ireland Ltd. Keywords: Caged glycosphingolipids; Caged sphingolipids; Caged lyso-glycosphingolipids; Caged lyso-sphingolipids; Second messenger; Cellular modulator

1. Introduction

* Tel: + 972 8 9481914; fax: +972 8 9476189; e-mail: [email protected] 1 Dedicated to Roger W. Jeanloz on the occasion of his 80th birthday 2 Most of this work was carried out during a sabbatical (1994) of the author at the Division of Physical Biochemistry, National Institute of Medical Research, Mill Hill, London, UK.

Glycosphingolipids (GSLs) are of particular importance in cellular recognition and, alongside with other sphingosine (Sph) derivatives, in the modulation of cellular functions where they may serve as second messengers. Their effects as signaling molecules have been identified in the functional modulation of channels, enzymes (protein kinases), receptors and G-proteins; effects which subsequently change cellular events like growth,

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proliferation, differentiation, Ca2 + mobilization and apoptosis. The emerging picture is complicated inter alia by ‘cross talk’ of sphingolipids (SLs) and GSLs messengers with other second messengers and, on the other hand, the levels of SLs are modulated by growth factors (reviewed in Hakomori, 1993; Hakomori and Igarashi, 1995; Hakomori, 1996; Spiegel and Milstien, 1995, 1996; Divecha and Irvine, 1995). Light-sensitive protecting groups have been employed in synthetic chemistry to protect specific functional groups during synthetic steps and are removed by light once the protection is no longer required (reviewed in Zehavi, 1988). Furthermore, light-sensitive protecting groups are intensively used in the synthesis of caged compounds, a nomenclature suggested by Kaplan et al. (1978) for photosensitive, but normally inert molecules that fragment following irradiation with near ultraviolet light yielding biologically active molecules (reviewed in McCray and Trentham, 1989; Adams and Tsien, 1993; Corrie and Trentham, 1993). Examples of caged compounds include nucleotides, proteins, neurotransmitters and Ca2 + chelators. The goal of this work has been caging of lysoSLs and lyso-GSLs (lyso designates compounds possessing free amino functions rather than the usual amide ones). Compounds selected to be caged were: (a) Sph, which has pronounced mitogenic effects, is also a cell growth regulator, although a negative regulator (Spiegel and Merrill, 1996), and triggering apoptosis was reported as well; (b) dihydrosphingosine (DHS), that is known for a more effective induction of proliferation of Swiss 3T3 cells than that of Sph (Hauser et al., 1994); (c) psychosine (Psy, galactosylsphingosine), a naturally occurring lyso-GSL, that is known for its cytotoxic effects, inhibits cytochrome oxidase, b-glucosidase, protein kinase C and may undergo detoxification by N-acylation (Hannun and Linardic, 1993); (d) N-methylsphingosine (MeSph), which might be an active modulator based on analogy to N,N-dimethylsphingosine and N,N,N-trimethylspingosine (Hakomori and Igarashi, 1995). The intention in the present work was to cage these amino compounds by converting them to light-sensitive derivatised 2-nitrobenzyl urethans or 2-nitrobenzyl amines.

Irradiation of this new family of potentially caged compounds will release the biologically active, free amino compounds important to cellular modulation (depending on the type of the irradiation, even in ms or ms time scale). The release could be followed by cellular studies where the kinetics of cellular effects triggered by free SLs is to be investigated (Corrie and Trentham, 1993).

2. Materials and methods Sph.H2SO4 was isolated following hydrolysis from bovine brain (Carter et al., 1947; Thierfelder, 1905; Levene and Jacob, 1912). Glucose oxidase (from Aspargillus niger, EC 1.1.3.4), peroxidase (from horseradish, EC 1.11.1.7), DL-erythro-dihydrosphingosine, the only form of DHS used in this work, and Psy were purchased from Sigma. Galactose oxidase (from Dactylium dendroides) was a product of Boehringer Mannheim. Thin layer chromatography (TLC) was performed on Silica gel 60A KGF plates (Whatman) or Silica gel 60F254 sheets (Merck) and compounds were detected by viewing under ultraviolet light or by spraying with sulfuric acid and heating. MS spectra were obtained using a VG platform at 3.5 kV with negative or positive ion electrospray. 1H NMR spectra were recorded on a Bruker AM-400 or a Bruker AMX-400 (400 MHz) instrument in CDCl3. Ultraviolet absorbance was recorded on a Gilford 3AG linear transport instrument. All the compounds synthesized in this work are light-sensitive and were kept in the dark. 1-Methyl-3-(4-carboxymethyl-2-nitrobenzyloxycarbonyl)imidazolium chloride (1) and 2-N-(4carboxymethyl-2-nitrobenzyloxycarbonyl)-sphingosine (2, MS: m/z =535.0, calculated 535.3, [M−1] − ) were synthesized according to Zehavi et al. (1990).

2.1. N-(4 -carboxymethyl-2 -nitrobenzyloxycarbonyl) -DL -erythro-dihydrosphingosine (3) Compound 3 was prepared by a small scale modification of that for compound 2 (Zehavi et al., 1990). DHS (100 mg, 0.33 mmol) was vigorously stirred at 0°C in a mixture of tetrahydro-

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furan (0.2 ml) and 10% sodium hydrogen carbonate (0.5 ml). Compound 1 (prepared from 70 mg of methyl 4-hydroxymethyl-3-nitrobezoate) and ether (5 ml) were added in small portions during 1 h and stirring was continued at room temperature overnight. The ether phase was dried over sodium sulfate, evaporated and applied to a silica gel column (10 g, 1 cm in diameter) that was washed first with petroleum ether/ethylacetate 2:1 v/v (40 ml), and eluted with petroleum ether/ethylacetate 1:1 v/v (3.5 ml/fraction). Fractions 25 – 60 contained pure (TLC, same solvent) compound 3, a yellowish solid, 8.2 mg, 4.7% yield. MS: m/z = 536.9, calculated 537.3, [M−1] − . 1H NMR: d 8.74 (d, 1H, J3,5 1.2 Hz, H-3, aromatic), 8.29 (dd, 1H, J5,6 8.1 Hz, H-5, aromatic), 7.73 (d, 1H, H-6, aromatic), 5.83 (d, 1H, JNH,2 8.0 Hz, NH), 5.58 (s, 2H, benzylic), 4.05 (m, 1H, H-1), 3.98 (s, 3H, OCH3), 3.83–3.75 (m, 2H), 3.59 (m, 1H, H-2, collapses to a narrow m following deuterium exchange), 1.71–1.54 (m, 2H, CH2), 1.26 (m, 26H), 0.88 (t, J 6.5 Hz, CH3-18).

2.2. N-(2 -nitrobenzylation) of sphingosine deri6ati6es Dry dimethylformamide (1 ml) was added to a mixture of 2-nitrobenzyl bromide (72 mg, 0.33 mmol) and a sphingosine derivative (0.33 mmol), the mixture was stirred well at 0°C and 1,2,2,6,6pentamethylpiperidine (200 ml in the case of Sph.H2SO4 and 66 ml in other cases) was added. The stirring was continued for 1 h at 0°C and for 17 h at room temperature, it was then evaporated in vacuo (30°C bath) and applied to a silica gel column. The fractions of interest were pooled and evaporated to give amorphous solids. Starting from Sph.H2SO4, the column (13 g silica gel, 1 cm in diameter) was washed first with petroleum ether/ethylacetate 2:1 v/v (60 ml) followed by petroleum ether/ethylacetate 1:1 v/v. Fractions 39–60 (5 ml/fraction) contained 2-N-(2nitrobenzyl)sphingosine (4, pure by TLC, same solvent, 50 mg, 35% yield). MS: m/z= 435.3, calculated 435.3, [M +1] + . 1H NMR: d 7.95 (d, 1H, J3,4 8.6 Hz, H-3 aromatic), 7.59 – 7.57 (m, 2H, aromatic), 7.46–7.41 (m, 1H, aromatic), 5.80 – 5.73 (m, 1H, olefinic), 5.46 – 5.40 (m, 1H, olefinic),

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4.10 (app. d, 2H, J 1.5 Hz, benzylic), 3.70 (app. d, 2H, J 6.7 Hz, H-1, H-1%), 2.63–2.66 (m, 1H), 2.02–2.07 (m, 1H), 1.37–1.30 (m, 2H, CH2), 1.25 (m, 22H), 0.88 (t, 3H, J 7.0 Hz, CH3-18). Starting from DHS, the pure product, 2-N-(2nitrobenzyl)-DL-erythro-dihydrosphingosine (5, 119 mg, 83% yield) eluted from the column and migrated on TLC like compound 4. MS: m/z = 437.0, calculated 437.3, [M+ 1] + . 1H NMR: d 7.94 (d, 1H, J 7.8 Hz, H-3 aromatic), 7.61–7.57 (m, 2H, aromatic), 7.46–7.41 (m, 1H, aromatic), 4.16 (d, 1H, J 13.8 Hz, benzylic), 4.06 (d, 1H, benzylic), 3.81–3.70 (m, 3H, H-1, H-1%, H-3), 2.63–2.60 (m, 1H, H-2), 1.49–1.45 (m, 2H, CH2), 1.26 (m, 26H), 0.88 (t, 3H, J 6.8 Hz, CH3-18). Starting from Psy, the reaction was carried out as for compound 4 and the column was eluted with ethylacetate/methanol 7:1 v/v (2.8 ml/fraction). Fractions 4–9 contained compound 6a (pure by TLC, same solvent, 41 mg, 21% yield). MS: m/z= 597.5, calculated 597.4, [M+ 1] + . 1H NMR: d 7.67 (dd, 1H, J3,4 8.1 Hz, J 0.8 Hz, H-3, aromatic), 7.59 (d, 1H, J 7.6 Hz, aromatic), 7.40 (t, 1H, J 7.6 Hz, aromatic), 7.23 (dd, 1H, J 8.1 Hz, J 0.8 Hz, aromatic), 5.69–5.63 (m, 1H, olefinic), 5.52–5.46 (m, 1H, olefinic), 4.31 (d, 1H, J1Gal,2Gal 7.6 Hz, H-1Gal), 4.27 (m, 1H), 4.18–4.02 (m, including: 4.13 {m, 1H}, 4.10 {d, 2H, benzylic} and 4.06 {m, 1-H, J3Gal,4Gal 2.7 Hz, J4Gal,5Gal B 3 Hz, H-4Gal}), 3.95 (dd, 1H, J 2.6 Hz, J 10.3 Hz), 3.84 (AB, 2H, J 4.6 Hz, H-6Gal, H-6%Gal), 3.73 (t, 1H, J2Gal,3Gal 9.2 Hz, H-2Gal), 3.65 (dd, 1H, H-3Gal), 3.50–3.60 (m, 1H), 3.36–3.28 (m, 1H), 2.84–2.81 (m, 1H), 2.05–2.01 (m, 2H, CH2), 1.28 (m, 22H), 0.88 (t, 1H, CH3-18, J17,18 6.6 Hz); the assignment of the galactosyl hydrogens is based on double irradiation experiments and are marked with Gal. A procedure constituting a modification of a galactose oxidase, peroxidase, o-tolidine coupled system commonly used for the determination of galactose and several of its derivatives (Hjelm and de Verdier, 1984) was applied to lactose (i) and to compound 6a (ii). The buffer used was 0.1 M potassium phosphate, pH 6.0, containing 0.05% o-tolidine. The following were added in sequence into a 1 cm cuvette, mixed and A425 was recorded at 25°: (i) peroxidase (2 mg, 220 U, in 83 ml

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buffer), lactose (25 mg in 250 ml water), dimethylformamide (25 ml) and, following a delay of 4 min, galactose oxidase (0.5 mg, 10 U, in 100 ml buffer) were added; (ii) peroxidase (2 mg, 220 U, in 333 ml buffer), compound 6a (7 mg in 25 ml dimethylformamide) and, following a delay of 4 min, galactose oxidase (0.5 mg, 10 U in 100 ml buffer) were added. The initial velocities for lactose and for compound 6a were determined in paralel as 2.125 and 1.225 A425, respectively. Fractions 16–35 contained compound (6b, pure by TLC, same solvent, 101 mg, 51% yield). MS: m/z =597.5, calculated 597.4, [M+ 1] + . 1H NMR: d 7.93 (d, J3,4 8.0 Hz, 1H, H-3, aromatic), 7.60 –7.56 (m, 2H, aromatic), 7.42 – 7.38 (m, 1H, aromatic), 5.71–5.64 (m, 1H, olefinic), 5.30 – 5.25 (m, 1H, olefinic), 4.29 (d, 1H, J1Glc,2Glc 6.2 Hz, H-1Glc), 4.28–4.06 (m, 4H, containing: d, 2H, benzylic, J 14.1 Hz, and H-3Glc), 3.96 (m, 1H), 3.78 –3.54 (m, 3H, including: H-2Glc, and 3.78 – 3.77 {m, 1H}), 3.46 (m, 1H), 3.58 – 3.54 (m, 1H), 2.60 (m, 1H), 1.93–1.92 (m, 2H, CH2), 1.25 – 1.20 (m, 22H), 0.87 (t, 3H, J 6.7 Hz, CH3-18); the assignment of glucosyl residue hydrogens is based on double irradiation experiments and are designated as Glc. Compounds 6a and 6b (2 mg samples) were hydrolyzed in 1.1 M H2SO4 (in water)/dioxane, 3:1 (1 ml, 100°C, 2h), water (1.5 ml) was added, the solution was neutralized with barium carbonate, centrifuged, and filtered. No glucose was determined in the neutralized hydrolysate of compound 6a, glucose was determined, however, in the corresponding filtrate from compound 6b (42% of the theoretical) by a glucose oxidase – peroxidase test (Raabo and Terkildsen, 1960).

2.3. N-methylation of 2 -N-(2 -nitrobenzyl)sphingosine deri6ati6es 2.3.1. Monomethyl deri6ati6es Dry dimethylformamide (2.2 ml) was added to a mixture of compound 4 or 5 (0.33 mmol) and methyl iodide (213 ml), the mixture was stirred well at 0°C and 1,2,2,6,6-pentamethylpiperidine (140 ml) was added. The stirring was continued for 1 h at 0°C and for 15 h at room temperature, it was then evaporated in vacuo (30°C bath) and

applied to a silica gel column (13g, 1 cm in diameter) that was eluted with petroleum ether/ ethylacetate 2:1 v/v (2 ml/fraction) and, the fractions of interest were pooled and evaporated. Compound 4 (fractions 27–44) yielded 2-Nmethyl-2-N-(2-nitrobenzyl)sphingosine (7, yellowish oil, 56 mg, 37%). MS: m/z= 449.1, calculated 449.3, [M+ 1] + . 1H NMR: d 7.83 (dd, 1H, J 7.7 Hz, J 0.9 Hz, H-3 aromatic), 7.58–7.52 (m, 2H, aromatic), 7.42–7.38 (m, 1H, aromatic), 5.75– 5.71 (m, 1H, olefinic), 5.69–5.54 (m, 1H, olefinic), 4.30 (m, 1H), 4.10 (m, 1H), 3.84 (d, 2H, J 6.1 Hz, benzylic), 2.71 (m, 2H), 2.28 (s, 3H, N–CH3), 2.07 (m, 1H, H-2), 1.39–1.30 (m, 2H, CH2), 1.26 (m, 22H), 0.88 (t, 3H, J 6.5 Hz, C–CH3). Compound 5 gave 2-N-methyl-2-N-(2-nitrobenzyl)-DL-erythro-dihydrosphingosine (8, yellowish oil, 97 mg, 65%). MS: m/z= 451.1, calculated 451.3. Both compounds were pure by TLC (same solvent). 1H NMR: d 7.81 (d, 1H, J 7.9 Hz, H-3 aromatic), 7.50 (app. d, 2H, J 4.1 Hz, aromatic), 7.42–7.38 (dd, 1H, aromatic), 4.07 (d, 1H, J 14.6 Hz, benzylic), 4.06 (d, 1H, benzylic), 3.98–3.81 (m, 3H), 2.53 (dd, 1H, J 5.4 Hz, J 5.6 Hz, H-2), 2.25 (s, 3H, N–CH3), 1.48–1.43 (m, 2H, CH2), 1.26 (m, 26H), 0.88 (t, 3H, J 6.6 Hz, C–CH3).

2.4. Saponification of compounds 2 and 3 Compounds 2 and 3 (1–2 mg samples) were shaken in 0.66 M sodium hydroxide in water/tetrahydrofuran 2:1 (50 ml), at room temperature for 1 h. The reaction mixtures were applied to Amberlite IR 120 (H + ) columns (3 cm long, 4 mm in diameter) with some methanol and the columns were eluted with chloroform/methanol 1:1 (2 ml). The eluates were evaporated to give the TLC pure (ethylacetate or ethylacetate/methanol 5:1) carboxylic acid derivatives of sphingosine or DHS, respectively.

3. Results and discussion The present paper purposely introduces for the first time potentially caged SLs and GSLs. However, a few members of these families of compounds, where the amino functions were

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Scheme 1.

protected as light-sensitive 2-nitrobenzyloxycarbonyl derivatives were originally prepared by us as intermediates, potential intermediates or products in GSL synthesis on polymer supports. These includes protected Sph, lactosylsphingosine and lyso-Gb4 derived from native SLs (Zehavi et al., 1990) and protected Sph and lactosylsphingosine from total synthesis (Zimmermann et al., 1988; Zehavi et al., 1990a). In general, two approaches to caging were undertaken in the current work, the first leading to substituted N-2-nitrobenzyloxycarbonyl derivatives, the second to N-2-nitrobenzyl derivatives. Quantum yields (Qp) and rate constants (k) for the photolysis of related and frequently differently substituted caged amines, range between Qp 0.25 – 0.8 and k 17 – 1.7 × 104 s − 1 for the first group; Qp 0.02 – 0.075 and k 600 – 824 s − 1 for the second group (reviewed in Corrie and Trentham, 1993; Corrie and Papageorgiou, 1996). Particularly relevant to this work is the photolysis of 2-(2-nitrobenzylamino)propanediol with Qp 0.32 (D.R. Trentham, to be published). Compounds 2 (Zehavi et al. (1990), the MS data are presently reported) and 3 are N-2-nitrobenzyloxycarbonyl derivatives prepared starting from native Sph and synthetic DHS,

respectively (Scheme 1). Compound 2 was compared with the product of total chemical synthesis (Zehavi et al., 1990a); the two had identical migration in TLC (petroleum ether/ethylacetate 2:1 v/v) and very similar NMR spectra. Both compounds 2 and 3 undergo facile saponification of the methyl ester that enhances the polarity which could be helpful in delivery into the cell cytosol rather than the membranes. Considering all the afore mentioned compounds, subsequent photolysis should lead to efficient release of Sph or DHS (Zehavi, 1988). A second approach to caging SLs and GSLs possessing free amino functions was to N-alkylate these compounds with 2-nitrobenzyl bromide. Potentially caged compounds prepared in this fashion (4 and 5 starting from Sph and DHS, respectively) could subsequently be methylated (methyl iodide) yielding N-methyl-N-(2-nitrobenzyl)-sphingosine (7) and N-methyl-N-(2-nitrobenzyl)-dihydrosphingosine (8), respectively (Schemes 2 and 3). The 2-nitrobenzylation yield was substantially higher for compound 5 compared with that of compound 4, apparently due to impurities in the Sph preparation (evidenced by numerous by-products of the reaction) and, higher steric restraints in the Sph compared with the DHS case

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Scheme 2.

which is also observed in the methylation step. Steric hindrance affects both the Sph and the DHS derivatives, di-N,N-(2-nitrobenzylation) was not observed and it was even difficult to demonstrate di-N-methylation. N-2-nitrobenzylation of Psy surprisingly led to two products, a Psy (galactosylsphingosine) one (6a) and a glucosphingosine one (6b), a result that must be due to a large glucosylsphingosine contamination of the commercial Psy (galactosyl sphingosine, Scheme 3). The structural assignment of the last two compounds is based on MS and NMR (supported by decoupling experiments). The MS data correspond nicely to the theoretical values; very significantly the coupling constants in the NMR, J1Glc,2Glc 6.2 Hz and J1Gal,2Gal 7.6 Hz for compounds 6a and 6b indicate that the two are b- anomers and in the case of compound 6a, J3Gal,4Gal 2.3 Hz and J4Gal,5Gal B3 Hz are compatible with a galactosyl residue. The glucose determination, following acid hydrolysis, supported the assignment of compound 6b as a glucosylsphingosine derivative (no glucose was found in the hydrolysate of compound 6a). Being a substrate of galactose oxidase is compatible with the presence of a galactosyl moiety in compound 6a. While signaling and modulatory effects of SLs may frequently be attributed to the sphingoid backbone and natural variations are known (Hannun and Linardic, 1993; Bruzik, 1986; Bruzik and Tsai, 1987; Bruzik, 1988), one has to be particu-

larly alert to the possibility of crude preparations. The preparation of 2-(4-carboxymethyl-2-nitrobenzyloxycarbonyl) and of 2-(2-nitrobenzyl) derivatives of Sph, for instance, results in a number of ultraviolet absorbing spots when checked by TLC. In every case, the compound corresponding to the major spot was carefully purified by column chromatography (compounds 2 and 4) and checked again by TLC. Suitable derivatives of Sls, including the ones used in this work, could be helpful in the purification of SLs and SL derivatives. The following is an encouraging example, suggesting that the compounds synthesized in this paper could serve as valuable, caged SLs in cellular signaling experiments. Compound 4, a DHS derivative, was applied to the intracellular envioronment of dorsal root ganglion neurons from neonatal rats using the cell patch clamp technique. While no effect was noticed with unphotolysed, caged DHS (4), irradiation and intracellular photorelease of DHS activated an inward Ca2 + -dependent current suggesting that it may involve mobilization of Ca2 + from a ryanodine-sensitive intracellular store (Ayar et al., 1996).

Acknowledgements The author is grateful to Dr D.R. Trentham for the invitation to his laboratory at the National

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Scheme 3.

Institute of Medical Research (NIMR), his encouragement and discussions. The author thanks Mrs M. Herchman for her assistance, Dr S. Howell for the MS and Mr J.S. Craik for the NMR spectra obtained using the facility at the MRC Biomedical and NMR Centre. This work was supported in part by the Scho¨nbrun Fund.

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