Synthesis, Inhibitory Effects on Nitric Oxide and Structure-Activity Relationships of a Glycosphingolipid from the Marine Sponge Aplysinella rhax and Its Analogues

The novel glycosphingolipid, β-D-GalNAcp(1→4)[α-D-Fucp(1→3)]-β-D-GlcNAcp(1→)Cer (A), isolated from the marine sponge Aplysinella rhax has a unique structure, with D-fucose and N-acetyl-D-galactosamine moieties attached to a reducing-end N-acetyl-D-glucosamine through an α1→3 and β1→4 linkage, respectively. We synthesized glycolipid 1 and some non-natural di- and trisaccharide analogues 2-6 containing a D-fucose residue. Among these compounds, the natural type showed the most potent nitric oxide (NO) production inhibitory activity against LPS-induced J774.1 cells. Our results indicate that both the presence of a D-Fucα1-3GlcNAc-linkage and the ceramide aglycon portion are crucial for optimal NO inhibition.


Introduction
Carbohydrates in the form of glycoconjugates, for example glycoproteins, glycolipids and proteoglycans, play an important role in many intracellular and extracellular events including cell-cell OPEN ACCESS adhesion, cell differentiation, signal transduction, cancer metastasis and immune responses [1]. The majority of these studies have focused on higher animals and relatively little is known about the functions of glycoconjugates in lower animals [2]. In order to study the biological properties of glycans in glycoconjugates, over the past decade we have synthesized novel glycolipid and glycoprotein derivatives found in various invertebrates [3][4][5][6][7][8][9][10][11][12]. Organic synthesis is a powerful method to explore structure activity relationships by providing access to large amounts of homogeneous and structurally defined oligosaccharides including not only natural compounds, but also non-natural compounds [13]. Recently, Zollo et al. isolated and characterized a novel neutral glycosphingolipid (A, Figure 1) from the marine sponge Aplysinella rhax which features a D-fucose and an N-acetyl-Dgalactosamine attached to a reducing-end N-acetyl-D-glucosamine through a α1→3 and a β1→4 linkages, respectively [14]. This was the first report on glycolipids containing D-fucose. Furthermore, these glycolipids have been found to exhibit significant inhibitory activity on LPS-induced nitric oxide (NO) release by J774.1 macrophages. In order to study the structure-activity relationships of these compounds inhibiting NO release, we previously reported the synthesis of β-D-GalNAcp(1→4)[α-D-Fucp(1→3)]-β-D-GlcNAcp(1→)aglycon trisaccharide analogues, containing a 2-branched fatty alkyl residue and a 2-(trimethylsilyl)ethyl (TMS-Et) residue, respectively [6]. Moreover, biological evaluation of these novel glycosphingolipid analogues using an LPS-induced NO release assay demonstrated that the presence of D-fucose is crucial for the NO inhibitory effect, while structural modifications at the aglycon moiety appeared to have little to no effect on LPS-induced NO release [6]. In this study, we describe for the first time the total synthesis of glycosphingolipid 1 and its structural analogues 3-6 to elucidate the structure activity relationships on LPS-induced NO production in more detail ( Figure 1).   2-(Trimethylsilyl)ethyl β-D-galactopyranosyl-(1→4)-[α-D-fucopyranosyl-(1→3)]-2-acetamido-2deoxy-β-D-glucopyranoside (3) was selected to explore how the presence of a terminal β-D-galacto-pyranosyl linkage instead of a 2-acetamido-2-deoxy-β-D-galactopyranosyl linkage affects the biological effect. Disaccharide-based regioisomers 4 and 5 were selected to explore differences in the connectivity of the α-D-fucopyranosyl moiety to the-β-D-GlcNAc portion while trisaccharide 6 was chosen to study the effect of two α-D-fucopyranosyl linkages linked to the core -D-GlcNAc moiety. The NO-inhibitory affect of commercially available ceramide 7 and known trisaccharide 2 was included in these experiments for comparison.

Chemical synthesis
Synthesis of glycosphingolipid 1: Glycosylation of phytoceramide acceptor 9 [15] with the glycosyl imidate 8 [6] was carried out in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) [16] and 4 Å molecular sieves (MS4 Å) to obtain the desired glycolipid derivative 10 in 33% yield with complete β-steroselectivity. Deprotection of the Troc group was achieved with Zn in a mixture containing acetic anhydride and acetic acid, followed by catalytic hydrogenolysis over 10% Pd/C in MeOH/THF to provide 11 in 47% yield. Deacetylation of 11 using Zemplén conditions and purification by column chromatography on Sephadex LH-20 afforded target glycolipid 1 quantitatively (Scheme 1). Syntheses of oligosaccharides 3-6: Glycosylation of known disaccharide acceptor 12 [3] with the known D-fucopyranosyl donor 13 [6] in the presence of N-iodosuccinimide (NIS), trifluoromethanesulfonic acid (TfOH) [17] and MS4 Å in dichloromethane provided the desired α-glycoside 14 in 88% yield with complete α-stereoselectivity. The newly formed α-glycosidic linkage was confirmed by 1 H-NMR spectroscopy. The anomeric proton of the fucose moiety in 14 appeared at 4.85 ppm as a doublet with a homonuclear proton-proton coupling constant of 3.7 Hz (H-1 of Fuc, δ = 4.85 ppm, J H1,H2 = 3.7 Hz). Deprotection of the Troc group in 14 was achieved with Zn in a mixture containing acetic anhydride and acetic acid, followed by catalytic hydrogenolysis over 10% Pd-C in MeOH and acetylation to provide 15. Zemplén deacetylation and purification by column chromatography on Sephadex LH-20 produced trisaccharide 3 quantitatively (Scheme 2). The synthesis of disaccharides 4 and 5 and trisaccharide 6 is outlined in Schemes 3-5. Glycosylation of known glycosyl acceptors 16, 19 [6] and 22 [18] with the D-fucopyranosyl donor 13 in the presence of NIS, TfOH and MS4 Å in dichloromethane gave the desired α-glycosides 17 (68%), 20 (78%) and the trisaccharide 23 (42%) with complete α-steroselectivity, respectively. The newly formed αglycosidic linkage was confirmed by 1 H-NMR spectroscopy. The Troc-protecting group of 17 was converted into an acetamido group by reduction with Zn-AcOH followed by debenzylidenation and debenzylation with catalytic hydrogenolysis over 10% Pd/C in MeOH-AcOH and acetylation to afford 18 in 44% yield. Finally, standard deacetylation and purification by column chromatography on Sephadex LH-20 furnished disaccharide 4 in 86% yield (Scheme 3). Disaccharide 5 was synthesized from the disaccharide 20 in a six steps deblocking/blocking procedure (Scheme 4). At first, the chloroacetyl protecting group in 20 was deblocked with thiourea in an ethanol/pyridine solvent mixture before conversion of the Troc group into an acetamido group using standard conditions. Debenzylation using catalytic hydrogenolysis followed by acetylation provided protected disaccharide 21 in 45% yield which was deprotected using standard conditions to provide disaccharide 5 (Scheme 4).

Inhibitory Effects of Synthetic Compounds on NO Production
The synthetic compounds were evaluated for their ability to inhibit nitric oxide (NO) production by LPS-induced macrophage-like J774.1 cells [19] (Figure 2). NO, a short living mediator is synthesized by a family of enzymes termed NO-synthase. Two types of NOS are recognized: constitutive isoforms and inducible isoforms (iNOS). iNOS is regulated by inflammatory mediators (LPS, cytokines) and the excessive production of NO by iNOS has been implicated in the pathogenesis of the inflammatory response [14]. The glycolipid 1 showed comparable NO inhibitory activity in high concentration (100 μM) to N G -monomethyl-L-arginine (L-NMMA) used as the positive control. Related compounds having the common D-Fucα1-3GlcNAc structure (i.e. 2, 3 and 4) also showed significant inhibitory activity resulting in a 20% reduction of NO release at 50 μM and 100 μM concentrations. However, very little or no inhibition of NO release were seen at these concentrations for disaccharide 5 and trisaccharide 6 bearing an unnatural D-Fucα1-4GlcNAc linkage. Interestingly, glycosphingolipid 1 showed stronger activity than 2, suggesting that the ceramide-based aglycon contributes to the inhibition of NO release more efficiently than a 2-(trimethylsilyl) ethyl-based aglycon. Moreover, commercial ceramide 7 showed inhibitory activity at higher concentration (100 μM). However, the activity of ceramide is strongly enhanced by glycosylation to the trisaccharide β-D-GalNAcp(1→4)[α-D-Fucp(1→3)]-β-D-GlcNAc indicating that both the trisaccharide and ceramide-based aglycon portion of the glycosphingolipid contribute to the inhibition of cellular nitric oxide release.

General
Optical rotations were measured with a Jasco P-1020 digital polarimeter. 1 H-and 13 C-NMR spectra were recorded with JMN A500 and ECP 600 FT NMR spectrometers with Me 4 Si as the internal standard for solutions in CDCl 3 and CD 3 OD. MALDI-TOFMS was recorded on an Applied Biosystems Voyager DE RP mass spectrometer. High-resolution mass spectra were recorded on a JEOL JMS-700 under FAB conditions. TLC was performed on Silica Gel 60 F254 (E. Merck) with detection by quenching of UV fluorescence and by charring with 10% H 2 SO 4 . Column chromatography was carried out on Silica Gel 60 (E. Merck). The compounds 3,4,6-Tri [18] were prepared as reported. Benzylceramide 9 was prepared by the conventional four-steps procedure [15] from phytosphingosine, which was purchased from Degussa (The Netherlands).  (11). To a solution of 10 (31mg, 16.8 μmol) in acetic anhydride (2 mL) and AcOH (2 mL) was added zinc powder (100 mg). The reaction mixture was stirred for 16 h at room temperature. After completion of the reaction, the solids were filtered off and the filtrate was concentrated with toluene. The solution of the product and Pd/C (10%, 100 mg) in 1:1 MeOH/THF (2.0 mL) was stirred for 16 h at room temperature under H 2 , then filtered and concentrated. The product was purified by silica gel column chromatography using 2:1 toluene acetone as eluent to give 11 as an amorphous powder (11 mg, 47%

2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-[2,3,4-tri-O-benzyl-α-Dfucopyranosyl-(1→3)]-6-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy-carbonylamino)-β-D-glucopyranoside (14)
. To a solution of 12 (99 mg, 0.11 mmol) and 13 (89 mg, 0.17 mmol) in dry CH 2 Cl 2 (1.5 mL) was added powdered MS 4Å (200 mg), and the mixture was stirred for 2 h at room temperature, then cooled to -60 °C. NIS (57 mg, 0.03 mmol) and TfOH (1.5 μL, 0.01 mmol) were added to the mixture, which was stirred for 3 h at -60 °C, then neutralized with Et 3 N. The solids were filtered off and washed with CHCl 3 . The combined filtrate and washings were successively washed with aq Na 2 S 2 O 3 and water, dried (MgSO 4 ), and concentrated. The product was purified by silica gel column chromatography using 3:1 hexane-EtOAc as eluent to give 14 (128 mg, 88%  (15). To a solution of 14 (107 mg, 0.08 mmol) in acetic anhydride (6 mL) and AcOH (6 mL) was added zinc powder (150 mg). The reaction mixture was stirred for 12 h at 40 °C. After completion of the reaction, the solids were filtered off and the filtrate was concentrated with toluene. The solution of the product and Pd/C (10%, 100 mg) in MeOH (2.0 mL) was stirred for 16 h at room temperature under H 2 , then filtered and concentrated. The residue was acetylated with acetic anhydride (2 mL) in pyridine (3 mL) for 16 h at room temperature. The reaction mixture was poured into ice-water and extracted with CHCl 3 . The extract was washed sequentially with 5% HCl, aq NaHCO 3 and water, dried (MgSO 4 ), and concentrated. The product was purified by silica gel column chromatography using 5:1 toluene-acetone as eluent to give 15 (37 mg, 46%) as an amorphous powder.   (17). To a solution of 16 (329 mg, 0.61 mmol) and 13 (479 mg, 0.91 mmol) in dry CH 2 Cl 2 (1.5 mL) was added powdered 4Å MS (800 mg), and the mixture was stirred for 2 h at room temperature, then cooled to -60 °C. NIS (307 mg, 1.37 mmol) and TfOH (16 μL, 0.18 mmol) were added to the mixture, which was stirred for 3 h at -60 °C, then neutralized with Et 3 N. The solids were filtered off and washed with CHCl 3 . The combined filtrate and washings were successively washed with aq Na 2 S 2 O 3 and water, dried (MgSO 4 ), and concentrated. The product was purified by silica gel column chromatography using 7:1 hexane-EtOAc as eluent to give 17 (394 mg, 68%  (18). To a solution of 17 (113 mg, 0.12 mmol) in acetic anhydride (7 mL) and AcOH (7 mL) was added zinc powder (150 mg). The reaction mixture was stirred for 12 h at 40 °C. After completion of the reaction, the solids were filtrered off and the filtrate was concentrated with toluene. The solution of the product and Pd/C (10%, 150 mg) in 3:1 MeOH-AcOH (2.0 mL) was stirred for 12 h at room temperature under H 2 , then filtered and concentrated. The residue was acetylated with acetic anhydride (6 mL) in pyridine (10 mL) for 12 h at room temperature. The reaction mixture was poured into ice-water and extracted with CHCl 3 . The extract was washed sequentially with 5% HCl, aq NaHCO 3 and water, dried (MgSO 4 ), and concentrated. The product was purified by silica gel column chromatography  (20 (21). To a solution of 20 (296 mg, 0.29 mmol) in EtOH (2.5 mL) was added pyridine (1.5 mL) and thiourea (173 mg, 2.32 mmol). The reaction mixture was stirred for 6 h at 80 °C. The mixture was diluted with CHCl 3 , washed with aq 5%HCl, aq NaHCO 3 and brine, dried (MgSO 4 ) and concentrated. The solution of the residue in AcOH (2 mL) was added zinc powder (350 mg). The reaction mixture was stirred for 12 h at 60 °C. After completion of the reaction, the solids were filtered off and the filtrate was concentrated with toluene. The residue was acetylated with acetic anhydride (4 mL) in pyridine (7 mL). The reaction mixture was poured into ice-water and extracted with CHCl 3 . The extract was washed sequentially with 5% HCl, aq. NaHCO 3 and water, dried (MgSO 4 ), and concentrated. The solution of the product in MeOH (1.5 mL) and THF (0.5 mL) was hydrogenolysed under hydrogen in the presence of 10% Pd/C (150 mg) for 16 h at room temperature, then filtered and concentrated. The residue was acetylated with acetic anhydride (3 mL) in pyridine (5 mL). The reaction mixture was poured into ice-water and extracted with CHCl 3 . The extract was washed sequentially with 5% HCl, aq NaHCO 3 and water, dried (MgSO 4 ), and concentrated. The product was purified by silica gel column chromatography using 5:1 toluene-acetone as eluent to give 21 as an amorphous powder (86 mg, 45%

Conclusions
We have succeeded for the first time in carrying out the total syntheses of D-fucose-containing glycosphingolipids found in invertebrate species. Both the presence of a D-Fucα1-3GlcNAc-linkage and the ceramide aglycon portion resulted in a significant enhancement of their ability to inhibit NO production by LPS-induced macrophage-like J774.1 cells. The prepared glycolipids are easily-accessible target compounds in the field of carbohydrate chemistry and may serve as chemical probes to explore glycosphingolipid-mediated anti-inflammatory processes in biology and medicine.