Synthesis of α-L-rhamnosyl ceramide and evaluation of its binding with anti-rhamnose antibodies

Abstract

An α-L-rhamnosyl ceramide (1, α-L-RhaCer) has been prepared that was recognized by anti-L-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-L-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-L-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acetyl-1-(2,2,2-trichloroethanimidate)-α-L-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition-fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-L-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-ovalbumin (Rha-Ova) with Sigma Adjuvant System (SAS) and the anti-L-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-L-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies.

Keywords: Anti-cancer vaccine; Anti-rhamnose antibody; Glycolipid; Rhamnosyl-ceramide.

Figure 1

Structure of α-L-rhamnosyl ceramide (1) and model for anti-Rha-antibody-mediated uptake of TAA in tumor cells

Figure 2

Anti-rhamnose antibody titer of Balb/CJ mice immunized with rhamnose ovalbumin conjugate (after 1^st boost) Rha-BSA: Rhamnose conjugated with bovine serum albumin (BSA). Anti-Rha Serum: Serum isolated from mice immunized with rhamnose-ovalbumin. Nonimmunized serum: Serum isolated from nonimmunized mice.

Figure 3

Flow cytometry of EL4 tumor cells FL1-A = FITC (A) EL4 with α-L-RhaCer and anti-rhamnose antibodies (B) EL4 with α-L-RhaCer and non-rhamnose antibodies (C) Black peak: EL4 cells with α-L-RhaCer and non-Rha antibodies; blue peak: EL4 cells with Cer and anti-Rha antibodies; red peak: EL4 cells with α-L-RhaCer and anti-Rha antibodies.

Scheme 1

Synthesis of glycosyl donors 4 and 6 Reagents and Conditionsi. Ac₂O, pyridine, DMAP, RT, 16 h, quant.; ii. p-thiocresol, BF₃·OEt₂, CH₂Cl₂, 0 °C – RT, 18 h 52%; iii. hydrazine acetate, DMF, 55 °C, 4 h, 91%; iv. CCl₃CN, DBU, CH₂Cl₂, 0 °C – RT, 2 h, 85%.

Scheme 2

Synthesis of intermediates 14 and 21 Reagents and Conditions: i. p-thiocresol, 90% TFA, 55 °C for 15 min, then RT for 0.5 h, quant.; ii. 2,2-DMP, p-TSA, ACN, RT, 3 h, 45%; iii. HgO (yellow), HgCl₂, acetone/H₂O, 12 h, 55 °C, 98%; iv. p-TsNHNH₂, MeOH, 12 h, RT, 80%; v. C₁₃H₂₇MgBr, Et₂O, 0 °C for 0.5 h, then RT overnight, 52%; vi. BzCl, pyridine, 0 °C – RT, 12 h, 77%; vii. 3:1:1 AcOH-THF-H₂ O, 55 °C, 13 h, 89%; viii. AllocCl, 3:2 CH₂Cl₂-pyridine, 7minus;10 °C, 3 h, 92%; ix. 3:1:1 AcOH-THF-H₂O, 55 °C, 12 h, 64%.

Scheme 3

Preparation of the sphingosine derivative acceptor Reagents and Conditions: i. TIPSCl, imidazole, THF, 0 °C, 24 h, 91%; ii. PPh₃, DIAD, HN₃, THF, 4 Å, 0 °C, 2 h, 92%; iii. 1M TBAF, THF, 0 °C – RT, 10 min, 80%; iv. HF-pyridine, THF, RT, 24 h, 94%

Scheme 4

Synthesis of α-L-rhamnosyl ceramide (1) Reagents and Conditions: i. Donor 6, BF₃· OEt_2, CH₂Cl₂, 4 Å MS, −20 °C, 0.5 h, 98.6%; ii. (a) PPh₃, H₂O, THF, 45 °C, 3 h; (b) C₁₅H₃₁COOH, EDC· HCl, HOBt, Et₃N, CH₂Cl₂, RT, 10 h, 84% over two steps; iii. NaOMe, MeOH-CH₂Cl₂, pH=9, RT, 10 h, 93%.

Scheme 5

Synthesis of ceramide control 24 Reagents and Conditions: i. (a) PPh₃, H₂O, THF, 45 °C, 3 h; (b) C₁₅H₃₁COOH, EDC· HCl, HOBt, Et₃N, CH₂Cl₂, RT, 10 h, 50% over two steps; ii. (a) 1M TBAF, THF, 0 °C – RT, 10 min; (b) NaOMe, MeOH-CH₂Cl₂, pH=9, RT, 10 h, 86.5% over two steps.

Scheme 6

Attempted glycosylation of S-tolyl donor and sphingosine acceptor. Reagents and Conditions: i. Acceptor 14, NIS, TMSOTf, 4 Å MS, −45 °C; ii. Acceptor 14, DMTST_, CH₂Cl₂, 4 Å MS, 0 °C.