Retinoic acid-mediated antibody response

Supplementary Information

Materials. Amino acid building blocks, resins and coupling agents were obtained from Novabiochem (Darmstadt, Germany), Anaspec (San Jose, CA) or ChemPep (Miami, FL). Cholesterol, dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Distearoylglycerol (DSG; LP-R4-029) was obtained from Genzyme Pharmaceuticals (Cambridge, MA). Anhydrous solvents of 99.8% or greater purity were obtained from Acros Organics (Geel, Belgium). Monophosphoryl lipid A derived from Escherichia coli (MPL; #L6638), all-trans retinoic acid (ATRA; #R2625) and 13-cis retinoic acid (13-cis RA; #R3255) were obtained from Sigma-Aldrich. Unless otherwise specified, all other reagents were obtained from Sigma-Aldrich.

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Lipopeptide synthesis. N-MPR peptide (NEQELLELDKWASLNGGK) was synthesized on NovaPEG resin in an automated solid phase synthesizer (ABI 433A, Applied Biosystems, Foster City, CA) with standard fluorenylmethyloxycarbonyl/o-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate/n-hydroxybenzotriazole (FMOC/HBTU/HOBT) protocols. An orthogonally protected lysine (Fmoc-Lys(1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl)-OH; Fmoc-Lys(ivDde)-OH) was incorporated at the C terminus for on-resin conjugation of lipids or biotin. The N terminus was Boc-protected. Removal of the ivDde group was accomplished by 3 x 15 minute treatments of the peptidyl resin with 2% hydrazine hydrate in dimethylformamide (DMF; 10 mL per g resin). The resin was washed in DMF (3 x 10 mL) and dichloromethane (DCM; 3 x 10 mL) and dried under vacuum.

Lipid conjugation was accomplished via amidation of a carboxylated lipid and a deprotected lysine e-amine at the C terminus. A carboxyl group was introduced to DSG via reaction of an available alcohol with succinic anhydride. Briefly, 1.8 mmol DSG was dissolved in 5 mL anhydrous DCM and combined with 3.6 mmol succinic anhydride in 10 mL anhydrous pyridine. The mixture was refluxed at 60 ^oC overnight. The reaction was continued to completion as monitored by thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS; Voyager DE, Applied Biosystems, Foster City, CA) in para-nitroaniline matrix. The product was washed twice with 1M hydrochloric acid (HCl), dried over sodium sulfate and stored dry until use. Carboxylated lipids were obtained in approximately 90-100% yield. The molecular weight and TLC R_F value were as follows: DSG-Suc, 749.6 (+ Mg²⁺) Da, R_F 0.08 (DCM/acetone, 20/1).

Lipidation was accomplished by activation of 270 mmol carboxylated lipid with 270 mmol each of HBTU, HOBT and diisopropylethylamine (DIEA) in anhydrous DMF/DCM (DCM as needed for lipid solubilization) for 30 min at room temperature followed by addition of 67.5 mmol resin and continued reaction under argon for 24h at room temperature. Following the reaction, the resin was washed with DMF (4 x 10 mL) and DCM (4 x 10 mL) to remove unreacted lipids and dried under vacuum. The lipopeptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% water, 2.5% ethanedithiol and 1% triisopropylsilane for 4 hours under argon. The lipopeptide was precipitated into cold ethyl ether, pelleted by centrifugation at 3000 rpm (RT6000, Sorvall, Waltham, MA) and washed once with cold ethyl ether. The ether was poured off and the pellet was re-dissolved in methanol (MeOH), transferred to a round bottom flask, dried by rotary evaporation under reduced pressure and further dried under high vacuum. Stock lipopeptide solutions were prepared MeOH/CHCl₃ and stored at -20 ^oC. The molecular weight was as follows: N-MPR-DSG 2963.2 Da.

Biotinylated N-MPR peptide was prepared for use in ELISA by an analogous method. Biotin was attached to the deprotected C terminal amine by activation of 500 mmol D-biotin with 500 mmol HBTU/HOBT/DIEA in 1.65 mL anhydrous 1:1 DMF/dimethylsulfoxide (DMSO) for 30 min followed by addition of resin and continued reaction under argon for 24h at room temperature. Following the reaction, the resin was washed with 1:1 DMF/DMSO (3 x 10 mL), DMF (3 x 10 mL) and DCM (3 x 10 mL) and dried under vacuum. Biotinylated peptides were cleaved and purified as described above. Molecular weight of N-MPR-biotion was 2455.1 Da as determined by MALDI-MS. Biotin content was quantified by 4´-hydroxyazobenzene-2-carboxylic acid dye exclusion (Sigma #H2153) according to the manufacturer’s instructions.

 

RAL Synthesis. The synthesis of all-trans retinoic acid phospholipid (RAL) is depicted in Scheme S1. First, 1-O-octadecyl-2-O-benzyl-sn-glycerol was converted to phosphocholine by phosphorylation with phosphorus oxychloride and coupling to the choline tetraphenyl borate salt. Then the benzyl group was removed by catalytic transfer hydrogenation. Finally, all-trans retinoic acid was attached to the 2-hydroxy group with the typical DCC/DMAP method.

1-O-octadecyl-2-O-benzyl-sn-glycerol was from BACHEM (Torrance, CA). Other reagents were from Aldrich (Milwaukee, WI). TLC analyses were performed on 0.25-mm silica gel F₂₅₄ plates using a variety of developing systems. High performance flash chromatography (HPFC) was carried out on a Biotage (Charlottesville, VA) Horizon™ HPFC™ system with pre-packed silica gel columns (60 Ǻ, 40-63 mm). Unless noted otherwise, the ratios describing the composition of solvent mixtures represent relative volumes. ¹H NMR spectra were acquired on a Varian 400 MHz instrument. Chemical shifts are expressed as parts per million using tetramethylsilane as internal standard. J values are in Hertz. MALDI-TOF mass spectra were obtained at the Mass Spectrometry Facility, University of California San Francisco.

 

1-O-octadecyl-2-O-benzyl-sn-glycero-phosphate (1): A solution of 1-O-octadecyl-2-O-benzyl-sn-glycerol (10 g, 23 mmol) and anhydrous pyridine (3.72 mL, 2 equiv.) in anhydrous tetrahydrofuran (40 mL) was added dropwise to the freshly distilled phosphorus oxychloride (2.36 mL, 1.1 equiv.) in tetrahydrofuran (20 mL) with stirring at 0 °C. Stirring was continued for 3 h at 0 °C. Then 10% sodium bicarbonate (60 mL) was added, and the mixture was stirred at 0 °C for 15 min. The solution was then poured on ice water (200 mL), acidified with conc. HCl (pH ca. 2), and extracted with diethyl ether (400 mL × 2). The ether extracts were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure, azeotropically dried with toluene twice, and used directly for next step reaction. Yield: 11.8 g, 100%. TLC: R_f = 0.05 (CHCl₃/MeOH/NH₄OH, 65/25/4).

 

1-O-octadecyl-2-O-benzyl-sn-glycero-phosphocholine (2): Compound 1(5.14 g, 10 mmol), choline tetraphenyl borate (8.46 g, 2 equiv.) and 2,4,6-triisoproylbenzene sulfonyl chloride (6.04 g, 2 equiv.) were dissolved in anhydrous pyridine (100 mL) with heating. The reaction mixture was stirred at 70 °C for 1h, then 3 h at room temperature. After the addition of water (10 mL), the solvents are removed by rotary evaporation. The residue was extracted with diethyl ether (250 mL × 2). The extracts were combined and evaporated. The crude product was purified by HPFC using a solvent gradient of 20% -35% MeOH-H₂O (25/4) in chloroform. Yield: 5.8 g, 96.7%. TLC: R_f = 0.17 (CHCl₃/MeOH/H₂O, 65/25/4). ¹H NMR (CDCl₃-CD₃OD), d 0.87 (t, J = 7.2, 3H); 1.27 (br, 30H); 1.57 (m, 2H); 3.15 (s, 9H); 3.36-3.60 (m, 7H); 3.83 (m, 1H); 4.04 (m, 1H); 4.16 (m, 2H); 4.68 (m, 2H); 7.31-7.45 (m, 5H). MALDI-MS calculated for C₃₃H₆₃NO₆P⁺ [M + H]⁺ 600.45, found 600.54.

 

1-O-octadecyl-2-hydroxy-sn-glycero-phosphocholine (3): To as solution of compound 2(5.77 g, 9.6 mmol) in 70 mL methanol, were added 10% Palladium on activated carbon (2 g) and ammonium formate (3.03 g, 48 mmol) under nitrogen atmosphere. The mixture was stirred vigorously under nitrogen for 10 h at 60 °C. The mixture was cooled to room temperature, filtered through Celite 545. The filtrate was concentrated, and applied to a flash40+M column, purified with the HPFC system using an elution gradient of 30% -50% MeOH-H₂O (25/4) in chloroform. Yield: 3.3 g, 67%. TLC: R_f = 0.05 (CHCl₃/MeOH/H₂O, 65/25/4). ¹H NMR (CDCl₃-TFA, 10:1), d 0.88 (t, J = 6.9, 3H); 1.27 (br, 30H); 1.63 (m, 2H); 3.25 (s, 9H); 3.64-3.85 (m, 6H); 4.30 (m, 1H); 4.58 (m, 1H); 4.68 (m, 2H); 5.03 (m, 1H). MALDI-MS calculated for C₂₆H₅₇NO₆P⁺ [M + H]⁺ 510.40, found 510.62.

 

1-O-octadecyl-2-all-trans-retinoyl-sn-glycero-phosphocholine (4; RAL): To a solution of compound 3 (0.6 g, 1.1 mmol) and all-trans retinoic acid (0.73 g, 2 equiv.) in dry ethanol-free chloroform, were added dimethylaminopyridine (0.15 g) and dicyclohexylcarbodiimide (0.5 g, 2.1 equiv.). The reaction mixture was kept in dark with stirring for 36 h. The precipitate was filtered and the filtrate was concentrated, applied to flash 25+M column, purified with the HPFC using a elution gradient of 10%-30% MeOH/28% NH₄OH (25/4) in chloroform. Yield: 626 mg, 72%. TLC: R_f = 0.52 (CHCl₃/MeOH/NH₄OH, 65/25/4). ¹H NMR (CDCl₃), d 0.89 (t, J = 6.9, 3H); 1.04 (s, 6H); 1.26 (br, 30H); 1.45-1.53 (m, 4H); 1.63 (m, 2H); 1.72 (s, 3H); 2.00 (s, 3H); 2.04 (m, 2H); 2.34 (s, 3H); 3.32 (s, 9H); 3.41 (m, 2H); 3.58 (m, 2H); 3.78 (m, 2H); 4.00 (m, 2H); 4.30 (m, 2H); 5.17 (m, 1H); 5.79 (m, 1H); 6.12-18 (m, 2H); 6.28 (m, 2H); 7.01 (m, 1H). MALDI-MS calculated for C₄₆H₈₃NO₇P⁺ [M + H]⁺ 792.60, found 792.84.

 

RAL digestion by phospholipase A₂. The cleavage of retinoic acid from RAL by phospholipase A₂ (PLA₂) was examined in vitro. 10 mmol RAL were dried to a thin film in a borosilicate glass tube by rotary evaporation under reduced pressure and following by further drying under high vacuum overnight. The film was hydrated in 2 mL containing 10 mM HEPES, 25 mM KCl, 10 mM CaCl₂ and 20 mM Triton-X, pH 8.8 and heated at 45 ^oC for 20 min. Fifty mL of a 100 U/mL solution of snake venom PLA₂ (Sigma #P7778) were added to 250 mL of the RAL solution and incubated for 30 min. When analyzed by TLC, incubation with PLA₂ resulted in total loss of the original RAL spot (R_f = 0.38) and appearance of two new spots, corresponding to free retinoic acid (R_f = 0.28) and lysolipid (R_f = 0.18; CHCl₃/MeOH/NH₄OH, 65/25/4).

 

Liposome preparation. Lipopeptides were formulated in liposomes composed of 15:2:3 DMPC:DMPG:Cholesterol with MPL, RA and lipopeptide as indicated. Prior to use, glassware was rinsed with MeOH and CHCl₃ and dried for at least 90 min at 150 ^oC to destroy pyrogens. Lipid solutions were combined in borosilicate glass tubes and dried to a thin film by rotary evaporation under reduced pressure. Films were further dried under high vacuum overnight. Lipids were hydrated in sterile PBS (UCSF Cell Culture Facility) by intermittent vortexing and bath sonication under argon for a brief period (approximately 15 seconds) to disperse the lipids into the buffer. Defined diameter vesicles were formed by extrusion 11 times through 400 nm polycarbonate membranes using a hand-held extruder (Avestin, Ottowa, Canada). To prevent contamination, the extruder was disassembled and thoroughly cleaned with MeOH and sterile PBS between samples. Vesicle size was characterized by dynamic light scattering and zeta potential was determined by electrophoretic mobility (Zetasizer 3000, Malvern, New Bedford, MA). Liposomes were prepared fresh prior to each injection and stored at 4 ^oC under argon until use.

 

Liposome association of N-MPR-DSG and RA. Liposomal association of antigen and retinoic acid was measured following sedimentation of liposomes by an ultracentrifugation method. Essentially, liposomes were formulated with antigen, MPL and RA exactly as described in Methods and centrifuged at 150,000g for 45 minutes in a Beckman TL-100 ultracentrifuge. When control liposomes containing fluorescently labeled lipids were centrifuged in this manner, greater than 90% of the fluorescence was consistently recovered in the liposome pellet. The liposome pellet was resuspended by pipetting and an aliquot was disrupted by mixing with 1.5% (v/v) C₁₂E₁₀ detergent. Antigen concentration in the pellet and supernatant was quantified by the absorbance of aromatic side chains at 280 nm and RA concentration was quantified by the absorbance at 350 nm (NanoDrop, Thermo Scientific, Wilmington, DE). The percent of liposome association was defined as (Pellet)/(Pellet + Supernatant) x 100.

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