Synthesis of Short Polyoxyethylene-Based Heterobifunctional Cross

Bioconjugate Chem. 1996, 7, 180−186

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ARTICLES Synthesis of Short Polyoxyethylene-Based Heterobifunctional Cross-Linking Reagents. Application to the Coupling of Peptides to Liposomes Benoıˆt Frisch,* Christophe Boeckler, and Francis Schuber Faculte´ de Pharmacie, Universite´ Louis Pasteur, 67400 Strasbourg-Illkirch, France. Received August 8, 1995X

We describe the synthesis of [2-[2-[2-[(2-bromoacetyl)amino]ethoxy]ethoxy]ethoxy acetic acid (7), [2-[2(2,5-dioxo-2,5-dihydropyrrol-1-yl)ethoxy]ethoxy] acetic acid (11), and [2-[2-(pyridin-2-yldisulfanyl)ethoxy]ethoxy] acetic acid (16), three new thiol-reactive heterobifunctional reagents, and the preparation of their corresponding dipalmitoylphosphatidylethanolamine derivatives (8, 12, and 17). Such phospholipid amide derivatives were aimed to be incorporated into the bilayers of liposomal constructs used for immunization with e.g. synthetic peptides. The spacer arms introduced by 8, 12, and 17 are hydrophilic polyoxyethylene chains of variable lengths that were expected to provide a good accessibility to their conjugates and have a lesser intrinsic immunogenicity than the spacer introduced by N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE), a classical reagent used for conjugation of ligands to the surface of liposomes. Such an immunogenicity might be prejudicial (e.g. carrier-induced epitopic suppression) to the development of synthetic vaccination formulations. Moreover, the derivatives 8, 12, and 17 allowed the coupling of peptides, bearing a thiol function, to their liposomal carrier via two types of linkages, i.e. stable thio ether (8 and 12) and bioreducible disulfide (17) bonds; this might be of importance in the mechanism of antigen presentation by competent cells. Using CG-IRGERA as a model peptide, the rate of coupling to 8, 12, and 17 was assessed as a function of pH.

INTRODUCTION

Synthetic peptide representatives of epitopes from e.g. microorganisms of transformed cells are useful for the development of therapeutic vaccines in infectious diseases or cancer (Arnon and Horwitz, 1992). However, most peptides of relatively small size are hampered by poor immunogenicity, and to trigger an immune response, they must be coupled to macromolecular carriers such as proteins or synthetic polymers (Defoort et al., 1992) or associated to particulate systems such as liposomes (Gregoriadis, 1994a,b) or immunostimulating complexes (e.g. ISCOMs, Lo¨vren and Larsson, 1994). In most cases, the presence of an adjuvant is also required. In this respect, to preserve the structural integrity of the peptide, it is important to design bioconjugation procedures that couple peptides in a chemically well-defined fashion to the carriers (Muller, 1988). Our group has elaborated an efficient strategy that confers a particularly important immunogenicity to small synthetic peptides, such as IRGERA which corresponds to the C-terminal of histone H3 (Frisch et al., 1991; Friede et al., 1993). It is based on the covalent coupling of the peptide to the surface of preformed small unilammellar vesicles (SUV) that carry an adjuvant such as monophosphoryl lipid A (MPLA) in their bilayer (Figure 1). When applied to a cyclic nine* To whom correspondence should be addressed at Laboratoire de Chimie Bioorganique, Unite´ de Recherche Associe´e au Centre National de la Recherche Scientifique (URA 1386), Faculte´ de Pharmacie, 74 route du Rhin, 67400 StrasbourgIllkirch, France. Telephone: (33) 88 67 68 54. Fax: (33) 88 67 88 91. X Abstract published in Advance ACS Abstracts, February 1, 1996.

1043-1802/96/2907-0180$12.00/0

Figure 1. Liposome-based construct for immunization with synthetic peptides. The peptide carrying a cysteine residue, e.g. at its N-terminus, is conjugated to the surface of preformed SUV containing a thiol-reactive phosphatidylethanolamine derivative and MPL as adjuvant.

residue peptide mimicking the A site of influenza virus hemagglutinin, over 70% protection was achieved in a murine vaccination model (Friede et al., 1994). The coupling of the peptides to liposomes was performed according to the strategy originally developed by Martin and Papahadjopoulos (1982) for the preparation of immunoliposomes. Thus the peptide, which was extended by a Cys-Gly spacer arm at its N-terminal, in the presence of preformed liposomes that contain N-[4(p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE), readily reacts with the maleimide moiety to give a stable thio ether linker linkage. This thiol-reactive phospholipid derivative is obtained by reaction between phosphatidylethanolamine and succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), a commercially available heterobifunctional coupling reagent. In the development of synthetic vaccines, this particular reagent, which also introduces a spacer arm between the peptide immunogen and the vesicle surface, has drawbacks. First, it is intrinsically immunogenic and therefore could engender © 1996 American Chemical Society

Heterobifunctional Cross-Linking Reagents

secondary immune responses that are prejudicial to a vaccination formulation (e.g. carrier-induced epitopic suppression). Such an immunogenicity is classical for structures that carry aromatic groups (Bernatowicz and Matsueda, 1986; Jones et al., 1989; Peeters et al., 1989). Second, the spacer introduced is fairly hydrophobic, and the possibility exists that it will partition into the bilayer of the liposome, thus limiting an optimal presentation of the antigen to the cells involved in the immune response. Since many commercial heterobifunctional reagents that are useful for such a purpose carry hydrophobic groups, we have decided to synthesize new reagents with polyoxyethylene spacers. The main advantage of this approach is that these more hydrophobic and flexible arms should be, a priori, less immunogenic (Zalipsky, 1995); moreover, their length can be easily modulated, thus allowing a good accessibility of the linked peptide. In the present article, we describe the synthesis of three new such heterobifunctional reagents 7, 11, and 16 (Schemes 1-3). One end of these reagents has been functionalized to react with a thiol group carried by a peptide to give a stable thio ether bond (maleimide or bromoacetyl moieties) or a disulfide linkage (dithiopyridine moiety) that can easily regenerate the free peptide by reduction or by thiol exchange reaction. Such a difference in the mode of attachment to the carrier is of importance in defining the mechanisms involved in the presentation of the peptide to the immune system (Friede et al., 1995). The other end bears a free carboxylic function destined to be coupled to the amine of PE. These thiol-reactive phospholipid derivatives have also been studied for their reactivity with the thiol function of a model peptide and for their immunogenicity (Boeckler et al., submitted for publication). EXPERIMENTAL SECTION

General. Reagent-grade solvents were used without further purification. Dicyclohexylcarbodiimide (DCC), bromoacetic acid, N-hydroxysuccinimide, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), methyl chloroformate, bromoacetic chloride, [2-(2-chloroethoxy)ethoxy]ethanol, and 2,2′-dipyridyl disulfide were purchased from Aldrich Chemical Co. (L'Isle d'Abeau Chesnes, France). Methanesulfonyl chloride and ninhydrin were obtained from Lancaster Synthesis (Bischeim, France). Tetra(ethylene glycol), triethylamine, and triphenylphosphine were from Janssen Chemica (Geel, Belgium). Sodium azide and aluminum oxide 90 active, basic were purchased from Merck (Darmstadt, Germany) and maleimide and Nmethylmorpholine from Fluka Chemical Co. (Buchs, Switzerland). Phosphomolybdenum blue and dithiothreitol were obtained from Sigma Chemical Co. Bio-Sil,HA was purchased from Bio-Rad (Ivry/Seine, France). (Benzotriazol-1-yl-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) was from Neosystem (Strasbourg, France), and thiourea was obtained from Prolabo (Paris, France). 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was a gift from D3F (Doullens, France). The peptide, CGIRGERA, was a kind gift from Dr. J. P. Briand (CNRS-IBMC, Strasbourg, France). Thin layer chromatography was performed on silica gel 60 F 254 plates (Merck, Darmstadt, Germany). Flash chromatography was carried out using silica gel 60, 4063 µm (230-400 mesh) (Merck). 1H-NMR spectra were recorded at 200 MHz on a Bruker WP-200 SY spectrometer. Mass spectra were recorded on a VG ZAB-HF mass spectrometer. Methanesulfonic Acid 2-[2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy]ethyl Ester (2). Methanesulfonyl chlo-

Bioconjugate Chem., Vol. 7, No. 2, 1996 181

ride (10 mL, 0.129 mol) was added dropwise to a solution of 25 g (0.129 mol) of tetra(ethylene glycol) (1) and 17.91 mL (0.129 mol) of triethylamine in 200 mL of tetrahydrofuran (THF) at 0 °C. The reaction mixture was stirred for 2 h at 0 °C and left overnight at room temperature (RT). After elimination of the precipitate by filtration, the solution was evaporated under reduced pressure. The residue was purified by chromatography on a silica gel column eluted with EtOAc/hexane (80:20 to 100:0), and the title product was obtained (8.79 g, 25% yield) as a colorless oil: Rf 0.2 (EtOAc); 1H-NMR (CDCl3) δ 4.404.35 (m, 2H, CH2OS), 3.78-3.57 (m, 14H, CH2OCH2, HOCH2), 3.07 (s, 3H, SCH3). 2-[2-[2-(2-Azidoethoxy)ethoxy]ethoxy]ethanol (3). A solution of 2 (8 g, 29.4 mmol) and sodium azide (2.8 g, 43 mmol) in 50 mL of acetonitrile was heated under reflux for 36 h. After return to room temperature, 50 mL of water was added and the mixture was extracted with CH2Cl2. The organic phase was then dried on MgSO4 and concentrated. The residue was chromatographed on a silica gel column eluted with EtOAc. Compound 3 was obtained as a colorless oil (5.78 g, 90% yield): Rf 0.5 (EtOAc); 1H-NMR (CDCl3) δ 3.73-3.57 (m, 14H, CH2OCH2, HOCH2), 3.37 (t, J ) 5.0 Hz, 2H, CH2N3). 2-[2-[2-(2-Aminoethoxy)ethoxy]ethoxy]ethanol (4). Compound 3 (4.88 g, 22.28 mmol), triphenylphosphine (6.43 g, 24.51 mmol), and water (600 mg, 33.42 mmol) were mixed with 25 mL of THF. After the solution was stirred for 4 h at room temperature, the solvent was eliminated under reduced pressure and the residual product was purified on a silica gel column that was eluted with CHCl3/CH3OH/Et3N (3:3:1). The title compound was obtained as colorless oil (4.07 g, 95% yield): Rf 0.4 (CHCl3/CH3OH/Et3N, 3:3:1); 1H-NMR (CDCl3) δ 3.72-3.44 (m, 14H, CH2OCH2, HOCH2), 2.79 (t, J ) 4.9 Hz, 2H, CH2N). 2-Bromo-N-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl]acetamide (6). Compounds 4 (193 mg, 1 mmol) and 5 (236 mg, 1 mmol) were dissolved in 10 mL of CHCl3 (0 °C), and the mixture was left at room temperature overnight. The solvent was then removed under reduced pressure, and the product was purified on a silica gel column that was eluted with CH2Cl2/CH3OH (95:5). A colorless oil was obtained (220 mg, 70% yield): Rf 0.2 (CHCl3:CH3OH, 95:5); 1H-NMR (CDCl3) δ 7.66 (s, 1H, NH), 3.87 (s, 2H, CH2Br), 3.82-3.42 (m, 16H, HOCH2, CH2OCH2, CH2N), 2.16 (s, 1H, OH). [2-[2-[2-[(2-Bromoacetyl)amino]ethoxy]ethoxy]ethoxy acetic Acid (7). Compound 6 (270 mg, 0.8 mmol) was dissolved in 10 mL of acetone. Under stirring, 0.5 mL of 2.67 M Jones reagent (26.72 g of chromic trioxide in 23 mL of concentrated sulfuric acid brought to a final volume of 100 mL of water) was added dropwise over 15 min. After an additional 30 min, a few drops of 2-propanol were added, followed by 10 mL of water to dissolve the precipitated chromium salts. The organic solvent was then removed under reduced pressure and the aqueous phase extracted two times with 20 mL of CH2Cl2. The combined CH2Cl2 extracts were dried on MgSO4 and concentrated, and the residue was chromatographed on silica gel column eluted with CH2Cl2/ CH3OH (80:20). The product was obtained as a colorless oil (180 mg, 64% yield): Rf 0.26 (CH2Cl2/MeOH, 80:20); 1H-NMR (CDCl3) δ 7.18 (s, 1H, NH), 4.17 (s, 2H, O2CCH2O), 3.88 (s, 2H, CH2Br), 3.74-3.49 (m, 12H, CH2CH2O); HRMS (FAB+) m/z calcd for C10H19O6NBr (M + H+) 328.0396, found 328.039. Hexadecanoic Acid 3-[[[2-[2-[2-[2-[2-[(2-Bromoacetyl)amino]ethoxy]ethoxy]ethoxy]acetylamino]ethoxy]hydroxyphosphoryl]oxy]-2-(hexadecanoyloxy)propyl Ester (8). DPPE (100 mg, 155 µmol) was added

182 Bioconjugate Chem., Vol. 7, No. 2, 1996

to a solution of 1 mL of anhydrous CH3OH and 10 mL of anhydrous CHCl3 containing 7 (80 mg, 244 µmol), DCC (50 mg, 243 µmol), and N-hydroxysuccinimide (5 mg, 43 µmol). The reaction was carried out overnight under argon at room temperature. Thin layer chromatography revealed a complete conversion of DPPE to a less polar product (Rf 0.6; CH2Cl2/CH3OH/H2O, 65:25:4). Solvents were then removed under reduced pressure and the reaction products redissolved in CHCl3. The organic phase was extracted twice with 1% NaCl to remove water soluble byproducts. The title compound was purified by chromatography on a 10 mL silica gel (Bio-Sil,HA) column that had been activated (150 °C, overnight) and prewashed with 100 mL of CHCl3. The products were eluted with an additional 20 mL of CHCl3 followed by 20 mL of CHCl3/CH3OH (40:1, 30:1, 25:1, 20:1, and 15:1) and 100 mL of CHCl3/CH3OH (10:1) and finally with 100 mL of CHCl3/CH3OH (10:2). The phosphate-containing fractions eluting with 10:2 CHCl3/CH3OH were concentrated under reduced pressure, affording a colorless oil (60 mg, 42% yield). Analysis by TLC (Rf 0.6; CH2Cl2/ CH3OH/H2O, 65:25:4) indicated a single phosphatepositive and ninhydrin-negative spot: 1H-NMR (CDCl3/ CD3OD, 1:1) δ 5.30-5.20 (m, 1H, CHCH2OP), 4.44-4.12 (m, 2H, CH2CHCH2OP), 4.10-3.90 (m, 6H, CHCH2OP, POCH2CH2N, COCH2O), 3.85 (s, 2H, NCOCH2Br), 3.723.38 (m, 14H, POCH2CH2N, OCH2CH2), 2.34-2.29 (m, 4H, CH2CO2), 1.70-1.50 (m, 4H, CH2CH2CO2), 1.28 (m, 48H, (CH2)24), 0.89 (t, J ) 6.6 Hz, 6H, CH3); MS (FAB+) m/z 1001.1 (M + H+), 1023 (M + Na+), 1039 (M + K+). [2-[2-(2,5-Dioxo-2,5-dihydropyrrol-1-yl)ethoxy]ethoxy]acetic Acid (11). [2-(2-Aminoethoxy)ethoxy]acetic acid (10) (760 mg, 4.55 mmol) in solution in 20 mL of a saturated solution of NaHCO3 was treated, at 0 °C under vigorous stirring, with finely ground 9 (723 mg, 4.66 mmol). After 5 min, the mixture was diluted with 75 mL of water and stirred at 20 °C for 35 min, after which the pH was brought to 6 with concentrated sulfuric acid. The reaction mixture was reduced to 20 mL by evaporation, acidified to pH 1-2 with 1 M sulfuric acid, and extracted 8-fold with 100 mL of EtOAc/HOAc (95:5). The organic phase was dried on MgSO4 and evaporated to dryness under reduced pressure. The residue was purified on a silica gel column eluted with CHCl3/ethanol/HOAc (60: 28:2). It afforded a yellowish solid (790 mg, 70% yield): Rf 0.44 (CHCl3/CH3OH/HOAc, 70:28:2); mp 65-67 °C; 1H-NMR (CDCl ) δ 6.73 (s, 2H, CHCH), 4.11 (s, 2H, 3 CH2CO2), 3.79-3.66 (m, 8H, CH2CH2O); HRMS (FAB+) m/z calcd for C10H14N1O6 (M + H+) 244.0821, found 244.0820. Hexadecanoic Acid 3-[[[2-[2-[2-[2-(2,5-Dioxo-2,5dihydropyrrol-1-yl)ethoxy]ethoxy]acetylamino]ethoxy]hydroxyphosphoryl]oxy]-2-(hexadecanoyloxy)propyl Ester (12). To a solution of DPPE (20 mg, 28 µmol) in 4 mL of anhydrous CHCl3 (40 °C) were added 11 (5 mg, 20 µmol), DCC (4.5 mg, 22 µmol), and Nethyldiisopropylamine (5 µL, 29 µmol). The reaction mixture was left under argon at room temperature for 15 h. N,N′-dicyclohexylurea was removed by filtration, and the organic phase was extracted twice with 1% NaCl. The title compound was purified by chromatography on a 10 mL silica gel (Bio-Sil,HA) column that had been activated (150 °C, overnight) and prewashed with 100 mL of CHCl3. The column was washed with an additional volume of 20 mL of CHCl3 followed by 20 mL of CHCl3/CH3OH mixtures (40:1, 30:1, 25:1, 20:1, and 15: 1) and finally with 60 mL of 10:1 CHCl3/CH3OH. Analysis by TLC (CHCl3/CH3OH/HOAc, 60:20:3) indicated a single phosphate-positive and ninhydrin-negative spot for the fractions eluting with 15:1 and 10:1 CHCl3/CH3OH.

Frisch et al.

These fractions were pooled and concentrated under reduced pressure to afford a yellowish oil (10 mg, 54% yield): Rf 0.67 (CHCl3/CH3OH/HOAc, 60:20:3); 1H-NMR (CDCl3/CD3OD, 1:1) δ 6.78 (s, 2H, COCHCHCO), 5.305.20 (m, 1H, CH2CHCH2), 4.59-4.15 (m, 2H, CH2CHCH2OP), 3.99 (s, 2H, NCOCH2O), 3.74-3.66 (m, 8H, CH2CH2O), 2.36-2.28 (m, 4H, CH2CO2), 1.65-1.57 (m, 4H, CH2CH2CO2), 1.25 (m, 48H, (CH2)12), 0.89 (t, J ) 6.0 Hz, 6H, CH3); MS (FAB+) m/z 936.6 (M + Na+). [2-(2-Chloroethoxy)ethoxy]acetic Acid (14). To a solution of [2-(2-chloroethoxy)ethoxy]ethanol (13) (15 g, 89 mmol) in 500 mL of acetone was added dropwise over 15 min, and under stirring, 45 mL of 2.67 M Jones reagent (see above). The solution was allowed to stir for an additional 30 min period, after which a few drops of 2-propanol were added followed by 300 mL of water. The aqueous phase obtained after evaporation of the organic layer was added to 150 mL of a saturated NaCl solution and extracted seven times with 200 mL of CHCl3. The combined extracts were dried on MgSO4 and evaporated to dryness. The residue was chromatographed on a silica gel column eluted with CHCl3/ethanol/HOAc (30:5:3), and a colorless oil was obtained (10.5 g, 64% yield): Rf 0.46 (CH2Cl2/CH3OH/HOAc, 25:1:1); 1H-NMR (CDCl3) δ 10.11 (s, 1H, OH), 4.18 (s, 2H, OCH2CO2), 3.82-3.58 (m, 8H, CH2CH2O). [2-(2-Mercaptoethoxy)ethoxy]acetic Acid (15). A solution of 14 (1.56 g, 8.6 mmol) and thiourea (2.7 g, 35 mmol) in 23 mL of water was heated under reflux for 4 h. After the addition of 30 mL of 2.5 M NaOH, the heating was resumed for an additional period of 4 h. After the mixture was cooled at room temperature, the pH was adjusted to 1 with concentrated HCl. The resulting solution was extracted seven times with 20 mL of CHCl3. The combined extracts were dried on MgSO4 and evaporated to dryness, leaving an oil which was chromatographed on an aluminum oxide column. The products were eluted with CH2Cl2/ethanol/HOAc (60:40:5). The title compound was obtained as a colorless oil (1.17 g, 75% yield): Rf 0.23 (CH2Cl2:ethanol, 3:2); 1H-NMR (CD3OD) δ 4.19 (s, 2H, OCH2CO2), 3.77-3.61 (m, 6H, CH2O, OCH2), 2.70 (t, J ) 6.5 Hz, 2H, SCH2). [2-[2-(Pyridin-2-yldisulfanyl)ethoxy]ethoxy]acetic Acid (16). 2,2′-Dipyridyl disulfide (2.7 g, 12.4 mmol) was dissolved in 15 mL of absolute ethanol containing 0.5 mL of HOAc, and 15 (1.12 g, 6.2 mmol) was added dropwise over 10 min. The mixture was stirred at room temperature under nitrogen for 24 h, and the solvent was evaporated under reduced pressure. The residue was chromatographed on a column of active aluminum oxide (activated at 150 °C overnight). The byproducts were eliminated with CH2Cl2/ethanol (60:40), and the desired reaction product was eluted with CH2Cl2/ethanol/HOAc (60:40:4). The presence of the dithiopyridine bond was assessed by a spectrophotometrical analysis. Aliquots (0.1 mL) of the chromatographic fractions were mixed with 3 mL of 0.1 M NaHCO3 containing a small amount of dithiothreitol, and the formed 2-thiopyridone was detected by its absorbance at 343 nm. The 2-thiopyridone-generating fractions were pooled and concentrated under reduced pressure, affording a colorless oil (740 mg, 50% yield): Rf 0.3 (CH2Cl2/ethanol, 3:2); 1H-NMR (CDCl3) δ 8.94 (s, 1H, CO2H), 8.47 (d, J ) 4.3 Hz, 1H, CHN), 7.79-7.63 (m, 2H, CHCHCHN), 7.11 (t, J ) 5.8 Hz, 1H, NCCH), 4.15 (s, 2H, OCH2CO2), 3.76-3.61 (m, 6H, CH2OCH2CH2O), 2.99 (t, J ) 6.2 Hz, 2H, CH2SS); HRMS (FAB+) m/z calcd for C11H16O4NS2 (M + H+) 290.0520, found 290.0517. Hexadecanoic Acid 2-(Hexadecanoyloxy)-3-[[hydroxy[2-[2-[2-[2-(pyridin-2-yldisulfanyl)ethoxy]eth-



oxy]acetylamino]ethoxy]hydroxyphosphoryl]oxy]propyl Ester (17). A solution of 16 (40 mg, 138.4 µmol) and BOP (73 mg, 165 µmol) in 5 mL of anhydrous CHCl3 was prepared at room temperature. After 30 min, 100 mg (144.5 µmol) of DPPE and 50 mL (276 µmol) of diisopropylethylamine, previously dissolved at 40 °C in 5 mL of anhydrous CHCl3, was added. The reaction mixture was stirred at room temperature for 24 h. The volume was then adjusted to 30 mL with CHCl3 and the organic phase washed successively four times with 20 mL of 1% citric acid and three times with 20 mL of water. Finally, the organic extract was dried on MgSO4 and evaporated to dryness. The residue was purified on a silica gel column eluted with CHCl3/CH3OH/HOAc (30: 5:1). The title product was dissolved in tert-butanol/ water (95:5) and was recovered after lyophilization as a white solid (84.7 mg, 64% yield): Rf 0.67 (CHCl3/CH3OH/ HOAc, 60:20:3); 1H-NMR (CDCl3/CD3OD, 1:1) δ 8.42 (d, J ) 4.8 Hz, 1H, CHNC), 7.85-7.74 (m, 2H, CHCHCHNC), 7.23 (t, J ) 5.9 Hz, 1H, NCCH), 5.30-5.20 (m, 1H, CHCH2OP), 4.44-4.12 (m, 2H, CH2CHCH2OP), 4.04 (s, 2H, NCOCH2O), 4.03-3.96 (m, 4H, CH2OPOCH2), 3.803.65 (m, 6H, CH2CH2OCH2), 3.54 (t, J ) 6.0 Hz, 2H, CH2NHCO), 3.05 (t, J ) 6.0 Hz, 2H, CH2SS), 2.36-2.29 (m, 4H, CH2CH2CO2), 1.65-1.56 (m, 4H, CH2CH2CO2), 1.28 (m, 48H, (CH2)24), 0.89 (t, J ) 6.6 Hz, 6H, CH3); MS (FAB-) m/z 961.4 (M - H+). Rate of Reaction of Thiol-Reactive Functions in 8, 12, and 17 with the Peptide CGIRGERA. Solutions of 8, 12, and 17 (100 nmol) in CHCl3 were evaporated under vacuum. The resulting lipidic film was resuspended, at room temperature, by sonication (2 min in a sonicator bath) in 800 µL of 50 mM sodium phosphate/ citric acid (pH 5.0, 6.0, and 7.0) or sodium borate (pH 8.0 and 9.0) buffers containing 5% (v:v) methanol. The suspensions were then put in a bath thermostated at 25 °C, and at time zero, CGIRGERA (200 nmol in 8.6 µL of water) was added. For 8 and 12, reaction progress was measured at given times by determination of residual thiols (Riddles et al., 1979) on 100 µL aliquots with 1 mL of Ellman’s reagent [2 mM DTNB and 1 mM ethylenediaminetetraacetic acid (EDTA) in 0.2 M sodium phosphate buffer (pH 7.27). The absorbance was read at 412 nm ( ) 14 150 M-1 cm-1). For 17, reaction progress was measured spectrophotometrically at 343 nm by quantification of the release of 2-thiopyridone ( ) 8080 M-1 cm-1) (Carlsson et al., 1978).

Scheme 1. Synthesis of Compound 7a

a Key: (a) triethylamine, methanesulfonyl chloride, THF, overnight, RT; (b) NaN3, CH3CN, reflux 36 h; (c) triphenylphosphine, H2O, THF, 10 h, RT; (d) CHCl3, overnight, RT; (e) Jones reagent, acetone, 1 h, RT.


a

Key: (a) NaHCO3, H2O, 5 min 0 °C; H2O, 35 min, 20 °C.


RESULTS AND DISCUSSION

The thiol-reactive heterobifunctional reagents 7, 11, and 16 (Schemes 1-3) were synthesized according to methodologies outlined in this paper. They contain flexible di- or tri(ethylene glycol) spacer arms and bear maleimide, bromoacetyl, or dithiopyridine moieties whose reactivities with the terminal cysteine of a model octapeptide were determined to evaluate the best coupling conditions. To investigate the immunogenicity of their spacer arms, these reagents were also conjugated, via their carboxylic function, to DPPE to yield respectively compounds 8, 12, and 17 (Figure 2) that could be incorporated into liposomes for immunological studies (Boeckler et al., submitted for publication). Synthesis of Compounds 7 and 8. Commercial tetra(ethylene glycol) was converted into monomesylate derivative 2 (Scheme 1) which in turn was converted into the azide 3 by reaction with sodium azide in acetonitrile under reflux. Compound 3 was conveniently reduced into the primary amine 4 with triphenylphosphine in the presence of water (Gololobov et al., 1981). A first attempt to transform 4 into 7 by oxidation of the alcohol function

a Key: (a) Jones reagent, acetone, 30 min, RT; (b) thiourea, H2O, reflux 4 h; (c) NaOH (2.5 M), reflux 4 h; (d) 2,2′-dipyridyl disulfide, ethanol, EtOAc, 24 h, RT.

into a carboxylic group followed by introduction of the bromoacetamide moiety proved unsuccessful. This was due to the very poor solubility of the intermediary amino acid in organic solvents and to side reactions with the carboxylic group in the last reaction. Therefore, 4 was first transformed, in good yields, into 6 by reaction with the N-hydroxysuccinimidyl ester of bromoacetate (5; Bernatowicz and Matsueda, 1986). Finally, the heterobifunctional reagent 7 was obtained by oxidation of the alcohol function with Jones reagent. In a final step, we wanted to activate the carboxylic function of 7 by formation of N-hydroxysuccinimidyl or p-nitrophenyl esters. Such esters proved to be very unstable and therefore difficult to purify. This is attributable to the polarity of the spacer arm which requires fairly polar solvents as eluents, e.g. methanol on silica gel and water on reverse

184 Bioconjugate Chem., Vol. 7, No. 2, 1996

Frisch et al.

Figure 2. Structure of the thiol-reactive DPPE derivatives 8, 12, and 17 and their coupling to the model peptide CG-IRGERA.

phase media, which are detrimental to these esters. Moreover, the intrinsic reactivity of polyoxyethylene succinimidyl esters has been documented, and the carboxymethyl-NHS derivatives were found to be the least stable (Shearwater, 1995). Compound 8 was readily obtained by reaction of the carboxylic function of 7 with the free amine of DPPE in the presence of DCC and N-hydroxysuccinimide. The latter reagent assists in the formation of the amide bond (Weygand et al., 1966; Mattson et al., 1993). Compound 8 was purified by chromatography on BioSil,HA; it is stable for several months when stored dry at -20 °C under argon. Synthesis of Compounds 11 and 12. The synthesis of compound 11 is outlined in Scheme 2. The known amino acid 10 was prepared according to Slama and Rando (1980), and to introduce the maleimide moiety, a variety of methods were tested without success. For example, the classical reaction of the free amine with maleic anhydride (Coleman et al., 1958) which should produce the maleimide after the elimination of a water molecule, e.g. in the presence of toluene in a Dean-Stark apparatus (Rich et al., 1975) or with anhydrous sodium acetate in the presence of acetic anhydride (Witter and Tuppy, 1960), gave very poor yields. Similar results were obtained with other compounds that also contained a carboxylic function (Keller and Rudinger, 1975). The best method was that of Keller and Rudinger (1975) which makes use of 2,5-dioxo-2,5-dihydropyrrole-1-carboxylic acid methyl ester (9). This reaction of 10 with this maleimide donor must be carefully controlled. The conversion of the reaction intermediate into 11 is very dependent on pH, temperature, and time. At pH 8.5, in the presence of bicarbonate ions which catalyze the cyclization reaction, the formation of the maleimide is in competition with its own hydrolysis. The optimal experimental conditions were found by monitoring spectrophotometrically the formation of the maleimide at 297 nm (this peak disappears in the presence of an excess of thiol). They involve two temperature stages (5 min at 0 °C followed by 35 min at 20 °C) and a final acidification of the reaction medium (pH 6) to stabilize the maleimide moiety. Under these conditions, compound 11 was obtained with an average yield of 70%. For the same reasons as above, the preparation of the N-hydroxysuccinimidyl ester of 11 failed and 12 was prepared directly by reaction with DPPE in the presence of DCC and N-hydroxysuccinimide. Preparation of Compounds 16 and 17. Commercial compound 13 (Scheme 3) was oxidized with Jones

reagent to give 14 in good yields. The introduction of the thiol group (compound 15) was not as straightforward as anticipated. Thus, reaction of 14 with NaSH gave poor yields. We have therefore adapted a reaction which is classically used for the preparation of e.g. S-glycosides (Chipowsky and Lee, 1973). The chloro bond in 14 was reacted with thiourea to give an intermediary isothiourea which was directly hydrolyzed, under basic conditions, into a free thiol. Compound 15 was purified by chromatography through basic aluminum oxide. The heterobifunctional reagent 16 was finally obtained by derivatization with 2,2′-dipyridyl disulfide, according to Carlsson et al. (1978); this reaction was followed spectrophotometrically by measuring the formation of 2-thiopyridone. The formation of the derivative 17, by reaction of DPPE with 16 in the presence of DCC, gave many side products that were difficult to eliminate during the purification step. We therefore used BOP, a reagent frequently used in peptide synthesis (Castro et al., 1975), as the coupling agent. The advantage of this approach was the relatively facile elimination of excess reagent and secondary products by extraction. Reactivity of the Thiol-Reactive Moieties in 8, 12, and 17 with the Cysteine Residue of a Model Peptide CGIRGERA. The aim of our study is to replace MPB-PE in our liposomal construct (see Introduction) with these newly synthesized thiol-reactive DPPE derivatives. To conjugate peptides to the surface of such preformed vesicles, it was important to define the experimental conditions (time and pH) which give quantitative coupling yields. We have therefore studied the rate of reaction of 8, 12, and 17 with CG-IRGERA, the model peptide we have been using for optimizing our immunization system (see Introduction) and which carries at the N-terminus a cysteine residue (Figure 2). The reactions were carried out at 25 °C and at fixed pH values, with a molar ratio of 2 in favor of the peptide. Reaction progress for 8 and 12 (i.e. formation of the thio ether derivatives) was determined by measuring residual free thiols with Ellman’s reagent, whereas for 17, the thiol exchange reaction was assessed by following spectrophotometrically the formation of 2-thiopyridone. Controls, under these different reaction conditions, allowed us to determine the spontaneous oxidation of CG-IRGERA (e.g. dimerization) and to correct the experimental data. The reaction progress curves are given in Figure 3A-C. As expected, the maleimide moiety of 12 (Figure 3B) reacted rapidly with the peptide, especially at basic pH due to great reactivity of the thiolate group (pKa thiol



i.e. on the length and the flexibility of the spacer arms (Figure 1), we have measured the distances between the carbon atom of the amide group of the DPPE derivatives 8, 12, 17, and MPB-PE and the sulfur atom of the conjugated peptide. In extended conformations, as calculated with the Personal CAChe program, the following distances, in angstroms, were found: 8, 16.1; 12, 13.1; 17, 11.3; and MPB-PE, 12.5. CONCLUSION

Figure 3. Rate of reaction between CG-IRGERA and the thiolreactive DPPE derivatives 8 (A), 12 (B), and 17 (C), at different pH values. The coupling reactions were performed at 25 °C in the presence of a 2-fold molar excess of peptide (see text), and reaction progress was determined by measuring residual thiols with Ellman’s reagent (A and B) or by following the formation of 2-thiopyridone (C).

of cysteine ≈ 8.5); however even at pH 6.0, which preserves the maleimide group, the coupling was essentially quantitative after 2 h. It should be noted that at pH 9.0 the reaction of the maleimide moiety with CGIRGERA is fast enough to keep its hydrolysis by hydroxyl ions marginal. It follows that, similar to MPBPE (Martin and Papahadjopoulos, 1982), the coupling of the thiol-bearing peptide to 12 can be obtained in excellent yields under mild (neutral) pH and temperature conditions, making this PE derivative suitable for our synthetic vaccine program. Comparatively, the reactivity of the bromoacetyl moiety of 8 was less pronounced (Figure 3A). Whereas a quantitative conjugation was observed at pH 9.0 within 50 min of reaction, at lower pH values, the coupling was found more sluggish. The thiol exchange reaction between the peptide and compound 17 proceeded smoothly and was essentially quantitative within a pH range from 6 to 9 (Figure 3C). The driving force here is the release of 2-thiopyridone. This coupling reaction can therefore be performed using similar experimental conditions as given above for the coupling to a maleimido group. Size of the Spacer in Peptide Derivatives of 8, 12, and 17. Since the immune response against the peptides conjugated to the surface liposomes could, in principle, depend on the separation of the peptide from its carrier,

Three new thiol-reactive polyoxyethylene-based heterobifunctional reagents were synthesized with the aim of providing spacer arms of relatively low immunogenicity in liposomal constructs used for eliciting an immune response against synthetic peptides. After coupling to DPPE, they allowed the conjugation, under defined conditions, of synthetic peptides e.g. to the surface of preformed liposomes (Frisch et al., 1991) via spacers of variable lengths. Moreover, two types of linkages between the peptides, which carry a cysteine residue, and the vesicles were introduced: i.e. stable thio ether and bioreducible disulfide bonds which might be of importance in the processing of such constructs by immunocompetent cells. As described elsewhere (Boeckler et al., submitted for publication), an important immune response was obtained against IRGERA, the model hexapeptide used as the immunogen (Figure 1), which did not depend on the length of the spacer or on the linkage type. The two spacer arms in 8 and 17 were also found to be less immunogenic than the one introduced with the classical MPB-PE derivative. These new heterobifunctional reagents should also find applications in other fields; for example, we have used this approach in the design of multiantennary glycoconjugates (Kichler and Schuber, 1995), and their corresponding phospholipid derivatives were found to strongly enhance gene delivery with neutral complexes of lipospermine (Kichler et al., 1995). LITERATURE CITED Arnon, R., and Horwitz, R. J. (1992) Synthetic peptides as vaccines. Curr. Opin. Immunol. 4, 449-453. Bernatowicz, M. S., and Matsueda, G. R. (1986) Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent. Anal. Biochem. 155, 95-102. Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. N-succinimidyl 3-(2-pyridyldithio) propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-734. Chipowsky, S., and Lee, Y. C. (1973) Synthesis of 1-thioaldosides having an amino group at the aglycon terminal. Carbohydr. Res. 31, 339-346. Coleman, L. E., Bork, J. F., and Dunn, H. (1959) Reaction of primary aliphatic amines with maleic anhydride. J. Org. Chem. 24, 135-136. Defoort, J. P., Nardelli, B., Huang, W., and Tam, J. P. (1992) A rational design of synthetic peptide vaccine with built-inadjuvant. Int. J. Pept. Protein Res. 40, 214-221. Friede, M. (1995) Liposomes as carriers of antigens. In Liposomes as tools in basic research and industry (J. R. Philippot and F. Schuber, Eds.) pp. 189-199, CRC Press Inc., Boca Raton, FL. Friede, M., Muller, S., Briand, J.-P., Van Regenmortel, M. H. V., and Schuber, F. (1993) Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing Monophosphoryl Lipid A. Mol. Immunol. 30, 539-547. Friede, M., Muller, S., Briand, J.-P., Plaue´, S., Fernandes, I., Frisch, B., Schuber, F., and Van Regenmortel, M. H. V. (1994) Selective induction of protection against influenza virus

186 Bioconjugate Chem., Vol. 7, No. 2, 1996 infection in mice by a lipid-peptide conjugate delivered in liposomes. Vaccine 12, 791-797. Frisch, B., Muller, S., Briand, J.-P., Van Regenmortel, M. H. V., and Schuber, F. (1991) Parameters affecting the immunogenicity of liposome-associated synthetic peptide antigen. Eur. J. Immunol. 21, 185-193. Gololobov, Y. U., Zhmurova, I. N., and Kasukhin, L. F. (1981) Sixty years of Staudinger reaction. Tetrahedron 37, 437-472. Gregoriadis, G. (1994a) The immunological adjuvant and vaccine carrier. Properties of liposomes. J. Drug Targeting 2, 351356. Gregoriadis, G. (1994b) Liposomes as immunoadjuvants and vaccine carriers: antigen entrapment. Immunomethods 4, 210-216. Gregory, J. D. (1955) The stability of N-ethylmaleimide and its reaction with sulfhydryl groups. J. Am. Chem. Soc. 77, 39223923. Jones, G. L., Edmundson, H. M., Spencer, L., Gale, J., and Saul, A. (1989) The use of maleimidocaproyloxysuccinimide to prepare malarial peptide carrier immunogens. J. Immunol. Methods 123, 211-216. Keller, O., and Rudinger, J. (1975) Preparation and some properties of maleimido acids and maleoyl derivatives of peptides. Helv. Chim. Acta 58, 531-541. Kichler, A., and Schuber, F. (1995) Versatile synthesis of biand tri-antennary galactose ligands: interaction with the Gal/ GalNAc receptor of human cells. Glycoconjugate J. 12, 275281. Kichler, A., Remy, J.-S., Boussif, O., Frisch, B., Boeckler, C., Behr, J.-P., and Schuber, F. (1995) Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids. Biochem. Biophys. Res. Commun. 209, 444450. Klausner, Y. S., and Bodansky, M. (1972) Coupling reagents in peptide synthesis. Synthesis, 453-463. Lo¨vgren, K., and Larsson, M. (1994) Conjugaison of synthetic peptides to carriers Iscoms: affecting the immunogenicity of the conjugate. J. Immunol. Methods 173, 237-243. Martin, F. J., and Papahadjopoulos, D. (1982) Irreversible coupling of immunoglobulin fragments to preformed vesicles. J. Biol. Chem. 257, 286-288.

Frisch et al. Martin, F. J., Heath, T. D., and New, R. R. C. (1989) Covalent attachment of proteins to liposomes. In Liposomes a practical approach (R. R. C. New, Ed.) Chapter 4, pp 163-182, IRL Press, Oxford University Press, Oxford, England. Mattson, G., Conklin, E., Desai, S., Nielander, G., Savage, M. D., and Morgensen, S. (1993) A practical approach to crosslinking. Mol. Biol. Rep. 17, 167-183. Muller, S. (1988) Peptide-carrier conjugation. In Synthetic polypeptides as antigens (M. H. V. Van Regenmortel, J. P. Briand, S. Muller, and S. Plaue´, Eds.) Vol. 19, pp 95-127, Elsevier, Amsterdam, The Netherlands. Peeters, J. M., Hazendonk, T. G., Beuvery, E. C., and Tesser, G. I. (1989) Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moities on the immunogenicity of the conjugates. J. Immunol. Methods 120, 133-143. Rich, D. H., Gesellchen, P. D., Tong, A., Cheung, A., and Buchner, C. K. (1975) Alkylating derivatives of amino acids and peptides. J. Med. Chem. 18, 1004-1010. Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5,5′-dithiobis(2-nitrobenzoic acid) a reexamination. Anal. Biochem. 94, 75-81. Shearwater Polymers Inc. Catalog (1995) March issue pp 4445, Huntsville, AL. Slama, J. S., and Rando, R. R. (1980) Lectin-mediated aggregation of liposomes containing glycolipids with variable hydrophilic spacer arms. Biochemistry 19, 4595-4600. Weygand, F., Hoffmann, D., and Wu¨nsch, E. (1966) Peptidsynthesen mit Dicyclohexylcarbodiimid unter Zusatz von NHydroxysuccinimid. Z. Naturforsch. 21B, 426-428. Witter, A., and Tuppy, H. (1960) N-(4-dimethylamino-2,5dinitrophenyl)maleimide: a coloured sulfhydryl reagent. Biochim. Biophys. Acta 45, 429-442. Zalipsky, S. (1995) Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates. Bioconjugate Chem. 6, 150-165.

BC950092V

Synthesis of Short Polyoxyethylene-Based Heterobifunctional Cross

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