842
Bioconjugate Chem. 1998, 9, 842−846
Convenient Polymer-Supported Synthetic Route to Heterobifunctional Polyethylene Glycols Thierry Bettinger, Jean-Serge Remy, Patrick Erbacher, and Jean-Paul Behr* Laboratoire de Chimie Ge´ne´tique associe´ CNRS/Universite´ Louis Pasteur de Strasbourg, UMR 7514, Faculte´ de Pharmacie, BP 24, 67401 Illkirch Cedex, France. Received April 12, 1998; Revised Manuscript Received June 22, 1998
Conventional synthesis of heterobifunctional poly(ethylene glycol) derivatives, especially of medium size, is a rather tedious task. A straightforward solid-phase methodology has been developed that is illustrated here by the synthesis of R-pyridyldithio-ω-hydroxy-poly(ethylene glycol)600. This derivative was prepared from resin-bound PEG600 with a global yield of 65% for 6 individual steps, i.e., with an average yield of 93%/step. Intermediate purification steps simply consisted of resin washing. Progress of each reaction toward completion could conveniently be monitored by 13C NMR of the resin-bound PEG derivatives. This example highlights both the versatility and efficiency of combining polymersupported synthesis with direct 13C-NMR characterization of the intermediate compounds.
INTRODUCTION
Poly(ethylene glycol) (PEG)1 conjugation chemistry has been developed recently as a consequence of the potential therapeutic interest of pegylated drugs (Herman et al., 1995; Zalipsky, 1995). Indeed it is now demonstrated that PEG-modified biological molecules can benefit from extented plasma lifetimes, from reduced uptake by the reticuloendothelial system (Allen, 1994; Papahadjopoulos et al., 1991; Woodle and Lasic, 1992) and more generally from a decrease of the undesired consequences of electrostatic and van der Waals interactions. Following the synthesis of PEG-protein conjugates, new developments have emerged such as nonimmunogenic drugs and nucleic acid vectors. One of the future directions toward nonviral gene therapy is the use of PEG-grafted synthetic vectors as long circulating carriers for receptor-mediated gene delivery (Blume et al., 1993; Maruyama et al., 1995). In this context, heterobifunctional poly(ethylene glycol) linkers are required that allow attachment of ligands to one extremity of PEG-grafted synthetic vectors. Protein modification with PEG does not require true heterobifunctional PEG derivatives. Methoxy-PEG (mPEG), carrying an inert ether end group, was used to make the chemical processing easier (Greenwald et al., 1996; Herman et al., 1995; Zalipsky, 1995). The chemistry of heterobifunctional PEG derivatives is not well developed, reaction yields are far from being quantitative, hence, cumbersome polymer purification techniques have to be used (Zalipsky, 1993). Most published procedures use classical derivatization chemistry in homogeneous * Author to whom correspondence should be addressed. Phone: 33(0)388 676983. Fax: 33(0)388 678891. E-mail:
[email protected]. 1Abbreviations: PEG 600, poly(ethylene glycol) MW 600; HOPEG600-SSPy R-hydroxy-ω-pyridyldithio-poly(ethylene glycol)600; HO-PEG600-COOH, R-hydroxy-ω-carboxy-poly(ethylene glycol)600; CH3OH, methanol; CH2Cl2, dichloromethane; DMF, N,Ndimethylformamide; THF, tetrahydrofurane; Et3N,N,N,N-triethylamine; TFA, trifluoroacetic acid; DCC, N,N′-dicyclohexylcarbodiimide; DCU, dicyclohexylurea; DTT, 1-4-dithio-Lthreitol; NHS, N-hydroxysuccinimide; Tos-Cl, toluene-4-sulfonyl chloride; TLC, thin layer chromatography.
solution (sometimes even in potentially reactive aqueous medium), and often the functionalized PEG cannot be fully separated from the starting polymer (Zalipsky et al., 1994). Another possibility is to choose suitable initiator and/or terminator reagents for the polymerization of ethylene oxide (Yokoyama et al., 1992). However, recent work showed that such strategies may be obscured by diol impurities (Nagasaki et al., 1997), and the polymerization process still remains difficult to control (ethylene oxide is toxic and explosive). In another register, poly(ethylene glycol) is also being widely used in solid-phase synthesis (Bayer, 1991). Indeed, improvement of polymeric supports for peptide synthesis has led to using PEG as a good solvent-swelling linker between the inert resin and the solid-supported reaction center. The PEG linker mobility is high (Bayer, 1990) and peptide yields are good, showing it to be possible to combine the advantages of a solid support with those of the liquid-phase methodology. According to this background, we decided to develop a new strategy for heterobifunctionalization of poly(ethylene glycol) based on solid-phase chemistry. A major aspect of PEG functionalization lies in the proper characterization of intermediate and final products. We used gel-phase 13C NMR which allows convenient and nondestructive monitoring of reaction progress directly on the resin (Bayer, 1990). This is possible because of the rather long 13C relaxation times which are a consequence of the high mobility of the fully solvated PEG (Bayer, 1990). The method is sensitive and allows us to accurately determine the end point of each reaction step (among others, the terminal carbons of PEG usually exhibit characteristic chemical shifts). To demonstrate the interest of this strategy, we have prepared a low molecular weight PEG derivative (MW 600), which is known to be difficult to purify by size exclusion chromatography or diethyl ether precipitation. Such small PEGs can act as very effective molecular scale filters for preventing binding to underlying liposome or particle surfaces (Needham et al., 1997). PEG was functionalized with a 2-pyridyldithio residue which is widely used for specific coupling with thiol groups (Carlsson et al., 1978; Woghiren et al., 1993) since coupling
10.1021/bc980039h CCC: $15.00 © 1998 American Chemical Society Published on Web 08/20/1998
Synthesis of Heterobifunctional Polyethylene Glycols Table 1.
13C-Nuclear
Bioconjugate Chem., Vol. 9, No. 6, 1998 843
Magnetic Resonance Peaks of Polymer-Grafted Poly(ethylene Glycol) Derivatives structure
(δ)a (ppm)
resin-PEG-O-CH2CH2-OTos resin-PEG-O-CH2CH2-S(CdS)O-CH2CH3b resin-PEG-O-CH2CH2-SH resin-PEG-O-CH2CH2-S-S-Pyridinec
69.3, 68.7, 145, 129.7, 128.1, 130.1, 21.8 69.8, 35.2, nd, 68.7, 13.7 72.6, 24 68.7, 38.2, nd, 119.4, 136.9, 120.4, 149.2
a The polymer-bound derivative (150 mg) was added to CDCl (0.5 mL). Chemical shifts were normalized relative to CDCl ) 77.0 ppm. 3 3 The large poly(ethylene glycol) backbone peak was at 70.3 ( 0.3 ppm. Chemical shifts are listed according to the structures shown in Figure 1. b The SCdS resonance was not observed. c The SCNCH resonance was not observed.
releases a chromophore which allows straightforward reaction monitoring. We further activated the HOPEG600-SSPy as the N-hydroxysuccinimidyl carbonate derivative (Zalipsky et al., 1992); coupling of this compound with an amine to give a carbamate led to higher yields than classical amide bond formation. EXPERIMENTAL PROCEDURES
General. Aminomethylated polystyrene-1% divinylbenzene was purchased from Novabiochem (Meudon, France). Toluene-4-sulfonyl-chloride (TsCl), potassium O-ethyl dithiocarbonate, propylamine, and poly(ethylene glycol) (MW 600) were from Fluka (St Quentin Fallavier, France). 2,2′-Dipyridyl disulfide was obtained from Lancaster (Strasbourg, France). Thin layer chromatography (TLC) was performed on Kieselgel 60 F254 plates (Merck). Preparative chromatography was carried out on Chromatotron 8924 (Harrison Research). PEG was revealed by TLC by the Dragendorf assay (Thoma et al., 1964). The Kaiser qualitative ninhydrin test was carried out specifically as described elsewhere (Sarin et al., 1981). As a reaction vessel, we used a glass column ended with a join connection and a glass frit (porosity n°2) on the upper and lower end, respectively. This allowed us to stir the resin on a rotary evaporator and to wash the resin after each reaction step. Solid-phase reactions were carried out at room temperature. After each step, the resin was stirred (20 min) and rinsed successively with CH2Cl2 (5 × 50 mL) and CH3OH (5 × 50 mL). The resin was dried in vacuo to allow analytical analysis (5 mg was taken for IR analysis and 150 mg for 13C NMR).1H- and 13C-NMR spectra were recorded on a Bruker DPX300 Avance instrument at 300 MHz (scan number ) 1000 with classical acquisition parameters). Infrared spectra (IR, KBr pellet) were recorded on a spectrophotometer FT-IR 1600 (Perkin-Elmer). Synthesis of Crude r-Hydroxy-ω-Carboxy Poly(ethylene Glycol) (HO-PEG600-COOH) (1). Poly(ethylene glycol) (28 g, 46.7 mmol, 600 g/mol) was dissolved in 40 mL of dry CH2Cl2. To this solution was added 10 mL of dry THF containing diglycolic anhydride (1.02 g, 8.8 mmol) and pyridine (720 µL, 9 mmol). After stirring overnight under argon at room temperature, the reaction was completed as indicated by TLC (CH2Cl2/ MeOH/Et3N 90:10:1) (no anhydride left and detection of a single polar PEG-compound). The monoacid derivative of poly(ethylene glycol)600 was used without purification. PEG600 Grafting to the Resin via Amide Bond Formation (2). The above HO-PEG600-COOH solution (ca. 8.8 mmol) was activated with 1.2 molar equivalent of N-hydroxysuccinimide (1.22 g, 10.6 mmol) and N,N′dicyclohexylcarbodiimide (2.19 g, 10.6 mmol). The solution was stirred for 2 h at room temperature under argon. The dicyclohexylurea (DCU) suspension was filtered off. Aminomethylated poly(styrene-co-1% divinylbenzene) resin (1.1 g, 1.2 mmol of amino group) was poured into the glass column (see General) with the activated carboxylic acid solution. The column was connected to a rotary
evaporator and stirred for 2 days at room temperature. The solvent was then filtered off and the resin beads were washed as described above. Two additional washes with DMF/CH3OH (10/3) were necessary to remove the remaining DCU as checked by IR. Complete amino group capping was indicated by a negative Kaiser test. The resin was dried in vacuo for IR and 13C-NMR characterization. At this stage, the resin did not stick anymore to the glass wall. IR spectrum of the resin showed the characteristic absorption band of PEG ether backbone (1100 cm-1) and absorption bands at 1750 and 1680 cm-1 for the ester and amide bond, respectively (CdO sym stretch). Reaction of Polymer-Bound r-Hydroxy-Poly(ethylene Glycol)600 with Tosyl Chloride (3). Twentyfold molar excess of toluene-4-sulfonyl chloride (4.6 g, 24 mmol) and 3.34 mL of Et3N (24 mmol) in 15 mL of CH2Cl2 were added slowly to the resin suspended in 30 mL of CH2Cl2. The mixture was stirred overnight at room temperature while the resin turned brown-yellow. The solvent was filtered off and the resin was washed as described above. The resin became white again after these washes. The IR spectrum showed the characteristic sulfonyl group absorption at 1177 cm-1 (SO2, sym stretch) and did not show any residual hydroxyl absorption (3200-3600 cm-1) of the starting polymer. See 13CNMR data in Table 1. Reaction of the Polymer-Bound r-Tosyl-O-Poly(ethylene glycol)600 with Potassium O-Ethyl Dithiocarbonate (4). Potassium O-ethyl dithiocarbonate (212 mg, 1.32 mmol) in 25 mL of THF/DMF (50/1) was added to the polymer. The mixture was stirred for 4 h at room temperature. The solvent was filtered off, and the resin was washed. IR spectrum of the resin confirmed the disappearance of the tosyl group (1177 cm-1). See 13CNMR data in Table 1. Synthesis of Polymer-Bound r-Thiol-poly(ethylene Glycol)600 (5). Thirty-fold molar excess of propylamine (3 mL, 36 mmol) in 25 mL of CH2Cl2 was added to the column and stirred for 1 h. The solvent was filtered off and the resin was washed as above. 13C NMR confirmed that no disulfide bond formation (PEG-S-SPEG) had occurred. See 13C-NMR data in Table 1. Activation of Polymer-Bound r-Thiol-poly(ethylene Glycol)600 with 2,2′-Dipyridyl Disulfide (6). Five fold molar excess of 2,2′-dipyridyl-disulfide (1.32 g, 6 mmol) in 30 mL of CH2Cl2 was added to the column and stirred overnight at room temperature. The resin turned yellow. The solvent was filtered off, and the resin was washed while turning white. Again, no disulfide bond formation had occurred as indicated by 13C NMR. See 13C-NMR data in Table 1. Cleavage of r-Pyridyldithio-ω-hydroxy-poly(ethylene Glycol)600 from the Solid Support (7). A mixture of 30 mL of THF/CH3OH/Et3N (1/1/1) was added to the resin and stirred for 3 days at room temperature. The solution was filtered and the resin was washed as usual. The combined organic fractions were concentrated
844 Bioconjugate Chem., Vol. 9, No. 6, 1998 Scheme 1
in vacuo, and Et3N was removed by rough chromatography on SiO2 with CH2Cl2/CH3OH (20/1) as eluent. A brown oil was recovered (550 mg, 0.78 mmol), which was identified by TLC and characterized by 1H- and 13C-NMR spectroscopy. The global yield was 65% as determined by quantitative released of 2-thiopyridone upon reduction with DTT (Carlsson et al., 1978). Rf ) 0.45 (CH2Cl2/CH3OH 10:1). 1H NMR (CDCl3): δ 3.01 (t, SSCH2CH2O, 2H), 3.54 (s, PEG, 48H), 3.66 (t, SSCH2CH2O, 2H), 7.17 (m, SCCHCH, 1H), 7.75 (m,SCCHCHCH, 2H), 8.46 (m, SCNCH, 1H). 13C NMR (CDCl3): δ 38.4 (SSCH2CH2), 61.7 (OCH2CH2OH), 68.9 (SSCH2CH2), 70.5 (CH2CH2O of PEG backbone), 72.5 (OCH2CH2OH), 119.6 (SCCHCH), 120.6 (SCCHCHCH), 137.1 (SCCHCH) 149.5 (SCNCH). RESULTS AND DISCUSSION
Stepwise conversion of PEG600 to the thiol-activated HO-PEG600-SSPy (7) was carried out according to Scheme 1. To avoid a tedious PEG monofunctionalization/isolation procedure, diglycolic anhydride was reacted with a
Bettinger et al.
large excess of PEG. In such conditions, the only carboxylic acid formed should be the compound of interest. This compound can be selectively activated and grafted to the resin via amide bond formation. Resin washing should remove all other compounds. In practice, HOPEG600-COOH (1) was activated with N-hydroxysuccinimide (NHS) and N,N′-dicyclohexylcarbodiimide (DCC). The reaction mixture was filtered in order to remove most of the dicyclohexylurea (DCU) formed. After PEG grafting onto the resin, IR spectroscopy revealed some residual DCU sticking to the resin (broad absorption band at 1800-1600 cm-1). The solid support was therefore further washed with DMF/CH3OH (7/3). An extended reaction time was required for PEG grafting to the resin, which could be explained by the waxy constitution of PEG solution at this concentration (1 M). Complete reaction was confirmed by a negative Kaiser test. IR spectroscopy revealed the characteristic absorption bands for PEG (CH2-O-CH2, ether 1100 cm-1) as well as for the connecting ester and amide functions (1750 and 1680 cm-1, respectively, CdO sym stretch). The glycolic ester bond being labile, thiol group formation with strong nucleophiles such as alcoholates (Woghiren et al., 1993) had to be avoided. Thiol derivatization was achieved by a modification of the protocol reported by Zalipsky (Zalipsky et al., 1987) which used milder conditions compatible with an ester linkage. The primary hydroxyl group of PEG was activated with tosyl chloride in standard conditions, and the tosylated resin (3) showed an IR spectrum with the expected strong absorption at 1177 cm-1 (SO2). The 13C-NMR spectrum contained the characteristic tosyl group resonances at 21.8 ppm and between 128 and 145 ppm, which correspond to the methyl and aromatic ring carbon atoms, respectively (Figure 1). The broad signals around 130 and 40 ppm were due to the phenyl and aminoethyl groups of the solid support. At this stage, the activated resin-grafted PEG is easy to handle as solid beads and is stable upon long-term storage (at least 4 months at -20 °C). It may be regarded as a convenient starting material for the preparation of a variety of PEGs. TosylPEG600 was reacted with a stoichiometric amount of potassium O-ethyl dithiocarbonate to give the diester 4. Preliminary experiments using excess reagent were shown by 13C NMR and IR to lead to complete cleavage of the PEG from the resin. Compound 4 could not be characterized by IR spectroscopy. However, 13C-NMR spectrum confirmed complete removal of tosyl group (21.8 ppm and 128-145 ppm) and the appearance of two new resonances at 13.7 ppm (O-CH2-CH3) and 35.2 ppm (OCH2-CH2-S) (Figure 1). The thiocarbonate was aminolyzed, but instead of glycine, we used propylamine to remain in organic solution and avoid disulfide bond formation. Several attempts to cleave the thiocarbonate in mild basic solution (
