Bioconjugate Chem. 1997, 8, 793−797
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ARTICLES Synthesis and Characterization of High-Molecular Mass Polyethylene Glycol-Conjugated Oligonucleotides Gian Maria Bonora,*,† Eugenia Ivanova,‡ Valentina Zarytova,‡ Barbara Burcovich,§ and Francesco Maria Veronese§ Pharmaco-Chemico-Technology Department, Via Ospedale 72, University of Cagliari, 09124 Cagliari, Italy, Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia, and Department of Pharmaceutical Sciences, University of Padova, Padova, Italy. Received December 30, 1996X
The use of synthetic oligonucleotides as possible drugs for human therapy is usually hampered by their low in vivo stability and capacity to achieve high concentrations at their cellular targets. To overcome this, it has been suggested that they be modified chemically. Among the various options, conjugation with short- and long-chain polyethylene glycols (PEGs) has several advantages: PEG is nontoxic and very soluble, reduces immunogenic reactions, and increases the stability of the conjugated molecules. PEG is generally joined to oligonucleotides while bound to the insoluble solid-phase supports, after their synthesis, which does not allow for their being easy scaled up. The use of the liquid-phase approach as an alternative synthetic process, utilizing the PEG polymer both as a soluble, inert synthetic support and a stabilizing agent of the oligonucleotide, is proposed. A new protocol has been set up, characterized by a stable phosphate bond between the support and the oligonucleotide. This method has been tested on a 12mer previously investigated as an antisense agent against HIV. The PEG-conjugated 12mer was efficiently synthesized and purified. It was found that the highmolecular mass PEG chain results in enzymatic stability and does not interfere with the formation of the duplex with its nucleic acid target.
INTRODUCTION
The use of synthetic oligonucleotides as new therapeutic agents is currently of widespread interest (1). These molecules interact with either specific single-stranded RNA messengers, as antisense, (2) or double-stranded genomic DNA, as antigene (3), inhibiting the expression of pathogenic genetic messages. Moreover, these molecules have recently been investigated as new, specific ligands of non-nucleic acid receptors, such as extracellular proteins (4). The utilization of these molecules as new drugs for human therapy creates new problems related to their low in vivo stability and capacity for achieving high concentrations in the biological targets, since they are rapidly degraded by endogenous nucleases and exhibit reduced cellular uptake (5). Their chemical modification, mainly of the sugar-phosphate backbone, is commonly performed (6) to avoid recognition by degradative enzymes and, by increasing their lipophilicity, to enhance cellular penetration. However, these modified oligonucleotides have some disadvantages such as low solubility in a physiological media, toxicity of their metabolites, or inhibition of the degradative process of duplexes brought about by RNase H. Another solution considers the conjugation of the oligonucleotides with molecules that mask their identity * Author to whom correspondence should be addressed. Phone: +39 70 6758570. Fax: +39 70 6758553. E-mail:
[email protected]. † University of Cagliari. ‡ Novosibirsk Institute of Bioorganic Chemistry. § University of Padova. X Abstract published in Advance ACS Abstracts, July 1, 1997.
S1043-1802(97)00082-7 CCC: $14.00
and confer a stronger hydrophobic character, such as long-chain alcohols (7), steroids (8), cholic acid (9), peptides (10), etc. Recently, the introduction of shortand long-chain polyethylene glycols (PEGs) has been investigated. In fact, it is well known (11) that PEG is a unique molecule that, when covalently bound to a substrate, increases the solubility in organic and aqueous solution, decreases the immunogenic effect, and extends the body lifetime. This nontoxic compound has been used for the preparation of a series of biologically active conjugates (12). In oligonucleotide chemistry, a short PEG chain has been used as a non-nucleosidic linker in triplex-forming molecules (13), synthetic ribozymes (14), loop-forming chains, and circular nucleic acid derivatives (15). High-molecular mass PEG has recently been introduced at the 3′- and 5′-end of the oligonucleotide chains; the 3′-position has been modified by a PEG-modified solid-phase support (16-18), while the 5′-end reacted with a reactive PEG chain when bound to the solid-phase support (19-22) or after its release (23-25). With these procedures, very often a limited amount of PEG conjugate can be produced, owing to the reduced capacity of the solid-phase processes and its difficult upscaling. Moreover, when a postsynthetic conjugation is performed with high-molecular mass PEGs, the viscosity of their solution hampers the condensation reaction, especially when the oligonucleotides are still bound to an insoluble support. This problem may be further compounded when large scale production of these conjugated derivatives is needed (26). A procedure which is based on a new method of oligonucleotide synthesis has recently been proposed to solve these problems (27). During this process, PEG has © 1997 American Chemical Society
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been used as a soluble polymeric support for the large scale synthesis of these molecules. In this liquid-phase procedure (called HELP), a PEG unit with a molecular mass between 5000 and 20 000 Da has been employed to produce oligonucleotide chains of up to 20 monomers. To obtain a useful PEG conjugate, that procedure has been modified by introducing a stable bond between the polymer and the growing chain. PEG was then utilized both as a synthetic handle, for the liquid-phase procedure, and as a biological carrier for the final oligonucleotide. The synthesis of a high-molecular mass PEG-conjugated 12mer is reported here. This sequence was an active antisense against HIV-1 (28). The synthetic protocol, the purification of the final derivative, and the first data on enzymatic stability and binding specificity are described. EXPERIMENTAL PROCEDURES
Functionalization of Polyethylene Glycol. One gram of monomethoxy polyethylene glycol (MPEG, MW ) 9500), corresponding to 0.105 mmol of free OH groups, was coevaporated twice with a few milliliters of anhydrous acetonitrile (AcCN). Dimethoxytrityl (DMT) (2cyanoethyl)-N,N-diisopropylnucleoside phosphoramidite [0.26 mmol (2.5×)] and 1.05 mmol (10×) of tetrazole, dissolved in anhydrous AcCN, were added, under argon, to MPEG that had previously been dissolved in the minimum amount of anhydrous AcCN. The mixture was stirred under argon for 5 min and cooled in an ice bath, and the MPEG-nucleoside was precipitated by the slow addition of 5-6 volumes of tert-butyl methyl ether (TBME). The product was filtered, washed with ether, recrystallized from ethanol to eliminate any residual amount of reagents or soluble byproducts, and dried. The phosphite bond between the MPEG and the nucleoside was then oxidized to a phosphate one by dissolving the product in AcCN (1.0 g in 10 mL) and reacting it with 0.6 mL of tert-butyl hydroperoxide (TBHP), at 0 °C, under stirring, for 15 min. The product was precipitated with TBME, filtered, washed thoroughly with ether, and dried. Any unreacted OH groups of the MPEG were capped with a mixture of 2,6-lutidine, N-methylimidazole (NMI), and acetic anhydride (0.5 mL each for 1.0 g of MPEGnucleoside, dissolved in 5 mL of AcCN). The mixture was left under stirring at room temperature for 3 min; the DMT-5′-oligonucleotide-3′-MPEG was precipitated with TBME, filtered, washed with ether, and dried under vacuum over KOH pellets. Synthesis of MPEG-Conjugated Oligonucleotides. The synthesis of the MPEG-conjugated oligonucleotides was performed according to the phosphoramidite-based procedure previously applied in the liquid-phase synthesis of oligonucleotides (29) as follows. The fully protected DMT-5′-oligonucleotide-3′-MPEG was dissolved in a concentrated ammonia solution (20 mL for 100 mg of product) and left overnight at 60 °C. The ammonia was eliminated by evaporation with water (two or three times, 20 mL each) and the water solution extracted with ether (three times) and lyophilized. The 5′-DMT protecting group was eliminated by treatment with an acetic acid solution (4:1 v/v; 20 mL for 100 mg) for 30 min at room temperature. The water solution was extracted three times with ether and lyophilized. The deprotected MPEG-conjugated oligonucleotide was purified by anion-exchange HPLC. The chromatography was performed on a MonoQ HR 5/5 column, eluted with NaCl, the gradient ranging from 0.20 to 0.50 M, at pH
Bonora et al.
12.00, over 18 min (flow rate of 1.0 mL/min). Detection was by UV absorbance at 260 nm. The product was desalted by a gel-filtration procedure on Sephadex G25-F (2 × 10 cm), with MilliQ water as the eluent. An alternative desalification procedure was realized by dissolving the product in acetone or methanol (5 mL for 200 mg); after being stirred for 30 min, the solution containing the MPEG conjugate was filtered, or centrifuged, and dried. It was also possible to selectively extract the product from a water solution (5 mL for each gram) with dichloromethane (4 × 100 mL). Characterization of MPEG-Conjugated Oligonucleotide. To calculate the reaction yields, the absorption at 498 nm of the DMT group was determined spectrophotometrically (2 mg in 10 mL of a 70% perchloric acid solution in methanol) and the amount of MPEGbound nucleotide was measured by using the following equation:
A498 × milliliters of HClO4 solution × 14.3/(milligrams of MPEG-oligonucleotide) A Perkin-Elmer Lambda 5 UV/Vis spectrophotometer was used. 1H and 31P NMR spectra were recorded on a Bruker 400 AM spectrometer, utilizing deuteriochloroform (99.96%) or deuterium oxide (99.9%) as a solvent and TMS as an internal standard for protons and 85% phosphoric acid for phosphorus. To ascertain the complete removal of reagents, the MPEG conjugates were analyzed by thin-layer chromatography on precoated silica gel sheets 60 F254 with the eluant ethyl acetate/acetone/water (5:10:1). The purified compound was analyzed by RP HPLC on a Vydac C18 column (eluant: 0.1 M triethylammonium acetate at pH 7.0; gradient of acetonitrile from 10 to 80% over 60 min) and by IE HPLC on a MonoQ (eluant: gradient of NaCl from 0.05 to 0.5 M at pH 12.00 over 30 min). A WATER 600E apparatus, equipped with a 717 autosampler and a 490E UV/Vis detector, was used. The starting MPEG, and the final conjugate, were analyzed by GPC on a Biosil Sec-125 column (300 × 7.8 mm) eluted with 0.1 M Na2SO4/0.1 M NaH2PO4 at pH 6.8 (flow rate, 0.9 mL/min). A JASCO 880 PU apparatus, equipped with an ERMA Erc-7512 refraction index detector, was utilized. Thermal Denaturation Studies. The thermal denaturation of oligonucleotide complexes was performed by following the A260 variations of solutions containing equimolar amounts of free and MPEG-bound oligonucleotide and complementary sequence. The samples were 1.3 × 10-5 M in 10 mM sodium cacodylate (pH 7.4) containing 0.1 M NaCl and 1 mM EDTA. The temperature inside the cell was increased at a ratio of 0.5 °C/min. More than 600 experimental points for each optical melting curve were collected, with 10 points/(°C step); the curves were fully reversible. The UV detector of a Millichrom chromatograph with a specially designed thermoregulated cell was used (30). The first derivatives of the melting curves were calculated using a linear approximation gradient by 10 experimental points. Enzymatic Degradation. The enzymatic hydrolysis of the oligonucleotides was performed with a mixture of phosphodiesterase and nucleotidase from snake venom in 0.01 mL of a buffer containing 0.1 optical unit of the compound dissolved in 15 mM MgOAc2/0.2 mM EDTA/ 0.1 M Tris-HCl at pH 8.8, and 37 °C. The changes in concentration were followed by HPLC analysis of the samples taken from the mixture on a time scale.
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High-Molecular Mass PEG-Conjugated Oligonucleotides Scheme 1. Liquid-Phase Synthesis of MPEG-Conjugated Oligonucleides
Figure 1. 1H NMR spectrum of the starting DMT-5′-(Nbenzoyl)deoxycytidine-3′-MPEG in CDCl3. (Inset) Partial 31P NMR spectrum of the same solution (no further signals up to 150 ppm). MPEG methylene signals were suppressed by selective irradiation. Table 1. Synthetic Cycle (1.0 g of MPEG12000-OH) stepa
RESULTS AND DISCUSSION
Scheme 1 illustrates the liquid-phase method applied for the synthesis of the high-molecular mass MPEGconjugated oligonucleotides. The OH end groups of the MPEG unit were conjugated with 5′-DMT-protected phosphoramidite nucleosides, using the procedure adopted for the synthesis of the oligonucleotide chains (23). The MPEG-bound product, purified by precipitation from the reaction mixture and filtration, was again left to react with the next nucleotide. In comparison with the procedures based on insoluble supports, this method had the advantages previously observed in the traditional liquid-phase synthesis of oligonucleotides. Since all the reactions are performed in the solution state, the phase homogeneity may allow for the easy scaling up of the process; moreover, owing to the absence of diffusion problems inside the resin beads, a lower amount of reagent was generally required. It should be recalled that a reduced amount of conjugated product is obtained, when the PEG chain is introduced at the end of the oligonucleotide synthesis on the solid support, owing to the low reactivity of the PEG solutions in these heterogeneous conditions. This drawback, which was further underlined by increasing the PEG molecular mass, was obviously absent in this method which took place under homogeneous conditions. It is also worth noting that the spectral transparency and the great solubility of the PEG moiety, and of its conjugates, allows for the rapid and nondestructive analysis of the various synthetic steps and easy evaluation of the reaction products and its byproducts during the purification processes. To test the efficacy of this method, the 12mer conjugate with a monofunctional PEG chain, monomethoxy-substituted (MPEG), was synthesized. This oligonucleotide was previously employed to study its antisense activity against HIV (23). A MPEG molecule of a nominal 12 000 Da was used in this study, to obtain a better solubility of the final oligonucleotide chain and verify the effect of the higher molecular mass of the polymer on the biological properties of its conjugate. This polymer used was analyzed by GPC that indicated an average molecular mass of 9500 Da, with low polydispersity.
reagents
detritylation coupling
TCA solutionb AcCN
capping oxidation
0.1 M phosphoramidite 0.5 M tetrazole capping solutionb tBHP solutionb
quantity 20 mL minimum to dissolve 5.0 mL 4.0 mL 10.0 mL 10.0 mL
time (min) 15
5 5 15
a After each step, the sample is precipitated with 5-6 volumes of TBME, at 0 °C; a further recrystallization with 50 mL of ethanol may be required at the end of each cycle. b See Experimental Procedures.
The 1H and 31P NMR spectra of the polymer conjugated to the first nucleotide of the planned oligo chain are reported in Figure 1. Within the limits of instrumental errors, a quantitative reaction of the MPEG was obtained under the reported conditions; the integration values of the nucleotide signals were in a 1:1 ratio with those of the polymeric unit. Moreover, in the 31P NMR spectrum, only the signal from the phosphate bond at -2.2 ppm was visible, which demonstrated the efficacy of the oxidation procedure. From the UV/vis analysis of the sample, for each gram of MPEG, 95 µmol of nucleotide was measured which corresponded very well with the expected 97 µmol/g. The stability of the phosphate bond between the MPEG and the first nucleoside was evaluated by NMR and TLC analysis of a sample subjected to the same reaction conditions required for the synthesis and the final deblocking of the oligonucleotide chain. The assay indicates that no measurable release of the nucleoside molecule occurred under these conditions. The MPEG-12mer was obtained following the synthetic protocol reported in Table 1. In all the precipitation steps, TBME was used, instead of diethyl ether (23), because this ether absorbed a lower amount of water, which has an adverse effect during the synthetic process, and also appeared to be more efficient for the complete precipitation of the products. The reaction data of each step of the synthesis of the MPEG-12mer anti HIV are listed in Table 2. A very good coupling yield was observed for each step, as judged from the A498 of the derivatives. The overall and the average yields were comparable to those of the solid-
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Table 2. Synthetic Data of MPEG-12mer anti HIV (1.0 g of MPEG12000-OH) nucleoside
oligonucleotide bound (µmol/g)a
step yield (%)b
dC dG dA dC dG dA dA dC dT dA dC dG
95 89 85 79 76 72 69 64 60 58 56 54
98 98 99 98 99 99 99 97 98 99 99 97
overall yield ) 82% average yield ) 98.5% total amount recovered ) 0.9 g (60%) (5815 OD/g after purification) a
From A498 of the DMT group. b Calculated on the basis of the increased MW of the conjugate.
Figure 3. Thermal denaturation profiles of unmodified (unbroken line) and MPEG-conjugated (dashed line) 12mer duplexes. (Inset) First derivatives of the thermal profiles.
Figure 2. IE HPLC of the crude MPEG conjugates: (a) MPEG-6mer-OH and (b) MPEG-12mer-OH. Area values of the main peaks are indicated.
phase synthesis of similar products. We are hopeful that the amount of the final collected oligonucleotide, which in this case was not maximized because of the high number of precipitation steps needed for the purification of the intermediates, could be increased with automation of the process (31). However, it should be recalled that the amount of collectable product was even lower when PEG was introduced with a postsynthetic modification of the oligonucleotide supported on a solid phase, especially if a high-molecular mass polymer is employed. The purification of the MPEG conjugates was achieved by HPLC initially using a reversed-phase column. Unfortunately, the separation of the final 5′-DMT-protected, fully deblocked, conjugated compounds from shorter sequences was unsuccessful, since the MPEG moiety was similar chromatographically to all the derivatives, and a single peak was always observed, despite the presence of a crude mixture of products. However, the different compounds in the mixtures could be separated by anionexchange chromatography, since the charge balance of the different oligonucleotide chains was not suppressed by the high-molecular mass MPEG unit. The chromatographic patterns of the crude, fully deprotected 6mer and 12mer are reported in Figure 2; the efficacy of this synthetic procedure was clearly seen. After purification and desalting, the final compound was further analyzed by GPC. From the elution behavior, the approximate average molecular mass was 13 000 Da, close to the calculated value (13 480 Da). The final MPEG-12mer was further characterized by thermal denaturation studies. The dissociation of duplexes formed from equimolar concentrations of the conjugated oligonucleotide and the complementary unmodified target was examined. The A260 temperature
Figure 4. Digestion of unmodified (unbroken line) and MPEGconjugated 12mers (dashed line). The concentration of the starting oligonucleotides, quantified by HPLC, is plotted versus incubation time.
variations, and their first derivatives, are reported in Figure 3. It has been clearly demonstrated that the introduction of a single, long chain of MPEG at one end of the oligonucleotide has very little, if any, adverse effect on the hybridization properties of the oligonucleotide itself. The effect of stabilization against the enzymatic degradation in the presence of the MPEG was finally investigated. The disappearance over time of both free and conjugated 12mers, incubated with a mixture of hydrolytic enzymes, is reported in Figure 4. It was possible to observe that, under these conditions, the unprotected oligonucleotide was completely digested in less than 30 min, while during the same period, at least 20-25% of the starting MPEG-12mer was still present, which demonstrated the stabilizing effect of the MPEG when bound to the oligonucleotide. These results are in agreement with those published on oligonucleotide conjugates with PEG chains lower molecular mass (19). To summarize, the utility of this new, modified liquidphase method for the preparation of oligonucleotides firmly bound to high-molecular mass PEG chains has been demonstrated. The intrinsic features of this procedure are especially suitable for the large scale production of these derivatives. The first biological data have confirmed that the high-molecular mass MPEG chain could increase the cellular lifetime of its conjugated
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molecules without interfering with their capability to interact with the specific target. The antisense properties of this derivative are now under investigation and will be reported in the near future. ACKNOWLEDGMENT
The authors thank Dr. Tula Saison-Behmoaras and Prof. Claude He´le`ne (Museum National d’Histoire Naturelle, Paris) for the stimulating discussions. They are also grateful to Drs. L. Lokhov and N. Komatova, (Novosibirsk Institute of Bioorganic Chemistry, Russia) for the help with the experiments of thermal denaturation and enzymatic degradation. This research was supported by the Progetto Strategico CNR (Italy) ST74 “Oligonucleotidi Antisenso” and by grants from MURST (Italy). LITERATURE CITED (1) Cohen, J. S. (1992) Oligonucleotide therapeutics. Tibtech 10, 87. (2) Gillespie, D. (1992) Perspectives for antisense nucleic acid therapy. DN&P 7, 389. (3) Thuong, N. T., and He´le`ne, C. (1993) Sequence-specific recognition and modification of double-helical DNA by oligonucleotides. Angew. Chem., Int. Ed. Engl. 32, 666. (4) Wyatt, J. R., Vickers, T. A., Roberson, J. L., Buckeit, R. W., Klimkait, T., Deboets, E., Davis, P. W., Rayner, B., Imbach, J. L., and Eckers, D. J. (1994) Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion. Proc. Natl. Acad. Sci. U.S.A. 91, 1356. (5) Heidenreich, O., Kang, S.-H., Xu, X., and Nerenberg, M. (1995) Application of antisense technology to therapeutics. Mol. Med. Today 1, 128. (6) Sanghvi, Y. S., and Cook, P. D., Eds. (1994) Carbohydrate modifications in antisense research. ACS Symposium Series 580, Maple Press, York, PA. (7) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates. Nucleic Acids Res. 18, 3777. (8) Boutorin, A., Guskova, L., Ivanova, E., Kobetz, N., Zarytova, V., Ryte, A., Yurchenko, L., and Vlassov, V. (1989) Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3′-terminus and their interaction with DNA within mammalian cells. FEBS Lett. 254, 129. (9) Manoharan, M., Johnson, L. K., Bennett, C. F., Vickers, T. A., Ecker, D. J., Cowsert, L. M., Freier, S. M., and Cook, P. D. (1994) Cholic acid oligonucleotide conjugates for antisense application. Bioorg. Med. Chem. Lett. 18, 3777. (10) Tung, C., Rudolph, M. J., and Stein, S. (1991) Preparation of oligonucleotide-peptide conjugates. Bioconjugate Chem. 2, 464. (11) Harris, J. M., Ed. (1992) Poly(ethylene glycol) chemistry. Biotechnical and biomedical applications, Plenum Press, New York. (12) (a) Zalipsky, S. (1995) Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates. Bioconjugate Chem. 6, 150. (b) Zalipsky, S. (1995) Chemistry of polyethylene glycol conjugates with biologically active molecules. Adv. Drug Delivery Rev. 16, 157. (13) Kessler, D. J., Petitt, B. M., Cheng, Y.-K., Smith, S. R., Jayaraman, K., Vu, H. M., and Hogan, M. E. (1993) Triple helix formation at distant sites: hybrid oligonucleotides containing a polymeric linker. Nucleic Acids Res. 21, 4810. (14) Thomson, J. B., Tuschl, T., and Eckstein, F. (1993) Activity of hammerhead ribozymes containing non-nucleotidic linkers. Nucleic Acids Res. 21, 5600.
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