Postsynthetic Conjugation of Biopolymers with High Molecular Mass

Synthesis and transdermal permeation of novel N 4 -methoxypoly(ethylene glycol) carbamates of cytarabine. Lesetja J. Legoabe , David D. N'Da , Jaco C...
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Bioconjugate Chem. 2003, 14, 1038−1043

Postsynthetic Conjugation of Biopolymers with High Molecular Mass Poly(ethylene glycol): Optimization of a Solution Process Tested on Synthetic Oligonucleotides Maurizio Ballico,§ Susanna Cogoi,# Sara Drioli,§ and Gian M. Bonora*,§ Department of Chemical Science, University of Trieste, Via Giorgieri, 1 - 34127 Trieste, Italy, and Department of Biological Science and Technologies, University of Udine, P.le Kolbe, 4-33100 Udine, Italy. Received February 13, 2003; Revised Manuscript Received May 22, 2003

The reaction of oligonucleotides with high molecular weight monomethoxy poly(ethylene glycol)s (MPEGs) has been tested to set up a convenient procedure for the postsynthetic conjugation in solution of biopolymers. A first oligonucleotide was previously modified in 5′, using a liquid-phase procedure, with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. Two procedures commonly utilized for the activation of the terminal OH groups of the MPEG were evaluated, that is, the reaction with pNO2-phenyl chloroformate and with N,N′-disuccinimidyl carbonate. Both water as well as organic solution conditions were employed and compared. In a second test, a 3′-amino modified, commercial 20-mer was also conjugated in a microscale condition to verify the effect of size and concentration of MPEG on the postsynthetic conjugation of these biopolymers under troublesome synthetic conditions.

INTRODUCTION

The pharmacological properties of synthetic oligonucleotides can be modulated by their conjugation with proper molecules (1). The rationale of these modifications is dictated by the necessity to get better cellular uptake, biostability, and pharmacokinetic properties. Among the molecules described in the literature, it is possible to distinguish between low and high molecular mass units. Within the first group, lipophilic conjugates, as cholesterol and other steroids, vitamins, and folic acid have been widely employed (2). Oligosaccharides and peptides have been considered for their ability to deliver oligonucleotides specifically to the targeted cells (3). Cleaving and cross-linking agents have been also proposed to improve the overall biological performances (4). Furthermore, large conjugating molecules as proteins and antibodies have been linked, and great attention turned to polyamines as polycationic carriers (5). Among the different biocompatible polymers, poly(ethylene glycol) (PEG) was extensively investigated on the basis of previous success achieved with the PEGylation of protein (6). In fact, this procedure is well on its way to becoming a standard component of the pharmaceutical tool box, since PEG possesses a unique set of properties, including analytical methods for conjugate characterization, absence of toxicity, immunogenicity, and antigenicity, low mass-dependent elimination via the kidney, and high solubility in water and other organic media. At the oligonucleotide level, the presence of high molecular weight PEGs has showed a minimal effect on the hybridization behavior, while a clear enhancement of in vivo stability and cellular permeation has been observed, without any adverse toxic effect (7). * To whom correspondence should be addressed. Tel: +390405583927; fax: +390405583903; e-mail: [email protected]. § University of Trieste. # University of Udine.

Additionally, taking advantage from the recent procedure capable of producing pure, selectively and reversibly protected bifunctional PEGs (8), it is easy to imagine the production of PEG conjugates carrying on the same chain an oligonucleotide and an additional molecule as a peptide, a steroid, an intercalator, or whatever can be devised to further improve their pharmacological properties. The coupling of PEG to a bioactive molecule is achieved by a polymeric derivative having an activated functional group at one or both termini (9), chosen on the basis of the reactive groups of the molecule to be PEG-conjugated. In case of oligonucleotides, since they offer only low nucleophilic functions as primary OH groups, is quite difficult to attain an extensive modification through a direct reaction with PEGs. Moreover, the introduction of a large PEG chain by classical solid-phase procedures, commonly used for much postsynthetic oligonucleotide modification, suffers from the phase heterogeneicity of the process that implies poor reactivity and unpredictable kinetic effects. An acceptable yield of these reactions has been described in the literature only using PEGs of lower molecular weight following the standard phosphoramidite conditions (10). However, the adverse effects are enhanced with the increasing of the mass of the conjugating polymer (11), as demanded due to its better biological performances observed with sizes up to 2040 kDa. An oligonucleotide conjugation using a larger PEG molecule in a classical solution reaction has been reported; a very large excess of the ester-activated form of polymer was employed, but the yield of condensation was not discussed (12). For all these reasons, we decided to investigate the postsynthetic PEG conjugation in solution of synthetic oligonucleotides to ascertain the optimum level of modification achievable through this procedure. To avoid the low reactivity of the terminal hydroxyl group, and to likely extend this procedure even at the peptide level, a

10.1021/bc034020c CCC: $25.00 © 2003 American Chemical Society Published on Web 08/21/2003

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terminal amino function was introduced on the oligonucleotides. The two most used chemical activations of the OH functions of PEG, namely, the N,N′-disuccinimidyl and the pNO2-phenyl carbonate derivatives, were used both in water as well as in organic solution, and compared. The effects of concentration and molecular size of PEG were also investigated. MATERIALS AND METHODS

Oligonucleotide Synthesis. The oligonucleotides were a fully thioate 15-mer synthesized in our laboratory and a commercial, partially thioate 20-mer, modified with the 3′-amino-modifyer C7 CPG linker (MWG-Biotech AG, Ebersberg (D)). Both were used without any further purification. The monomethoxy PEGs (MPEG)s of 5 and 10 kDa were obtained from Fluka, Bucks (Switzerland). Introduction of the Amino-Linker. A 5′-terminal primary amino group was introduced, following the described procedure, on the fully thioate 15-mer obtained by liquid-phase synthesis on PEG (HELP technique) (13) after standard detritylation of the crude product. Conjugation with the 5′-Amino-Modifier Phosphoroamidite, MMt-Protected. A total of 1.00 g of MPEG10000(3′)-15-mer-(5′)-OH (15.6 µmol of 5′-OH groups) was dehydrated by coevaporation with 3 × 5 mL of anhydrous AcCN in a three-neck vessel. Through the rubber septa, the MPEG-supported oligonucleotides were dissolved by injecting with a syringe 5.0 mL of anhydrous AcCN; successively, 100 µmol of the 2-[2-(4-monomethoxytrityl)aminoethoxyl]-ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoroamidite (Glen Research) as 5′-amino-modifier phosphoroamidite (6 times excess) and 400 µmol of 1Htetrazole (24 times excess) were injected simultaneously through the septa. The mixture was kept under stirring, at room temperature and under argon atmosphere, for 5 min. The MPEG-bound product was precipitated from the solution, kept in an ice bath, by the addition of 100 mL of TBME under vigorous stirring. A white solid was filtered, washed with ether, and collected. To remove the residual excess of reagents, the product was recrystallized from EtOH. A total of 0.98 g of product were collected and dried under vacuum. On the basis of the monomethoxytrityl (MMt) absorbance at 472 nm ( ) 51 900 in 70% HClO4/EtOH ) 3/2 (v/v)) a functionalization degree of 67% was measured. The product was reacted again using the reported conditions, but with a prolonged reaction time (15 min). A final 86.5% degree of functionalization was achieved. Oxidation of the Phosphate Bond. A total of 0.98 g of MPEG-(3′)-15-mer-(5′)-NH-MMt were dissolved in 7 mL of anhydrous AcCN. In an ice bath, and under stirring, 0.6 mL of tert-butyl hydroperoxide (80% in di-tert-butylhydroperoxide/water 3:2, Fluka, Bucks (Switzerland)) were added and the reaction was left under stirring at 0 °C for 15 min. The product was precipitated with 100 mL of TBME. A total of 0.97 g of a white powder were collected, washed with ether, and dried under vacuum over KOH pellets. Deprotection and Detachment of the Oligonucleotides. A total of 0.80 g of MPEG-(3′)-15-mer-(5′)-NH-MMt were dissolved in 200 mL of concentrated NH4OH and stored, in a tightly closed vessel, in an oven at 50 °C for 18 h. The solution was coevaporated to dryness from distilled water until the ammonia was completely eliminated. The residual was dissolved in distilled water (100 mL) and extracted with ether (4 × 100 mL). After lyophylization, 0.79 g of a white solid was collected. The MPEG was removed by dissolving the product in 8 mL of a 0.5%

solution of NH4OH and precipitating the oligonucleotides in an ice bath, under vigorous stirring, with 150 mL of acetone. After centrifugation of the sample (0 °C, 20 min, 4000 rpm), 0.30 g of 3′-OH-15-mer-(5′)-NH-MMt, as ammonium salt, was quantitatively recovered and lyophilized. The supporting MPEG was eventually recovered from the acetone solution. Deprotection of the Terminal Amino Group. A total of 0.30 g of the (3′)-OH-15-mer-(5′)-NHMMt were dissolved in 75 mL of a 4:1 (v/v) solution of glacial AcOH in water. The solution was left under stirring for 4 h at room temperature. A pale yellow color was developed. After addition of 8 mL of distilled water, the solution was extracted with ether (5 × 75 mL). The residual was dissolved in water and lyophilized. A total of 0.29 g of a white product was collected. Less than 0.05% of MMt was still present. Activation of Terminal OH Groups of MPEG. Reaction with pNO2-Phenyl Chloroformate (MPEG-pNO2Phenyl Carbonate). A total of 5.0 g of MPEG5000 (MW ) 5 kDa), equivalent to 1.0 mmol of terminal OH groups, were coevaporated from anhydrous toluene and connected to a rotatory pump for 30 min for a complete dehydration. The solid residual was dissolved in 20 mL of anhydrous CH2Cl2 and cooled in an ice-bath. Under stirring, 0.4 g of pNO2-phenyl chloroformate (2 times excess) was added, together with 1 equiv of TEA (0.28 mL). The reaction was left under stirring at room temperature; the pH was maintained around 8 with TEA. After 24 h, the product was precipitated from an ice-bath solution, under stirring, with TBME (200 mL), filtered, washed with iPrOH and ether, and dried under vacuum over KOH pellets. The collected product was recrystallized from EtOH. A total of 5.12 g was collected. Reaction with N,N′-Disuccinimidyl Carbonate (MPEGOSu Carbonate). A total of 5.0 g of MPEG5000, equivalent to 1.0 mmol of terminal OH groups, was coevaporated from anhydrous toluene and connected to a rotatory pump for 30 min for a complete dehydration. The residue was added with 1.0 mL of anhydrous pyridine, and the slurry suspension was dissolved with 5 mL of anhydrous CH2Cl2 and 2 mL of anhydrous AcCN. Under stirring, at room temperature, 0.64 g of N,N′-disuccinimidyl carbonate were added (2.5 times excess). The reaction was left to react overnight. The product was precipitated from an ice-bath solution, under stirring, with TBME (200 mL), filtered, washed with iPrOH and ether, and dried under vacuum over KOH pellets. The product was recrystallized from AcOEt. A total of 5.15 g was collected. Conjugation of the 5′-Amino-Oligonucleotide with MPEG. These experiments were performed with the HELP-synthesized 15-mer to set up the best conjugating conditions. Synthesis in Organic Media (Heterogeneous). A total of 0.1 g of MPEG5000-(5′) OH-activated (20 µmol) was dissolved in 1.0 mL of CHCl3. A total of 0.05 g of the OH(3′)-15-mer-(5′)-NH2 (10 µmol) and 0.8 µL of TEA was added. The suspension was kept under vigorous stirring at room temperature. The insoluble solid was a white powder collected by addition, in an ice-bath and under stirring, of 100 mL of TBME. The product was washed with iPrOH and ether, and dried under vacuum. Synthesis in Aqueous Media (Homogeneous). A total of 0.05 g of the OH-(3′)-15-mer-(5′)-NH2 (10 µmol) were dissolved in 1.0 mL of a buffer solution of Na2CO3/ NaHCO3 at pH ) 9.0. 0.1 g of MPEG5000-(5′) OH-activated (20 µmol) were added and left under stirring at room temperature. After 72 h, the solution was evaporated to dryness in a rotavapor. The residue was suspended in

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20 mL of CHCl3 and left under stirring for 10 min. The insoluble material was filtered, and the CHCl3 solution was dried with anhydrous Na2SO4. A white solid was collected by addition, in an ice-bath and under vigorous stirring, of 100 mL of TBME. The product was washed with iPrOH and ether, and dried under vacuum. Analytical Methods. The NMR spectra were collected in DMSO-d6 on a JEOL EX 400 spectrometer using TMS as internal standard. The RP chromatography was performed on a ProgelTSK OligoDNA (column 15 × 0.46 cm) with the following gradient: eluent A: TEAAc 0.05 M, pH ) 7.0; eluent B: TEAAc 0.05 M, pH ) 7.0/AcCN 20:80 (for 2 min: 10%B; from 2 to 40 min: 40% B). The GPC was performed on a PL aquagel-OH 30-8µm (column 30 × 0.75 cm) eluted with water milliQ. A HPLC Hewlett-Packard series 1100 system equipped with a Lambda-Max model 481 UV/Vis detector was used. The gel-electrophoresis was performed in a Mighty Small II cell (Amersham Pharmacia Biotech) equipped with a Power Pac 3000 power supply (Bio-Rad), in denaturing conditions (urea 7 M) on 20% polyacrylamide. Conditions: 2 h of prerunning at 55 °C, 600 V, 30 W, 50 mA; 45 min of running at 55 °C, 600 V, 30 W, 50 mA. After running, the gel was incubated overnight in a Stains All solution (0.01% Stains All in 50% formamide), washed with water, and then analyzed on a Phosphorimager (Bio-Rad). The amount of NH2 was calculated using the colorimetric TNBS test as follows: 250 µL of TNBS 0.03 M in borate buffer at pH ) 9.3 was added to 1 mg of sample. The solution was diluted to 10 mL with borate buffer at pH ) 9.3, stirred, and allowed to stand for 30 min at room temperature. Absorbance at 421 nm ( ) 12 860) was measured. RESULTS AND DISCUSSION

Introduction of the 5′-NH2 Terminal Linker. To overcome the low nucleophilicity of the terminal OH group of the oligonucleotides, and consequently to increase the expected yield of the postsynthetic conjugation of these molecules, a linker carrying a primary amino group is required. This was introduced on the HELPsynthesized thioate 15-mer using a reagent that allows the standard amidite procedure and ensures high condensation yields and fast reaction times. It is important to observe that, in principle, the same conjugation reaction between a high molecular mass polymer, as PEG, and a biomolecule will likely be extended, on similar reaction conditions, on peptides or other biologically active molecules provided with a proper amino function. This reaction was performed on the oligonucleotides supported on a MPEG polymer using a liquid-phase procedure to take advantage of the presence of the soluble supporting polymer. In fact, it is possible to drive the reaction to high yield values using an excess of reagents, but saving advantageous homogeneous conditions; any unreacted material is easily removed by the usual precipitation and filtration procedure at the end of the reaction. Successively, the hydrolysis of the MPEGsupporting bond will permit the recovery of free NH2terminal modified oligonucleotides. In Scheme 1, the structure of the NH2-terminal modified oligonucleotides is reported. Up to near 90% of modification was achieved on the MPEG-(3′)-15-mer-(5′)-OH. The removal of the terminal MMt amino-protecting group did not offer any drawback, while the final hydrolytic cleavage of the MPEG-

Scheme 1. Free 15-mer Thioate Carrying the Amino Linker at the 5′-Position

Table 1. Yields and Reaction Solvent of the MPEG-Conjugation Reactions activated MPEG5000 MPEG-pNO2 phenyl carbonate MPEG-Osu carbonate

reaction solvent

conjugation yield (%)

CHCl3 H2O CHCl3 H2O

10 10 15 65

supporting ester bond was obtained, as usually, during the terminal deprotection procedure of the oligonucleotides chain. From the starting MPEG-supported oligonucleotides, the expected amount of free 5′-NH2-oligonucleotide was eventually collected. Only the final, fullsequence oligonucleotides carried the reacting amino group, since the reaction was performed on a crude product subjected, at the end of the chain assembly, to a capping procedure that irreversibly modified any still unreacted groups. Hence, only the terminal DMTprotected nucleotide will be able to further react with the linker, once deprotected. The hydrolyzed, supporting MPEG was almost completely removed from the reaction mixture by dissolving in acetone that solubilizes only the polymer, while the insoluble oligonucleotide was filtered out and collected. An UV analysis of the aqueous and organic solutions confirmed these results, since less than 0.05% of the calculated total absorbance at 260 nm due to the oligonucleotide was measured in acetone solution, while the starting MPEG-OH was recovered after precipitation with ether. Conjugation with High Molecular Weight MPEGs. To obtain a stable linkage between MPEG and the conjugating molecule, the OH-terminal groups of polymer must be activated. Two quite common conditions were chosen, that is the reaction with the pNO2-phenyl chloroformate and that with the N,N′-disuccinimidyl carbonate. Both will give a reactive intermediate that will form a stable urethane linkage with the NH2 group of the biomolecule. The two reactions, very popular as MPEGmodifying procedures, were compared for their behavior toward the same oligonucleotides. In addition, the reactivity of the two activated MPEGs was verified both in organic as well as in aqueous conditions to investigate if it was possible to avoid the time-consuming dehydration of the final product obtained from water solution. All these experiments were performed using a 15-mer synthesized by a large scale, liquid-phase procedure using the PEG-supported procedure (13). The sequence of the fully phosphorothioated oligonucleotide was (5′)-NH2linker-d(TCTCAGT3G4T2)-OH-(3′) In CHCl3, the conjugation was obviously achieved in heterogeneous conditions, but a likely solubilization of the product due to the presence of the amphipilic, large molecular weight MPEG was expected. However, with the proceeding of the reaction, only a very low solubilization of the reaction mixture was observed. On the other hand, the reaction performed in aqueous solution, characterized by the homogeneity of the reaction mixture, will reasonably offer a better reactivity, even if some adjunctive workup will be demanded. In any case, the presence of MPEG on the final product granted the usual advantages given by the soluble polymer-supported reactions, as the easy purification procedures. The overall results

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Figure 1. 1H NMR in DMSO-d6 of the crude MPEG-(5′)-NH-15-mer-(3′)-OH obtained from the water solution of the OSu-activated polymer.

as a function of the reaction conditions are summarized in Table 1 where yields were calculated from 1H NMR spectra in DMSO-d6 By comparing the results it is easy to observe that the best reaction conditions are achieved from the conjugation with the MPEG OSu-activated in water solution, where less than 30% of the unreacted, starting amino groups were still present, as measured by the TNBS test. In case of the pNO2-phenyl carbonate, almost the same result was observed for both the organic and the aqueous reaction, that is, near 10% of conjugation yield; a similar value was achieved from the reaction of MPEG-OSu in CHCl3. A 2:1 ratio between MPEG and oligonucleotides was used in all these experiments, but a high conjugation yield can be predicted increasing the excess of activated polymer. In Figure 1 a representative 1H NMR spectra is reported, where the ratio between H(1′) and H(3′) protons of the oligonucleotides and the internal CH2 of the MPEG’s chain can be used to evaluate the reaction. The yields have been calculated taking into account that some free MPEG resulting from the oligonucleotide synthesis was still present in about 20% of the overall amount, as calculated from the chromatographic analyses of the crude, deblocked material. The final value was in line with the amount of unreacted, free NH2 of the starting oligonucleotide as evaluated from the TNBS colorimetric test. In addition, the intensity of residual signals due to the terminal OH group of MPEG, as well as those of the OH-activating moieties, confirms that the MPEG signals are almost due to the stable conjugate. In Figure 2 the RP-HPLC chromatograms of the starting, free oligonucleotide and of its MPEG-conjugated, obtained by a water-based, OSu-activated procedure, are reported. It is possible to recognize a broad, late-running peak due to the MPEG-oligonucleotide. The two first peaks can be originated by the intermolecular associations of the starting oligonucleotide, as confirmed by its analysis under more diluted conditions (data not shown), in which the decrease of intensity of the second peak is paralleled by an increase of the first one. However, some nonconjugated, shorter oligonucleotides were still present in the sample. In fact, a crude synthetic mixture was used in which all the wrong sequences were previously capped and, consequently, made unable to be modified by the introduction of the NH2 linker and, successively, of the MPEG moiety. Furthermore, the intensity of the MPEG-

Figure 2. RP HPLC of the starting 15-mer (A) and of its MPEG-conjugated form (B).

conjugated peak is lower than expected from 1H NMR results, likely due to the presence of the conjugated polymer that decreases the UV absorbance of the conjugated sample compared with the free oligonucleotide. The CHCl3 fraction of the same reaction, once chromatographed, showed only traces of the same materials, as expected. These results are supported by a GPC investigation, where the peaks were monitored using the UV absorbance and the refractive index. As reported in Figure 3, in this case it is possible to observe a new peak assignable to the PEG-conjugated oligonucleotide. Its low intensity is, as said, very likely due to the presence of the conjugating polymer, while only a single peak due to unreacted oligonucleotides is observed with this chromatographic support. As a conclusion, we can reasonably assume that the best reaction condition demands the use of MPEG-OSu carbonate in a basic, buffered aqueous solution. A further study investigated the effect of the molecular size of the conjugating MPEG on the condensation yield, as well as the excess of the reacting polymer. As a sample oligonucleotide a commercial 20-mer, partially thioated, was in this case employed to additionally evaluate the efficiency of this PEG-conjugating procedure in a very low-scale condition. The new sequence,

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Figure 3. GPC of the starting 15-mer (A) and of its MPEGconjugated form (B). The arrow indicates the elution time of a standard sample of the starting MPEG5000.

modification of the terminal NH2 moiety of the oligonucleotides, while an increased size of the polymer makes almost inefficient the conjugation reaction, at least on these conditions. As observed in lanes 3 and 4, the amount of the MPEG-conjugates is enhanced using an higher excess of polymer, as seen at the level B. The level A shows a larger derivative likely arising from the impurity of PEG10000 present in the starting MPEG5000, as observed from its GPC analysis (not shown); alternatively some aggregation of the MPEG-conjugate cannot be excluded. Some aggregation due to the oligonucleotide sequence can be also envisaged from the spots at level C; their disappearance in lane 4 is very likely due to the reaction with the polymer that reduced the amount of free 20-mer still present. At the level D are clearly recognizable the spots due to free oligonucleotides in the starting crude, commercial sample. These results are complementary to those obtained with the shorter oligonucleotide and are here included as an example of a postsynthetic PEG conjugation performed on a quite low scale using a commercial product. A similar downscaling was not performed on the fully thioated 15-mer, but a comparable result can be reasonably expected. CONCLUSIONS

Figure 4. Gel-electrophoresis of the commercial 20-mer and of its crude MPEG-conjugated derivatives (Lane 1, starting 20mer; lane 2, 20-mer + MPEG10000 ) 1:2; lane 3, 20-mer + MPEG5000 ) 1:2; lane 4, 20-mer + MPEG5000 ) 1:10).

(5′)-OH-d(GpsA2GApsAGApsAGAGpsA2GA2GpsAG)-NH2(3′), where ps ) phosphorothioate bond, was used as control in a recent anti-gene activity study (14). The NH2terminal group was present on the 3′-terminal position, as given by the standard synthetic procedure used for this commercial product that adopts a 2-DMT-6-Fmocaminohexane-1-succinoyl linker supported on a long chain alkylamino-CPG solid-phase support. Due to the low abundance of the starting material, the conjugation procedure was performed on a microscale, using 1/100 of the sample employed in the previous experiment. Less than 1.0 mg of oligonucleotide was utilized. The main problem arose from the manipulation of the reaction mixture since a minimum volume reaction was demanded to maintain the reagent concentration as high as possible. A MPEG10000 (10 kDa) OSu-activated was initially employed consequently to the increased size of the oligonucleotide, with a twice-excess with respect to the oligonucleotide. Successively, the same reactions were performed using a MPEG5000 with a 2:1 and with a 10:1 excess of polymer. The final purification was performed by a molecular sieves chromatography, using a Bio-Rad P-4 column, eluted with water milliQ. The MPEGoligonucleotide was collected with the void volume of the column. The small fragments of the oligonucleotide were mostly present in the late-running peak. The crude products were analyzed by a gel-electrophoresis on polyacrylamide. The results are reported in Figure 4. It can be observed that only a larger excess of the activated MPEG guarantees in this case an efficient

In conclusion, an efficient procedure for the conjugation in solution of oligonucleotides, and very likely of other biomolecules carrying a reactive NH2 function, can be set up using properly activated, high molecular weight PEGs in water solution. A main problem could arise from the low abundance of rare products that hampers an efficient procedure, owing to a difficult manipulation of samples. However, a technical solution to these drawbacks can be reasonably devised. From the chemical point of view, the reactivity of the OSu-activated MPEGs behaves efficiently in buffered, basic water solution. The same reaction in organic solution suffers for the phase heterogeneity that reduces dramatically the conjugation yield. A clear advantage derives from the amphiphilic properties of the polymer that allow for a convenient purification procedure and recovery of products. ACKNOWLEDGMENT

This work was supported by grants from Regione Friuli-Venezia Giulia (Italy), L.R. 3/98, and from MIURItaly (Cofin 2001). The authors thanks Prof. Luig Xodo (University of Udine-Italy) for the helpful discussions. LITERATURE CITED (1) Manoharan, M. (2001) Oligonucleotide conjugates in antisense technology. In Antisense Drug Technology (Crooke, S., Ed.) pp 391-469, Marcel Dekker, Inc. (2) MacKellar, C., Grahham, D., Will, D. W., Burgess, S., and Brown, T. (1992) Synthesis and physical properties of antiHIV antisense oligonucleotides bearing terminal lipophilic groups. Nucleic Acids Res. 20, 3411-3417. (3) Garcia de la Torre, B., Albericio, F., Saison-Behmoaras, E., Bachi, A., and Eritja, R. (1999), Synthesis and binding properties of oligonucleotides carrying nuclear localization sequences. Bioconj. Chem. 10, 1005-1012. (4) Magda, D., Wright, M., Crofts, S., Lin, A., and Sessler, J. L. (1997) Metal complex conjugate of antisense DNA which displays ribozyme-like activity. J. Am. Chem. Soc. 119, 69476948.

Bioconjugate Chem., Vol. 14, No. 5, 2003 1043 (5) Markiewicz, W. T., Godzina, P., Markievicz, M., and Astriab, A. (1998) Synthesis of a polyamino-oligodeoxyribonucleotide combinatorial library, Nucleosides Nucleotides 17, 18711880. (6) Kodera, Y., Matsushima, A., Hiroto, M., Nishimura, H., Ishii, A., Ueno, T., and Inada, Y. (1998) PEGylation of proteins and bioactive substances for medical and technical applications. Prog. Polym. Sci. 23, 1233-1271. (7) Pang, S. N. J. (1993) Final report on the safety assessment of poly(ethylene glycol)s. J. Am. Coll. Toxicol. 12, 429-456. (8) Drioli, S., Benedetti, F., and Bonora, G. M. (2001) Pure, homobifunctional poly(ethylene glycol) orthogonally protected: synthesis and characterization. React. Funct. Polym. 48, 119-128. (9) Zalipsky, S. (1995) Chemistry of poly(ethylene glycol) conjugates with biologically active molecules. Adv. Drug Delivery Rev. 16, 157-182. (10) Tarasow, T. M., Tinnermeier, D., and Zyzniewski, C. (1997) Characterization of oligodeoxyribonucleotide-poly(ethylene glycol) conjugates by electrospray mass spectrometry. Bioconj. Chem. 8, 89-93.

(11) Ja¨schke, A., Fu¨rste, J. P., Nordhoff, E., Hillenkamp, F., Cech, D., and Erdmann, V. A. (1994) Synthesis and properties of oligodeoxyribonucleotide-poly(ethylene glycol) conjugates. Nucleic Acids Res. 22, 4810-4017. (12) Wlotzka, B., Leva, S., Eschgfaller, B., Burmeister, J., Kleinjung, F., Kaduk, C., Muhn, P., Hess-Stumpp, H., and Klussmann, S. (2002) In vivo properties of an anti_GnRG Spiegelmer: an example of an oligonucleotide-based therapeutic substance class. Proc. Natl. Acad. Sci. U.S.A. 99, 88998902. (13) Bonora, G. M., Rossin, R., Zaramella, S., Cole, D. L., and Ravikumar, V. T. (2000) A liquid-phase process suitable for large-scale synthesis of phosphorothioate oligonucleotides. Org. Process Res. Dev. 4, 225-231. (14) Cogoi, S., Ballico, M., Bonora, G. M., Quadrifoglio, F., and Xodo, L. E. (2003) Inhibition of Ki-ras gene by a triple helixforming oligonucleotide conjugated to high-molecular weight poly(ethylene glycol) in carcinoma pancreatic cells, submitted for publication.

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