Synthesis of Linear Polyether Polyol Derivatives As New Materials for

Mar 10, 2009 - Linear polyether polyol (PEP) consisting of glycidol as repeating units ... the pendant hydroxyl groups on PEP and prepared a series of...
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Bioconjugate Chem. 2009, 20, 780–789

Synthesis of Linear Polyether Polyol Derivatives As New Materials for Bioconjugation Zhongyu Li and Ying Chau* Department of Chemical and Biomolecular Engineering, the Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China. Received December 3, 2008

Linear polyether polyol (PEP) consisting of glycidol as repeating units is a flexible hydrophilic aliphatic polymer. The polyether main chain is similar to the widely used, biocompatible polymer poly(ethylene glycol) (PEG). While linear PEG has one or two terminal hydroxyl group(s), linear PEP distinguishes itself by the large number of pendant hydroxyl groups along the polyether main chain. We propose that this property of PEP represents a major advantage over PEG, namely, by providing multiple anchorage points and increasing the possibility for introducing different functional groups. As a first step to establishing PEP as a bioconjugation material, we modified the pendant hydroxyl groups on PEP and prepared a series of mono- and heterobifunctional derivatives with the potential to join various drug entities and biomolecules. The synthesis methods and the results of characterization are reported here.

INTRODUCTION Poly(ethylene glycol) (PEG) is one of the most widely used bioconjugation materials nowadays in pharmaceutical and biomedical applications (1-10). It consists of ethylene oxide as repeating units, with one terminal hydroxyl group in the monomethoxy form (mPEG) or two in the unmodified form of PEG. PEG is useful for bioconjugation due to the following reasons: (i) lack of immunogenicity, antigenicity, and toxicity; (ii) high solubility in water and most organic solvents; (iii) extensive hydration and high flexibility of the main chain, which is at the basis of its antifouling property; and (iv) approval by FDA for human use. However, the presence of only one or two reactive groups at the ends of PEG chain means that only one or two entities (drug, peptide, protein, nucleic acid, and other biomolecules) can be attached onto PEG. This severely limits not only the loading capacity of such conjugate but also the design options for PEG-based drug-carrier systems. One solution is to arrange PEG chains into a branched architecture, such as star-like, hyperbranched, arborescent, and dendrimeric PEG (11-16). Alternating polyurethanes have also been prepared by polymerization of bis-succinate-PEG with amino acid containing three functional groups, such as lysine (17-19). However, these strategies require complicated synthesis procedures, and the number of reactive groups are still low. Here, we take a different approach and start with linear polyether polyol (PEP). Composed of glycidol (and possibly ethylene oxide) as monomeric units, PEP is a flexible hydrophilic aliphatic polymer with multiple pendant hydroxyl groups along the main chain (Figure 1). The similarity in the polymer backbone with the extensively used PEG suggests that these two polymers may share the same characteristics of being hydrophilic, water-soluble, biocompatible, and resistant to protein adsorption (2, 18). In fact, it has been shown that both linear and hyperbranched polyglycidol are nontoxic and safe in vitro and in vivo (20-22). From the point of chemical synthesis, PEP with low polydispersity and well-defined structure can be readily synthesized by anionic polymerization (23-25). By adapting existing * Corresponding author. Tel.: +852-2358 8935; Fax: +852-2358 0054; E-mail address: [email protected].

Figure 1. Polyether polyol derivatives and PEG derivatives.

methods of hydroxyl group modification, we convert the pendant hydroxyl groups into different functional groups to increase their reactivity (2). Since the number of hydroxyl groups can be controlled by the ratio of monomers during polymerization, we can readily vary the drug loading density on PEP at will. We also use orthogonal chemistry to synthesize a number of heterobifunctional PEPs to further increase the versatility of this polymer. The chemical varieties of the derivatives mean that different chemical agents and biomolecules including peptides and nucleic acids are amenable for attaching to PEP. The increased number of functional groups along the main chain enables this polymer to be more effective as a bioconjugation carrier than PEG, since (i) the payload is higher; (ii) multivalency is possible; (iii) colocalized delivery of different drugs and simultaneous presentation of biospecific ligands are allowed.

MATERIALS AND METHODS Materials. All starting compounds were used as received without additional purification except for those specified. Chemicals were purchased from Aldrich unless otherwise indicated. THF (Merck, 99%) was refluxed over sodium wire and distilled from sodium naphthalenide solution. Diethylene glycol monomethyl ether (Fluka, 99%) and DMSO (Merck, 98%) were distilled over CaH2 under reduced pressure just before use. Ethylene oxide (EO, 99.7%) was purchased from Hong Kong Special Gas Company. Ethoxyethyl glycidyl ether (EEGE) and diphenylmethylpotassium (DPMK) were prepared

10.1021/bc900036f CCC: $40.75  2009 American Chemical Society Published on Web 03/10/2009

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Figure 2. Structures and names of PEP derivatives that have been successfully synthesized and characterized.

as described elsewhere (24-26). An ultrafiltration separator and poly(ether sulfone) (PES) membranes with molar mass cutoff at 10k, 20k, 30k, and 50k g/mol as calibrated by globular proteins were purchased from the Shanghai Institute of Applied Physics, Chinese Academy of Sciences. Methods. 1H NMR and 13C NMR spectra were obtained on a DMX 500 MHz spectrometer with tetramethylsilane (TMS) as the internal standard and CDCl3 as the solvent. For poly(EEGE) and poly(EEGE-co-EO), gel permeation chromatography (GPC) was performed on a Waters HPLC system with a G1310A pump and a G1362A refractive index detector, using tetrahydrofuran (THF) as eluent at 35 °C with an elution rate of 1.0 mL/min. Two Styragel columns (HR 3 THF and HR4E THF, Waters) were calibrated by polystyrene standards (Polymer Source). For poly(Gly-co-EO), polyglycidol, and their derivatives, size exclusion chromatography (SEC) was performed in 0.1 M NaNO3 at 40 °C with an elution rate of 0.5 mL/min on the same HPLC system. Ultrahydrogel 250 (Waters) and Ultrahydrogel 1000 (Waters) columns in series were calibrated by PEG standards (Polymer Source).

Synthesis of Polyether Polyol. Polyether polyol was synthesized in two steps similar to the published methods (23-25), except that EEGE molecules were used as monomers in addition to EO. PEEGE and poly(EEGE-co-EO) were first prepared using anionic polymerization. Acid deprotection of the precursor macromolecule afforded the homopolymer polyglycidol and the random copolymer poly(Gly-co-EO). Poly(EEGE-co-EO). 1H NMR (ppm) (CDCl3): 1.19 (m, CH3CH2-), 1.33 (d, -O-CH(CH3)-O-), 3.38 (s, CH3-O-), 3.45-3.85 (m, -CH2CH2O- and -CH2CHO- of polyether polyol main chain and -CH2-O-CH(CH3)-), 4.69 (q, -O-CH(CH3)-O-). Poly(Gly-co-EO). 1H NMR (ppm) (CDCl3): 3.38 (s, CH3-O-), 3.45-3.85 (m, -CH2CH2O- and -CH2CHO- of polyether polyol main chain and the pendant -CH2-OH groups). 13C NMR (ppm) (CDCl3): 58.3 CH3-O-, 70.5-71.3 -CH2CH2O- and -CH2CHO-, 80.4-CH2CHO-, 60.7 and 63.9 -CH2-OH. Synthesis of Monofunctional Derivatives of Polyether Polyol. PEP derivatives are numbered according to Figure 2. Synthesis of the monofunctional derivatives of polyether polyol

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Figure 3. Chemical reactions and structures of the critical intermediate and final products resulting from the synthesis of monofunctional derivatives PEP. Representative PEP derivatives of each reaction type are shown as examples. Besides what is shown above, polyether polyol derivatives 2-6 were also synthesized by Williamson reaction, 8 and 9 by esterification between hydroxyl groups and acyl halide, and 11 by esterification between hydroxyl groups and anhydride groups.

(1-18) are summarized in Figure 3. The synthesis routes for PEP derivatives 1-12 can be classified into four categories: (1) Williamson reaction, (2) esterification between hydroxyl groups and acyl halide, (3) esterification between hydroxyl groups and anhydride, and (4) esterification between hydroxyl groups and carboxyl groups. PEP derivatives 13-18 are obtained by further reactions of PEP derivatives 1, 7, and 9, respectively. Williamson Reaction. Carboxymethyl (CM)-PEP (1), carboxyethyl(CE)-PEP (2), ethyl acetate (EA)-PEP (3), alkynylPEP (4), allyl-PEP (5), and epoxide-PEP (6) were synthesized by Williamson reaction to form ether bonds between the polymer and the residues (27-31). PEP derivatives 1-5 were synthesized in a homophase system, in which PEP reacted with 2-chloroacetic acid, 3-bromopropionic acid, ethyl bromoacetate, propargyl bromide, and allyl chloride respectively in THF with NaH (27). Synthesis of CM-PEP (1) shown in Figure 3, is described in detail here as a typical example. One gram PEP (Mn ) 15.4 × 103 g/mol, 1.25 mmol hydroxyl groups) with moisture removed by azeotropic distillation with toluene just before use was reacted with 0.6 g NaH (2.5 mmol) in 10 mL anhydrous tetrahydrofuran (THF) at room temperature for 20 min, and then 0.43 g 2-chloroacetic acid (4.6 mmol) was added under vigorous stirring at room temperature for 24 h. After neutralization by hydrochloride solution, the solvent was removed by rotary evaporation. The product was purified by ultrafiltration. Filtered aqueous solution was concentrated to dryness, dissolved in dichloromethane (DCM), and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried in vacuo at 50 °C for 2 days, and a white product was obtained. Epoxide-PEP (6) was synthesized in a heterophase system using tetrabutylammonium bromide (TBAB) as a phase transfer catalyst (PTC) (29). 0.5 g polyether polyol (Mn ) 15.4 × 103 g/mol, 0.62 mmol hydroxyl groups), 0.018 g TBAB (0.055 mmol), and 0.248 g sodium hydroxide (6.2 mmol) in 5 mL of distilled water were mixed and reacted with 0.23 g epichlorohydrin (2.2 mmol) under vigorous stirring at 50 °C for 48 h. After the addition of 30 mL DCM, the organic phase was separated from the aqueous phase,

dried over MgSO4, filtered, and concentrated. The polymer was dissolved in water and purified by ultrafiltration. The filtered aqueous solution was concentrated to dryness, dissolved in DCM, and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried under vacuum at 50 °C for 2 days to afford a white product. Esterification between Hydroxyl Groups and Acyl Halide. Tosylate (Tos)-PEP (7), p-nitrophenyl formate (pNP)-PEP (8), and 2-bromoisobutyryl-PEP (9) were synthesized by the esterification between PEP and p-toluenesulfonyl chloride(TsCl), p-nitrophenyl chloroformate, and 2-bromoisobutyryl bromide, respectively, in DCM with triethylamine (TEA) as a catalyst (24, 32-34). Synthesis of Tos-PEP, as shown in Figure 3, is described here in detail as a typical example. Two grams of PEP (Mn ) 15.4 × 103 g/mol, 2.5 mmol hydroxyl groups) with moisture removed by azeotropic distillation with toluene was reacted with 0.629 g p-toluenesulfonyl chloride (3.3 mmol) in the presence of 0.3 mL TEA in 10 mL anhydrous DCM at 0 °C for 24 h. The solvent was removed by rotary evaporation. The product was purified by ultrafiltration. The white precipitate of triethylamine hydrochloride was filtered off and washed with DCM. Filtered aqueous solution was concentrated to dryness, dissolved in DCM, and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried in vacuo at 50 °C for 2 days to yield a white product. Esterification between Hydroxyl Groups and Anhydride Groups. Succinate-PEP (10) and maleate-PEP (11) were synthesized by the esterification (35-37) between PEP and succinic anhydride and maleic anhydride, respectively, in DCM with TEA as a catalyst. Synthesis of Succinate-PEP, as shown in Figure 3, is described here in detail as a typical example (35). 0.5 g PEP (Mn ) 15.4 × 103 g/mol, 0.62 mmol hydroxyl groups) with moisture first removed by azeotropic distillation with toluene was reacted with 0.177 g succinic anhydride (1.77 mmol) in the presence of 0.1 mL TEA in 10 mL DCM at room temperature or 24 h. The solvent was removed by rotary evaporation. The product was purified by ultrafiltration. Filtered aqueous solution was concentrated to dryness, dissolved in

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Figure 4. Synthesis of heterobifunctional polyether polyol derivatives containing alkynyl groups. (Similar methods are fit for synthesis of heterobifucntional PEP derivatives containing allyl groups).

DCM, and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried in vacuo at 50 °C to yield a white product. Esterification between Hydroxyl Groups and Carboxyl Group. 3-Mercaptopropionate-PEP (12) was synthesized by the esterification (38, 39) between PEP and 3-mercaptopropionic acid. A typical experiment (Figure 3) was as follows: 2 g PEP (Mn ) 15.4 × 103 g/mol, 2.5 mmol hydroxyl groups) was dissolved in 20 mL toluene; then, 0.318 g 3-mercaptopropionic acid (MPA) (3 mmol) along with HfCl4 · 2THF (2 mg, 0.004 mmol) were added and stirred at 50 °C under nitrogen. The reaction flask was equipped with an azeotropic distillation apparatus, and the mixture was refluxed at 110 °C under nitrogen for 24 h. Toluene was removed under reduced pressure. The crude product was dissolved in ethanol and was purified by an ultrafiltration membrane to remove low molar mass impurities under the pressure of nitrogen. Finally, ethanol was removed from the product by distillation and the remains were dried in vacuo at 50 °C for 2 days to afford a white product. Other Reactions. N-Succinimidyl carbonate (SC)-PEP (13) was derived from CM-PEP (1) using a procedure adapted from Zalipsky et al. (40) Two grams CM-PEP (Mn ) 16.5 × 103 g/mol, 2.3 mmol carboxyl groups) was reacted with 0.824 g N,N′-dicyclohexylcarbodiimide (DCC, 4 mmol) and 0.46 g N-hydroxysuccinimide (NHS, 4 mmol) in the presence of 0.2 mL TEA in 10 mL DCM at room temperature for 24 h. The crude product was concentrated and filtered to remove the byproduct 1,3-dicyclohexylurea (DCU). The filtered solution was precipitated in diethyl ether. The product was redissolved in DCM and precipitated again in diethyl ether. This step was repeated twice, and the final product was dried in vacuo at 50 °C. N-Cysteinyl formamide methyl-PEP (14) was derived from SC-PEP (13) using a procedure adapted from Zalipsky et al. (40) 0.39 g cystine hydrochloride (2.5 mmol) dissolved in 20 mL pH 8.0 phosphate buffer was reacted with 0.5 g SC-PEP (Mn ) 19.3 × 103 g/mol, 0.497 mmol SC groups), which was added in small aliquots. The solution was stirred vigorously at room temperature overnight. The product was purified by ultrafiltration. Filtered aqueous solution was concentrated to dryness, dissolved in DCM, and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried in vacuo at 50 °C. Amino-PEP (17) was derived from Tos-PEP (7) via the formation of phthalimide-PEP (16). The method is modified from the synthesis route of amino PEG by Mutter et al. (41) 4 g PEP (Mn ) 15.4 × 103 g/mol, 5.0 mmol -OH groups) and 3.1 g phthalimide potassium (16.7 mmol) were dissolved in 50 mL DMF and heated at 40 °C with stirring for 18 h. The mixture

was cooled and evaporated and dissolved in 200 mL DCM. The organic solution was washed with water (2 × 50 mL) and dried over magnesium sulfate. After filtration and evaporation under reduced pressure, phthalimide-PEP was precipitated in diethyl ether and dried in vacuo at 50 °C. 0.5 g phthalimide-PEP (16) (Mn ) 17.5 × 103 g/mol, 0.55 mmol phthalimide groups) was dissolved in ethanol (200 mL), and ammonia hydrate (40 mL) was added. The mixture was stirred at 30 °C for 24 h and then evaporated under reduced pressure. The crude product was dissolved in DCM and stirred for 24 h at room temperature. The solution was filtered and washed with water (2 × 50 mL) and dried over magnesium sulfate. The product, amino-PEP, was precipitated in diethyl ether and dried in vacuo at 50 °C. Azido-PEP (15) and 2-azidobutyryl-PEP (18) were derived using similar methods from Tos-PEP (7) and 2-bromoisobutyrylPEP (9), respectively. Synthesis of azido-PEP (15) illustrated in Figure 3 follows a method adapted from Mutter (41). 0.5 g Tos-PEP (Mn ) 20.1 × 103 g/mol, 0.478 mmol tosylate groups) was dissolved in 10 mL DMF, and then sodium azide was added under vigorous stirring at room temperature for 6 h. The product was purified by ultrafiltration. Filtered aqueous solution was concentrated to dryness, dissolved in DCM, and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried in vacuo at 50 °C to yield a white product. Synthesis of Heterobifunctional Derivatives of Polyether Polyol. Synthesis of heterobifunctional derivatives 19-23 containing alkynyl groups is illustrated in Figure 4. As an example, the synthesis of alkynyl succinate-PEP is described here in detail. Alkynyl-PEP was obtained by Williamson reaction: 0.5 g alkynyl-PEP (Mn ) 16.2 × 103 g/mol, containing 0.25 mmol alkynyl groups and 0.59 mmol hydroxyl groups) with moisture removed was then reacted with 0.177 g succinic anhydride (1.77 mmol) in the presence of 0.1 mL TEA in 10 mL DCM at room temperature for 24 h. The solvent was removed by rotary evaporation. The product was purified by ultrafiltration. Filtered aqueous solution was concentrated to dryness, dissolved in DCM, and dried over anhydrous MgSO4. The filtrate was distilled under vacuum to remove DCM and dried in vacuo at 50 °C to obtain a white product. Synthesis methods for heterobifunctional derivatives 24-27 containing allyl groups follows similar routes, except that allyl chloride was first used to react with PEP to obtain allyl-PEP.

RESULTS AND DISCUSION Synthesis and Characterization of Polyether Polyol (PEP). We have synthesized a number of polyether polyols which vary in their molar mass (from 6k to 20k g/mol) and

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Table 1. Characterization of Polyether Polyol and Their Precursors

A B C D E F G

feed ratioa EEGE:/EO

measured ratiob on copolymer EEGE/EO

Mn (×10-3) designedc (g/mol)

precursord Mn (×10-3) (g/mol), [polydispersity]

PEPe Mn (×10-3) (g/mol), [polydispersity]

NOHf

1:0 1:0 1:0 1:19 1:19 1:9 1:5

1:0 1:0 1:0 1:18.4 1:19.2 1:8.6 1:5.3

5 10 20 20 15 10 5

4.95 [1.06] 10.1 [1.08] 19.6 [1.05] 17.4 [1.06] 13.5 [1.08] 11.7 [1.05] 5.8 [1.07]

2.23 [1.08] 5.01 [1.11] 9.76 [1.09] 15.4 [1.09] 11.1 [1.10] 10.3 [1.08] 4.7 [1.12]

34.2 69.5 134 19.2 14.5 23.1 16.5

a The feed ratio of EEGE to EO. b The molar ratio of EEGE to EO in copolymer poly(EEGE-co-EO) measured by 1H NMR. c The designed number-average molar mass of precursor polymer. d Number-average molecular weight and polydispersity of precursor polymer are determined by GPC. Polydispersity is equal to the ratio of weight-average molar mass and number-average molar mass. e Number-average molar mass and polydispersity of PEP are determined by SEC. f The number of hydroxyl groups in polyether polyol: NOH ) NEEGE + 1, in which NEEGE was calculated by the integration of the peak in shift 1.19 ppm (m, CH3CH2-) and the integration of the peak in shift 3.38 ppm (s, CH3-O-) of 1H NMR data of poly(EEGE-co-EO).

ratio Gly/EO (from 1:0 to 1:19) (Table 1). The molar mass of these polymers can be well controlled by the input amount of initiator and monomer, and the resulting polymers are uniform in size. This feature is essential for bioconjugation materials. The polymer size influences the pharmacokinetics and biodistribution (42, 43). We expect PEP to behave similarly to PEG: the small molar mass PEG is cleared rapidly from the body, while PEG larger than 20k g/mol exhibits prolonged circulation in the body. The size also affects the extravasation into tissues and the uptake by the reticuloendothelial system (44). The number of hydroxyl groups present on PEP dictates the number of anchorage points for drug conjugation. This can be designed by the ratio Gly/EO, which is in turn controlled by the feed ratio EEGE/EO in making the precursor of PEP (Table 1). Compared to linear PEG, which has one or two linkage points, PEP is a more efficient carrier in terms of higher number of drug molecules per polymer. The ability to control the hydroxyl density means that we can optimize the drug loading density in the polymer design stage, and this will introduce more opportunities to improve drug potency by taking advantage of the multivalency effects (45, 46). Polyether polyol has a main chain similar to that of PEG and pendant hydroxyl groups similar to those on polyvinyl alcohol (PVA). It therefore possesses the same advantage of high water solubility as these two polymers. Each oxygen atom on the polyether main chain is a good hydrogen bond acceptor capable of interacting with two water molecules, while the pendant hydroxyl groups act as hydrogen donors for the surrounding water molecules. This property of polyether polyol means that it is a good drug delivery carrier of normally waterinsoluble drug molecules (47). In addition to confirming the good solubility of PEP in water, we have also assessed the solubility of polyether polyol in common organic solvents, since these properties influence the choice of reaction types, conditions, and purification methods in the subsequent preparation of PEP derivatives. While the solubility in polar solvent such as methanol is not greatly affected by the Gly/EO ratio, the solubility in nonpolar solvents such as THF, toluene, and DCM increases with decreasing Gly/EO ratio. This is expected from the decreasing hydrophilicity due to hydroxyl groups on the polymer. When the ratio Gly/EO is 1:19.2 or lower, PEP has the same solubility as PEG in these nonpolar solvents.. A simple procedure very often used to isolate PEG is ether precipitation. The efficiency of precipitating PEP in diethyl ether decreases with increasing Gly/EO ratio: a viscous gum was observed when Gly/EO > 1:5, sparing precipitate when Gly/EO ) 1:9, and white powder precipitate when Gly/EO < 1:19. Synthesis and Characterization of Derivatives of PEP. We have successfully prepared a series of PEP derivatives: 18 monofunctional derivatives and 9 heterobifunctional derivatives (Figure 2, Tables 2 and 3). They cover a variety of chemical reactivities commonly used for bioconjugation. The derivatives

have been analyzed by NMR to confirm the identity of the functional groups (Supporting Information Tables S1 and S2). The most appropriate synthesis route for each type of derivative was identified and adapted from existing methods on hydroxyl group modification. Most of the reaction yield and conversion efficiency (from hydroxyl groups to the indicated functional groups) were satisfactory (Table 2). Carboxyl-PEP and Its ActiVe Esters. Amines commonly found on peptides and drug molecules are available for covalent attachment to a polymeric carrier (2, 5). The widely used chemistry is via the formation of amide linkage with carboxyl or an active ester. We have prepared a number of derivatives in this group, which vary in the strength of chemical reactivity, length of linker, stability in water, and the presence of an additional functional group (Table 2). Although oxidation of hydroxyl groups on PEP by permanganate can easily generate carboxyl groups (30), we chose not to use this method due to the likelihood of polyether chain degradation. We have produced carboxyl-PEP via Williamson reaction (1 and 2) (Figure 3 and Table 2). For example, CMPEP was generated with 2-chloroacetic acid. The conversion efficiency of this type of reaction, however, only reached 88%. This is because the carboxyl group on 2-chloroacetic acid depletes the anionic ion in the reactive chain, and the anionic carboxyl group lowers the activity of the neighboring halogen atom. This disadvantage was overcome by first preparing ethyl acetate-PEP (3), and CM-PEP was produced by subsequent saponification. The conversion efficiency improved to 99% (Table 2 and Supporting Information Table S1). Modification of the polymer with succinic, glutaric, or maleic anhydride is a straightforward way to synthesize carboxyl polymer. The reaction can be readily driven to completion using an excess of anhydride as confirmed by 1H NMR (Supporting Information Table S1). Unlike the Williamson reaction, this route produces carboxyl-PEP with a hydrolyzable ester linkage instead of the noncleavable ether linkage. Depending on whether the therapeutic molecules to be attached to PEP need to be released in an aqueous environment, one can choose the linkage of desired stability. The reactivity toward amino group is higher for active ester than carboxyl group. SC-PEG (13) was prepared by condensing NHS with CM-PEP using DCC as the coupling agent. The conversion efficiency was only about 80%. A more efficient approach we found was by esterification with acyl halide. Easy and quantitative conversion was achieved by transforming hydroxyl groups into p-nitrophenyl formate groups (8) and tosylate groups (14). It should be pointed out that tosylate-PEP is also an important intermediate for producing azido-PEP (Figure 2). Mercapto-PEP and Epoxide-PEP. This group of PEP derivatives can join covalently with the thiol groups on biomolecules or small drugs. The thiol linkage has been widely adopted in

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Table 2. Summary of Reaction Yield, Conversion Efficiency, and Reactivity of Monofunctional PEP Derivatives

a Yield (%) ) Wproduct/Wprecursor × 100%. b Conversion efficiency (%) ) [functional group]product/[OH]precursor × 100%. c CM-PEP is synthesized by PEP reacting with 2-chloroacetic acid directly. d CM-PEP is synthesized by hydrolysis of ethyl acetate (EA)-PEP 5.

attaching PEG to therapeutic molecules and proteins (48, 49). Epoxide-PEP is an electrophile of mild reactivity toward hydroxyl groups and amino groups and better reactivity toward thiols (50). In our early attempts, 3-mercaptopropionate-PEP was prepared by esterification between hydroxyl groups and 3-mercaptopropionic acid in the presence of concentrated sulfuric acid as catalyst (51). We also tried to catalyze this reaction with DCC and DMAP. Both methods resulted in conversion efficiency of less than 10% according to 1H NMR. The successful reaction reported in this paper used HfCl4 · 2THF, a recently developed and highly efficient catalyst, for the esterification between carboxyl groups and hydroxyl groups (38, 39).A gel was formed as a result of

cross-linking between thiol groups in 3-mercaptopropionatePEP. When the gel was treated with DL-dithiothreitol (DTT), sodium borohydride, or lithium aluminum anhydride, the disulfide bonds were reduced to thiol groups. Amino-PEP. Compared with hydroxyl groups, primary amino groups have higher reactivity in nucleophilic substitution reactions, making them useful for conjugating with carboxyl groups and halide (2). The transformation of hydroxyl groups to amino groups was difficult, and the conversion efficiency was the lowest among all PEP derivatives. The reaction involved three steps: the conversion of hydroxyl group to tosylate group, followed by the formation of phthalimide, and finally the transformation of phthalimide into amino group. While the efficiency of the first step was

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Table 3. Summary of Reaction Yield, Conversion Efficiency, and Reactivity of Heterobifunctional PEP Derivatives

a R1functional group is -OCH2CCH, the conversion efficiency is 30%. b The conversion efficiency here is the [R2]/[OH]; therein, [R2] is the number of -R2 group in heterobifunctional PEP; [OH] is the total number of the -OH group in the original PEP. c R1 functional group is -OCH2CHCH2; the conversion efficiency is 41%.

close to 100% according to 1H NMR, the efficiency of subsequent steps suffered from the generation of a large amount of salt and high viscosity as a result of excess phthalimide potassium insoluble in methanol. Azido-PEP and Alkynyl-PEP. Azido groups are able to react with alkynyl groups in cycloaddition to form a stable triazole linkage (52, 53). Since this “click chemistry” occurs in mild conditions, and the reaction is highly specific, it has found many applications in bioconjugation (53-57). This group of derivatives was prepared with high yield and conversion efficiency. Dual ReactiVities. The maleate and N-cysteinyl formamide methyl pendant groups provide dual reactivities, which make the PEP derivatives useful for conjugating with two different entities. This feature may be useful for developing drug carriers for combination therapy. The carboxyl groups and double bonds on maleate-PEP are reactive toward aminoand thiol-containing compounds, while the thiol and carboxyl groups on N-cysteinyl formamide methyl-PEP are reactive toward thiol- and amino-containing compounds. Although both N-cysteinyl formamide methyl-PEP and 3-mercaptopropionate-PEP are thiol-reactive, the former has a more stable linker (comprising an amide bond) than the latter (comprising an ester bond). Heterobifunctional Polyether Polyol DeriVatiVes. Heterobifunctional polymers provide more options for designing drug carriers. They are also useful spacers between surfaces and various ligands (58-61). Compared to heterobifunctional PEG, the synthesis and purification of heterobifunctional PEP derivatives are much simpler. Furthermore, controlling the ratio of the different functional groups is feasible. If PEG diol is modified to generate heterobifunctional groups, the separation among the modified PEG, the macromolecular intermediates, and the starting PEG is challenging, owing to the similarity in physical properties including size (62-64). Thus, heterobifunctional PEG is mostly prepared starting from anionic polymerization using a heterofunctional initiator (65-72). Since PEP carries multiple hydroxyl groups, they can be transformed into heterofunctional derivatives by successive

modification steps. Separation between PEP derivatives and the small chemical reagents for modification can be readily achieved, for example, by membrane purification. We have demonstrated this approach by synthesizing two groups of heterobifunctional polyether polyol derivatives, one containing alkynyl groups and the other containing allyl groups (Figure 2, Table 3). The presence and the amount of different functional groups were characterized using NMR (Supporting Information Tables S1 and S2), which were tunable by the number of hydroxyl groups on PEP and the feeding amount of the modifying chemicals. These heterobifunctional derivatives of polyether polyol are potentially useful as drug carriers for two different drugs. They can also be used to bind to a surface or coat a particle with one type of functional groups, leaving the other type of functional groups to couple with drugs, peptides, proteins, DNA, or imaging probes. The alkynyl series were designed to conjugate with azide-containing drugs or azide-modified peptides and proteins, and the allyl series to conjugate with thiol-containing compounds by the radical addition reaction in the presence of 2,2′-azobisiosobutyronitrile (AIBN) (66, 67).

CONCLUSION A series of polyether polyol derivatives have been successfully prepared as materials for conjugating with drugs and biomolecules. The number of functional groups can be wellcontrolled. Heterobifunctional derivatives of polyether polyol can be readily prepared, and they should offer more flexibility in designing the next generation of polymer-drug conjugates.

LIST OF ABBREVIATIONS AIBN CM CE DCC DCM DCU

2,2′-azobisiosobutyronitrile carboxymethyl carboxyethyl dicyclohexylcarbodiimide dichloromethane 1,3-dicyclohexylurea

Polyether Polyol Derivatives for Bioconjugation DMSO DMAP DMF DTT EA EEGE EO Gly HfCl4 · 2THF PEG mPEG mTEG MPA NHS PG PEP Poly(Gly-co-EO) PBS pNP PTC SC SEC GPC TEA THF TMS TsCl Tos

dimethyl sulfoxide (dimethylamino) pyridine N,N-dimethylformamide DL-dithiothreitol ethyl acetate ethoxyethyl glycidyl ether ethylene oxide glycidol hafnium chloride · tetrahydrofuran poly(ethylene glycol) monomethoxyl poly(ethylene glycol) triethylene glycol monomethyl 3-mercaptopropionic acid N-hydroxysuccinimide polyglycidol polyether polyol poly(glycidol-co-ethylene glycol) phosphate buffered saline p-nitrophenyl formate phase transfer catalyst succinimidyl carbonate size exclusion chromatography gel permeation chromatography triethylamine tetrahydrofuran tetramethylsilane p-toluenesulfonyl chloride tosylate

ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Hong Kong Research Grant Council (Earmarked Research Grant HKUST 6407/06M). Supporting Information Available: Table S1 and Table S2 are 1H NMR and 13C NMR data of synthetic PEP derivatives and their precursors. This material is available free of charge via the Internet at http://pubs.acs.org.

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