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Bioconjugate Chem. 2004, 15, 424−427
TECHNICAL NOTES Synthesis of Heterotelechelic Poly(ethylene glycol) Derivatives Having r-Benzaldehyde and ω-Pyridyl Disulfide Groups by Ring Opening Polymerization of Ethylene Oxide Using 4-(Diethoxymethyl)benzyl Alkoxide as a Novel Initiator Yoshitsugu Akiyama,† Yukio Nagasaki,‡ and Kazunori Kataoka*,† Department of Materials Science, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, and Department of Material Science & Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan. Received September 30, 2003; Revised Manuscript Received January 15, 2004
New heterotelechelic PEG-containing benzaldehyde and 2-pyridyldithio endgroup (CHO-Bz-PEG-SSpyl) was synthesized with high efficiency and high selectivity. An R-benzylacetal-ω-methansulfonyl PEG was prepared as the first step to CHO-Bz-PEG-SSpyl through the ring-opening polymerization of ethylene oxide (EO) initiated by potassium 4-(diethoxymethyl)benzyl alkoxide (PDA), followed by the successive conversion of the end-alkoxide group to a methanesulfonyl group and then to dithiocarbonate derivative. Further, deprotection of the dithiocarbonate derivative and subsequent conversion to the 2-pyridyldithio group at the ω-end was successfully performed through a one-step reaction to form R-benzylacetal-ω-2-pyridyldithio PEG (aceBz-PEG-SSpyl). The aceBz-PEG-SSpyl was then treated with an aqueous HCl solution (pH 5.0) to generate the benzaldehyde group at the R-end. Molecular functionalities of the benzaldehyde and the 2-pyridyldithio end group of the heterotelechelic PEG (CHO-Bz-PEG-SSpyl) thus prepared were characterized by 1H and 13C NMR, showing that the reaction proceeded almost quantitatively. The benzaldehyde end group is available to conjugate various ligands having a primary amino group by forming the pH-sensitive imine linkage (-NdCHC6H4-).
Heterotelechelic poly(ethylene glycol) (PEG) derivatives possessing different functional groups at the R- and ω-chain ends have received increasing interest as PEGylation reagents (1-4) in such areas as drug delivery systems (DDS) (5-7) and diagnostics (8-11). For example, protein conjugates with heterotelechelic PEG are expected not only to have a propensity of decreased immunogenicity due to steric repulsion effects, but also to possess the availability of further conjugation of the ligand moiety to the distal end of PEG. Although several types of heterotelechelic PEGs have already been prepared based on the derivatization of R,ω-dihydroxy-PEG (12-15), there are still many obstacles such as complicated reaction steps with low yield and limited selectivities. These obstacles in the heterotelechelic PEG synthesis may be resolved by taking the alternative approach of the direct ring opening polymerization of ethylene oxide (EO) using a metal alkoxide initiator with a protected functional group (16-20). As a novel heterotelechelic PEG with high potential utility in bioconjugation field, we focused on the one with R-benzaldehyde and ω-2-pyridyldithio groups. The benzaldehyde group is able to react with the primary amino * To whom correspondence should be addressed. Professor Kazunori Kataoka: Tel: +81-3-5841-7138, Fax: +81-3-58417139, e-mail:
[email protected]. † The University of Tokyo. ‡ Tokyo University of Science.
group under physiological condition (pH 7.4), generating a stable imine linkage (-NdCHC6H4-) which needs no further reductive amination. The imine linkage is also known to be hydrolyzed at acidic condition (21, 22). Furthermore, self-condensation of PEG due to an aldol reaction, which is a major side reaction in the utilization of aldehyde-functionalized PEG derivatives, does not occur in the benzaldehyde-functionalized PEG due to a lack of R-proton. The 2-pyridyldithio group at the ω-end can be utilized for the selective PEGylation at the cysteine residue of natural as well as recombinant protein molecules (23, 24). Note that the R-benzaldehyde-ω2-pyridyldithio PEG is also useful for the functional PEGylation of gold and silver substrates used in bioassay systems, including surface plasmon resonance and colloidal colorimetry (25). In this study, a novel synthetic route was established for R-benzaldehyde-2-pyridyldithioPEG (CHO-Bz-PEG-SSpyl) through the ring opening polymerization of EO initiated with potassium 4-(diethoxymethyl)benzylalkoxide (PDA) as summarized in Scheme 1. 4-(Diethoxymethyl)benzyl alcohol was prepared by the reduction of 4-(diethoxymethyl)benzaldehyde with sodium borohydride (NaBH4) in dry methanol according to the literature with a slight modification (26). Conversion to the 4-(diethoxymethyl)benzyl alcohol was confirmed by 1H and 13C NMR (JEOL EX 300 spectrometer) analysis (S-1,2; Supporting Information). Potassium 4-(diethoxy-
10.1021/bc0341775 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/26/2004
Bioconjugate Chem., Vol. 15, No. 2, 2004 425 Scheme 1
methyl)benzylalkoxide (PDA) as an initiator for EO polymerization was then prepared by metalation of 4-(diethoxymethyl)benzyl alcohol with potassium naphthalene. The synthesis of the heterotelechelic PEG possessing benzylacetal and methansulfonyl endgroups (aceBzPEG-OSO2CH3) was done as the first step to CHO-BzPEG-SSpyl as follows: THF solutions of 4-(diethoxymethyl)benzyl alcohol (0.19 g, 0.90 mmol) and potassium naphthalene (0.90 mmol) were mixed into dry THF (30 mL) under an argon atmosphere to form PDA. After the mixture was stirred for 10 min, 4.5 g (102 mmol) of liquid EO, cooled below 0 °C, was added to the solution via a cooled syringe. After the mixture was allowed to react for 2 days at room temperature, potassium naphthalene (0.4 mmol) and triethylamine (0.82 g, 8.1 mmol) were added to the stirring polymer solution. Then, the solution was slowly added dropwise into methanesulfonyl chloride (0.72 g, 6.3 mmol) in dry THF (10 mL), to introduce methanesulfonyl groups at one of the PEG ends without any side reactions, and then added to the chloroform followed by washing with saturated NaClaq for purification. The organic layer was dried with anhydrous MgSO4. After evaporation of excess solvent, samples were reprecipitated into diethyl ether to obtain the polymer as a white precipitate. The recovered polymer was dried in vacuo and then freeze-dried from benzene. The yield of the obtained polymer after purification was 82% (3.69 g). From the GPC diagram (TOSOH HLC-8220: a TSKgel column (G4000HHR and G3000HHR)), the number-average molecular weight (Mn) and the molecular weight distribution (MWD) were determined to be 5410 and 1.03, respectively (S-3, Supporting Information). The polymer has a very narrow MWD, and its Mn is close to the calculated value derived from the monomer/initiator ratio, indicating that the polymerization of EO from PDA as an initiator proceeds without any remarkable side reactions. The end-group analysis was then done by 1H NMR spectroscopy according to the assignments of PEG and 4-(diethoxymethyl)benzyl alcohol (S-4, Supporting Information). The molecular weight determined from the intensity ratio of the oxymethylene peak of the PEG signals (OCH2CH2) with the methyl proton peaks of benzylacetal ((CH3CH2O)2CH) was 5820, which is in a good agreement with the Mn determined by GPC analysis (Mn 5410). To convert a methanesulfonyl group to a O-ethyldithiocarbonate group, the aceBz-PEG-OSO2CH3 was reacted with potassium O-ethyldithiocarbonate in the mixture of THF and DMF under an argon atmosphere for 4 h. The ω-end conversion from methanesulfonate to the ethyl-
dithiocarbonate moiety was confirmed to be quantitative based on the 1H NMR data (S-5, Supporting Information). The ω-end conversion to the 2-pyridyldithio group was then performed by the deprotection of the dithiocarbonate moiety in THF as follows: AceBz-PEG-S(CdS)OCH2CH3 (100 mg, 0.0186 mmol) and 2-pyridyl 2-disulfide (410 mg, 1.86 mmol) were dissolved in dry THF (15 mL), and n-propylamine (1.75 mL) was slowly added. The mixture was stirred for 3 h at room temperature and then dialyzed against MeOH followed by the complete evaporation of the solvent under vacuum. The polymer was purified by ether reprecipitation from CHCl3 solution. Finally, the obtained polymer was freeze-dried from benzene (yield: 89 mg (89%)). Figure 1 shows the 1H NMR spectrum of the obtained aceBz-PEG-SSpyl. The signals of the methyl (1.4 ppm) and methylene (4.6 ppm) protons of the O-ethyldithiocarbonate moiety had completely disappeared, while new peaks appeared at 7.1, 7.7, 7.8, and 8.5 ppm assignable to the pyridyl moiety. The ω-end conversion of O-ethyldithiocarbonate to the 2-pyridyldithio group was confirmed to be 99% based on the peak ratio of pyridyl to benzylacetal in the 1H NMR spectrum. The aceBz-PEG-SSpyl was then treated with an aqueous HCl solution (pH 5.0) to generate the benzaldehyde group at the R-end through the deprotection of the benzylacetal moiety under the following conditions:
Figure 1. 1H NMR spectrum of aceBz-PEG-SSpyl in CDCl3 at room temperature.
426 Bioconjugate Chem., Vol. 15, No. 2, 2004
Figure 2.
13C
NMR spectra in CDCl3 at room temperature of (a) aceBz-PEG-SSpyl and (b) CHO-Bz-PEG-SSpyl.
aceBz-PEG-SSpyl (70 mg, 0.013 mmol) was dissolved in an aqueous HCl solution (pH 5.0; 10 mL) and allowed to react for 6 h at room temperature. Dialysis against distilled water was then carried out followed by freezedrying. Figure 2 shows 13C NMR spectra of the polymer sample before and after the deprotection of the benzylacetal group. Carbon signals of the benzylacetal group, such as CH3CH2O and CH3CH2O appearing at 15.0 and 60.8 ppm, respectively, completely disappeared after the treatment with the aqueous HCl solution (pH 5.0) (Figure 2b). On the other hand, the deprotected sample has a new peak in the 13C NMR spectrum appearing at 191.8 ppm, assignable to the aldehyde of the benzaldehyde group. These results clearly indicate the successful conversion of the R-benzylacetal group to the benzaldehyde group, which was further confirmed by 1H NMR analysis as shown in Figure 3. Signals of the methyl and methylene groups of the benzylacetal group completely disappeared, while a new peak appeared at 10.06 ppm (s, 1H), which was attributable to the benzaldehyde proton at the end of the polymer chain. The R-end conversion from the benzylacetal to benzaldehyde moiety was again confirmed to be almost quantitative (90%) based on the peak intensity ratio of the aldehyde proton of the benzaldehyde group (10.06 ppm) and the methylene protons next to the 2-pyridyl disulfide group (3.0 ppm) in the 1H NMR, indicating an almost quantitative conversion of the R-end to the benzaldehyde group. It should be noted that the intensity of the signals derived from the pyridyl end groups did not change throughout the deprotection process under the acidic conditions, indicating that the obtained PEG contains benzaldehyde (R-end) and 2pyridyldithio (ω-end) groups. Worth mentioning is the fact that the benzaldehyde group has no active proton on the R-carbon to undergo aldol condensation. Indeed, the peak for dimerization products was not detected (data not shown). Consequently, the obtained heterotelechelic
Figure 3. 1H NMR spectrum of CHO-Bz-PEG-SSpyl in CDCl3 at room temperature.
CHO-Bz-PEG-SSpyl and its precursor, aceBz-PEG-SSpyl, have a wide potential utility as selective heterolinkers in various bioconjugation reactions as well as surface modifiers of gold and silver substrates to construct reactive PEG-brush layers useful for biosensing, such as surface plasmon resonance (SPR) sensor technology. Research in this direction is now underway in our laboratory, and the results will be reported elsewhere. ACKNOWLEDGMENT
This work was financially supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science
Bioconjugate Chem., Vol. 15, No. 2, 2004 427
and Technology of Japan (MEXT) as well as by the Core Research Program for Evolutional Science and Technology (CREST) from the Japan Science and Technology Corporation (JST). Supporting Information Available: Experimental procedures for the synthesis of 4-(diethoxymethyl)benzyl alcohol, aceBz-PEG-OSO2CH3, and aceBz-PEG-S(CdS)OCH2CH3, as well as GPC and 1H NMR of new polymers. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Harris, J. M., Ed. (1992) Poly(ethylene glycol) Chemistry, Biotechnical and Biomedical Applications, Plenum Press, New York. (2) Harris, J. M., and Zalipsky, S., Ed. (1998) Poly(ethylene glycol): Chemistry and Biological Applications, ACS Symposium Series 680, American Chemical Society, Washington, DC. (3) Anne, A., Demaille, C., and Mirous, J. (1999) Elastic bounded diffusion, dynamics of ferrocene-fabeled poly(ethylene glycol) chains terminally attached to the outermost monolayer of successively self-assembled monolayers of immunoglobulins. J. Am. Chem. Soc. 121, 10379-10388. (4) Wen, X., Wu, Q.-P., Lu, Y., Fan, Z., Charnsangave, C., Wallace, S., Chow, D., and Li, C. (2001) Poly(ethylene glycol)conjugated anti-EGF receptor antibody C225 with radiometal chelator attached to the termini of polymer chains. Bioconjugate Chem. 12, 545-553. (5) Kataoka, K., Harada, A., and Nagasaki, Y. (2001) Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Deliv. Rev. 47, 113131. (6) Deguchi, Y., Kurihara, A., and Pardridge, W. M. (1999) Retention of biologic activity of human epidermal growth factor following conjugation to a blood-brain barrier drug delivery vector via an extended poly(ethylene glycol) linker. Bioconjugate Chem. 10, 32-37. (7) Greenwald, R. B., Yang, K., Zhao, H., Conover, C. D., Lee, S., and Filpula, D. (2003) Controlled release of proteins from their poly(ethylene glycol) conjugates: drug delivery systems employing 1,6-elimination. Bioconjugate Chem. 14, 395-403. (8) Otsuka, H., Nagasaki, Y., and Kataoka, K. (2001) Selfassembly of poly(ethylene glycol)-based block copolymers for biomedical applications. Curr. Opin. Colloid Interface Sci. 6, 3-10. (9) Kurihara, A., and Pardridge, W. M. (2000) Aβ1-40 reptide radiopharmaceuticals for brain amyloid imaging: [1]In chelation, conjugation to poly(ethylene glycol)-biotin linkers, and autoradiography with alzheimer’s disease brain sections. Bioconjugate Chem. 11, 380-386. (10) Unger, E. C., Shen, D., Wu, G., Stewart, L., Matsunaga, T. O., and Trouard, T. P. (1999) Gadolinium-containing copolymeric chelates-a new potential MR contarast agent. Magn. Reson. Mater. Phys., Biol. Med. 8, 154-162. (11) Torchilin, V. P. (1999) Polymeric micelles in diagnostic imaging. Colloids Surf. B. 16, 305-319. (12) Roberts, M. J., Bentley, M. D., and Harris, J. M. (2002) Chemistry for peptide and protein PEGylation. Adv. Drug Delivery Rev. 54, 459-476.
(13) Zalipsky, S. (1995) Functionalized poly(ethylene glycols) for preparation of biologically relevant conjugates. Bioconjugate Chem. 6, 150-165. (14) Kaiser, K., Marek, M., Haselgrubler, T., Schindler, H., and and Gruber, H. J. (1997) Basic studies on heterobifunctional biotin-PEG conjugates with a 3-(4-pyridyldithio)propionyl marker on the second terminus. Bioconjugate Chem. 8, 545551. (15) Bettinger, T., Remy, J. S., Erbacher, P., and and Behr, J. P. (1998) Convenient polymer-supported synthetic route to heterobifunctional poly(ethylene glycol)s. Bioconjugate Chem. 9, 842-846. (16) Nagasaki, Y., Kutsuna, T., Iijima, M., Kato, M., and Kataoka, K. (1995) Formyl-ended heterobifunctional poly(ethylene oxide): synthesis of poly(ethylene oxide) with a formyl group at one end and a hydroxyl group at the other end. Bioconjugate Chem. 6, 231-233. (17) Nagasaki, Y., Iijima, M., Kato, M., and Kataoka, K. (1995) Primary amino-terminal heterobifunctional poly(ethylene oxide), facile synthesis of poly(ethylene oxide) with a primary amino group at one end and a hydroxyl group at the other end. Bioconjugate Chem. 6, 702-704. (18) Nakamura, T., Nagasaki, Y., and Kataoka, K. (1998) Synthesis of heterobifunctional poly(ethylene glycol) with a reducing monosaccharide residue at one end. Bioconjugate Chem. 9, 300-303. (19) Yokoyama, M., Okano, T., Sakurai, Y., Kikuchi, A., Osako, N., Nagasaki, Y., and Kataoka, K. (1992) Synthesis of poly(ethylene oxide) with heterobifuntional reactive groups at its terminals by an anionic initiator. Bioconjugate Chem. 3, 275276. (20) Akiyama, Y., Otsuka, H., Nagasaki, Y., Kato, M., and Kataoka, K. (2000) Selective synthesis of heterobifunctional poly(ethylene glycol) derivatives containing both mercapto and acetal terminals. Bioconjugate Chem. 11, 947-950. (21) Chafi, N., Kolli, M., Vergnaud, J. M., and Montheard, J. P. (1991) Amine release from schiff bases polymers and diffusion from dosage forms with eudragit RL in acidic medium. J. App. Polym. Sci. 43, 1837-1847. (22) Kamogawa, H., Sakai, T., and Sohma, H. (1988) Chemical release control: Schiff bases of vinylbenzaldehyde and functional amines. J. Polym. Sci. Part A: Polym. Chem. 26, 13351341. (23) Stayton, P. S., Shimoboji, T., Long, C., Chilkoti, A., Chen, G., Harris, J. M., and Hoffman, A. S. (1995) Control of protein-ligand recognition using a stimuli-responsive polymer, Nature 378, 472-474. (24) Manjula, B. N., Tsai, A., Upadhya, R., Perumalsamy, K., Smith, P, K., Malavalli, A., Vandegriff, K., Winslow, R. M., Intaglietta, M., Prabhakaran, M., Friedman, J. M., and Acharya, A. S. (2003) Site-specific PEGylation of hemoglobin at Cys-93(β): Correlation between the Colligative Properties of the PEGylated Protein and the Length of the Conjugated PEG Chain. Biochonjugate Chem. 14, 464-472. (25) Otsuka, H., Akiyama, Y., Nagasaki, Y., and Kataoka, K. (2001) Quantitative and reversible lectin-induced association of gold nanoparticles modified with R-lactosyl-ω-mercaptopoly(ethylene glycol). J. Am. Chem. Soc. 123, 8226-8230. (26) Paolesse, R., Macagnano, A., Monti, D., Tagliatesta, P., and Boschi, T. (1998) Synthesis and characterization of mesotetraphenylporphyrin-corrole unsymmetrical dyads. J. Porphyrins Phthal. 2, 501-510.
BC0341775
