Bioconjugate Chem. 1992, 3, 275-276
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Synthesis of Poly(ethy1ene oxide) with Heterobifunctional Reactive Groups at Its Terminals by an Anionic Initiator Masayuki Yokoyama,' Teruo Okano, and Yasuhisa Sakurai Institute of Biomedical Engineering, Tokyo Women's Medical College, Kawada-cho, 8-1, Shinjuku-ku, Tokyo 162, Japan Akira Kikuchi, Nobuyuki Ohsako, Yukio Nagasaki, and Kazunori Kataoka' Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Science University of Tokyo, Yamazaki 2641, Noda-shi, Chiba 278, Japan. Received March 10, 1992 Much attention has been focused on utilization of poly(ethylene oxide) (PEO) for biological and biomedical fields. These applications include cell fusion for hybridoma production ( I ) , protein modification for decreased antigenicity (2) or for suppression of reaginic antibody response (3),drug targeting (4,5),and controlled drug release (6). In these applications, PEO was mixed with or bound to other components. Since ethylene oxide (EO) units possess no reactive functional groups, PEO is bound to the other Componentswith its end groups. Several kinds of terminalderivatized PEOs (7)have been synthesized for the binding of PEO to the other components. Most of these PEOs, however, are limited to homobifunctional polymers (e.g. PEO with primary amines at the both terminals) and polymers with one reactive terminal and one unreactive terminal (e.g. PEO with amine and methoxy groups at its terminals, respectively). For more functionalized conjugates containing PEO chains, PEOs with heterogeneous reactive groups a t its two terminals are warranted. S6pulchre et ai. (8) reported a polymerization of EO by an anionic initiator which contained a masked primary amino group binding to a bulky aliphatic group (3,5,5-trimethylcyclohexyl group). Huang et al. (9) reported a preparation of the PEO with a primary amine and a hydroxyl groups a t eachterminal up to polymerization degree of 33 (molecular weight = 1500). They used CsH&H=NCH*CHzONa for an initiator of polymerization of EO. This paper reports a facile synthetic method of PEO with a primary amino group and a hydroxyl group at each terminal using a commerciallyavailable reagent, potassium bis(trimethylsily1)amide([(CH3)3Si]2NK,l),as an initiator of EO polymerization. Characterization of the high molecular weights of the polymers obtained under these conditions suggests this method will be useful for the biological applications. As shown in Scheme I, EO was polymerized by 1 to obtain PEO with bis(trimethylsily1)amine and potassium oxide terminals (2). These terminals are changed into a primary amine and a hydroxyl group, respectively, by subsequent acid treatment. EO was dissolved in tetrahydrofuran (THF) at -79 "C. A solution of 1 a t a concentration of 0.50 M in toluene (Aldrich Chemicals, Inc.) was added, and the mixture was stirred in adegassed sealed glass tube at 20 "C. Quantities of the reagents are summarized in Table I. For run 1, the mixture was divided into five samples of almost equal quantity to follow the polymerization. After a defined period, the mixture was treated with acid (1 mL of 1 N HC1for 4 and 16 h, or 1 mL of 11 % (v/v) acetic acid solution in THF for 28,48, and 96 h) to prepare samples for gel permeation chromatography (GPC). For run 2, the mixture was poured into a 15-foldvolume of diethyl ether.
* Author to whom correspondence should be addressed.
Scheme I. Synthesis of Poly(ethy1ene oxide) with an Amino and a Hydroxyl Group at Each Terminal
Hi
*
~2
N+CHZCHZO~H 3
The precipitate was washed with diethyl ether and dried in vacuo. For run 3, 10 mL of the reaction mixture was poured into 120 mL of diethyl ether to obtain the precipitated polymer. The residual mixture (20 mL) was transferred to a pear-shape flask, and solvent was evaporated. Both polymers obtained in run 3 were dissolved in 30 mL of THF, followed by an addition of a few drops of 0.1 N hydrochloric acid. This solution was stirred for 3 min at room temperature and poured into 300 mL of diethyl ether to obtain the precipitated polymer. The polymerization of EO by the initiator (1)was found to have progressed successfullyby GPC analysis. As shown in Figure 1,the elution volume of the sample decreased as the reaction time increased from 4 to 28 h for run 1. No change in elution volume was observed between 28 and 48 h. This indicated that polymerization of run 1 was completed within 28 h. Therefore, the polymerizations of runs 2 and 3 with smaller monomer/initiator ratios and longer reaction times than run 1 were also considered complete. The elution volume of run 1 (for 48 and 96 h) was found to correspond to molecular weights of 5.2 X lo3 and 6.0 X lo3,respectively, using PEO standards. These values were almost identical with the molecular weight (4.8 X lo3) calculated from the monomedinitiator ratio. Run 2 also resulted in a polymer whose molecular weight was almost the same as the calculated value. Therefore, the polymerization of EO initiated by 1 proceeded with theoretical efficiency. The molecular weight distributions of these polymers, characterized by GPC, were very narrow with ratios of weight-average molecular weight and number-average molecular weight (&./MI.,) of approximately 1.1. This indicated that the initiator (1) produced polymerization without side reactions. Run 3 brought about two samples before the acid treatment; one by precipitation and the other by evaporation. These two samples were found to have the same molecular weight (2.7 X lo3)by GPC. This indicates that precipitation did not affect the molecular weight of the obtained sample. After the acid treatment, the two polymers were mixed, followed by purification on an ionexchange resin. The mixed polymer was dissolved in 40 mL of distilled water and applied to 200 mL of Diaion PK216 (S03H type). After washing the column with 400 mL of distilled water, 1000 mL of 5% ammonia aqueous solution was added and the eluent was collected. The
1043-1802/92/2903-0275$03.~QIQ 0 1992 Amerlcan Chemical Society
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Bloconlugete Chem., Vol. 3, No. 4, 1992
Table I. Polymerization of Ethylene Oxide (EO) by [(CHs)sSi]2NK
a
run
EO,g
1 2 3
15.31 3.18 6.64
quantity THF, mL
EOIinitiator (mol ratio)
40 10 20
reaction time, h
105
96
58 52
149 50
Ma (calculated) 6.0X lo3 (4.8X 103) 2.5 X 103 (2.8X lo3) 2.7 x 103 (2.5 x 103)
--
M,IMna
1.15 1.08 1.07
Determined by GPC using PEO standard.
reaction time
96h 48h
4h I
20
.
.
.
.
,
30
elution volume (ml) Figure 1. Gel permeation chromatogram of run 1. Gel per-
meation chromatographywas carried out using a ToyosodaModel HLC-8020equipped with TSK gel G2000Hx1,TSK gel G3000Hx1, and TSK gel G4000H.l columns (gel-exclusion molecular weight is 1 X lo4,6 X l(r,and 4 X 105, respectively) at 40 OC in THF at a flow rate of 1.0 mL/min. Detection was performed by refractive index. eluent was freeze-dried, the polymer was redissolved in benzene, and the solution again was freeze-dried. The yield of this purification was 94.1%. This high yield suggested successfulincorporation of amino terminals, and this was confirmed by acid-base titration in chloroform with HC104 in acetic acid using methyl violet as an indicator. The purified sample was found to contain 4.0 X lo-' mol equiv of base/g, approximately the same as 3.7 X 10-4,calculated from the molecular weight determined by GPC on the assumptionthat every polymer chain carries the amino group at one terminal. Insummary, this letter reports a facile method to obtain
poly(ethy1ene oxide)s possessing a primary amino and a hydroxyl group at each terminal with the molecular weight well controlled by the monomer/initiator ratio as well as with narrow molecular weight distributions. In conventional methods which substitute hydroxyl terminals of PEO with other functional groups, substitution yields are dependent on a molecular weight of PEO; therefore, the more difficult it is to obtain high yields for the higher molecular weight of PEO. Furthermore, for preparation of the heterobifunctional(both reactive) PEO, yields must be low due to separation procedures from unsubstituted PEO and substituted PEO at both the terminals. On the other hand, this more convenient method introduces a primary amine to the polymer by an anionic initiator. Indeed, this method can provide 100% incorporation of an amino group even for high molecular weight polymers. Thus obtained heterobifunctional PEO is considered very useful for biological applications. For one example, this PEO may be utilized to design the directional drug-carrier systems which bind drugs and targeting moieties at each terminal of the PEO, respectively. LITERATURE CITED (1) Kdhler, G., and Milstein, C. (1975)Nature 256,495. (2) Abuchowski, A.,van Ea,T., Palczuk, N. C., and Davis, F. F. (1977)J. Biol. Chem. 252,3582. (3) Lee, W.Y., and Sehon, A. H. (1977)Nature 267,618. (4) Yokoyama, M., Okano, T., Sakurai, Y., Ekimoto, H., Shibazaki, C., and Kataoka, K. (1991)Cancer Res. 51, 3229. (5) Yokoyama, M., Miyauchi, M., Yamada, N., Okano,T., Sakurai, Y., Kataoka,K., and Inoue, S. (1990)Cancer Res. 50,1693. (6) Graham, N. B.,and McNeil, M. E. (1984)Biomaterials 427. (7) Harris, J. M., Struck, E. C., Case, M. G., Paley, S., Yalpani, M., van Alstine, J. M., and Brooks, D. (1984)J. Polym. Sci., Polym. Chem. Ed. 22,341. (8) SBpulchre,M., Paulus, G., and JBrome,R. (1983)Makromol. Chem. 184,1849. (9) Huang, Y.-H., Li, Z.-M., Morawetz, H. (1985)J.Polym. Sci., Polym. Chem. Ed. 23, 795. Registry No. 3, 32130-27-1;[(CH3)3Si]zNK,40949-94-8.
