TECHNICAL NOTE pubs.acs.org/bc
Synthesis of X(Y)-(EO)n-OCH3 Type Heterobifunctional and X(Y)-(EO)nZ Type Heterotrifunctional Poly(ethylene glycol)s 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
bS Supporting Information ABSTRACT: A synthetic route to prepare acetal-protected heterobifunctional poly(ethylene glycol), allyl(1-ethoxyethoxy)-PEG-OH (allyl(EE)-PEG-OH), was successfully established using a newly synthesized initiator, trimethylolpropane allyl (1-ethoxyethoxy) ether (TMPAEEE). Heterobifunctional allyl(OH)-mPEG and heterotrifunctional allyl(OH)-PEG-alkyne were obtained, respectively, after modification from this precursor polymer. The polymers were characterized by SEC, 1H NMR, and MALDI-TOF mass spectroscopy. This approach is applicable for synthesizing a wide variety of X(Y)-(EO)n-OCH3 type heterobifunctional and X(Y)-(EO)n-Z type heterotrifunctional PEGs.
’ INTRODUCTION PEGylation, which refers to the process of the attachment of poly(ethylene glycol) (PEG), has been widely investigated in the fields of pharmaceutical and biomedical engineering because the polymer is nontoxic and nondegradable and has excellent solubility and extremely low immunogenicity and antigenicity.1-3 A large number of PEGs and their derivatives with different structures have been synthesized successfully.4-10 Among them, heterobifunctional PEG with the structure of X-PEG-Y, where X and Y are different functional groups, has been synthesized with different approaches in the last two decades.11-25 It is widely used in drug delivery and biosensing for the modification of biomacromolecules and functionalization of nanoparticles.11-18 It also finds applications in liquid-phase organic synthesis.19-21 Nevertheless, neither PEGs with heterobifunctional groups on the same end with the structure X(Y)-(EO)n-OCH3 nor heterotrifunctional PEGs with the structure X(Y)-(EO)n-Z have been reported up until now. According to the structures, these types of PEG derivatives maybe have potential applications in fluorescence resonance energy transfer (FRET).26 The chromophores linked to X and Y groups can quench the signals prior to release due to the short distance between X and Y groups. Furthermore, heterotrifunctional X(Y)-PEG-Z gives an opportunity for the targeted and combined delivery of multiple drugs, which is a promising therapy for the treatment of cancer.27 For example, this PEG can conjugate with a small molecular weight drug and a protein via the X and Y group, respectively, while linking a targeting moiety such as folic acid with the Z group. These PEGs also have potential applications in synthesis of block copolymers with heterobifunctionalized terminal groups on PEG. r 2011 American Chemical Society
Inspired by the successful synthesis of heterobifunctional PEG initiated from allyl alcohol22,23 and from acid-labile (acetal) protected initiators,24,25 we devise a simple method to synthesize X(Y)-(EO)n-OCH3 type heterobifunctional PEG, which has two different functional groups on the same chain end, and X(Y)-(EO)n-Z type heterotrifunctional PEG, which has two different functional groups on one chain end and one additional functional group on the other end. Our approach is based on a new initiator, trimethylolpropane allyl (1-ethoxyethoxy) ether (TMPAEEE).
’ RESULTS AND DISCUSSION An initiator with an allyl group, a 1-ethoxyethyloxy (EE)protected hydroxyl group, and a free hydroxyl group was synthesized by an electrophilic addition between trimethylolpropane allyl ether (TMPAE) and ethyl vinyl ether with p-toluenesulfonic acid (p-TsOH) as the catalyst (Scheme 1).28 The reaction was conducted in diethyl ether solution in an ice-water bath. Ethyl vinyl ether in a slight excess of hydroxyl is needed to improve the transformation efficiency. A low temperature is needed because the boiling point of both diethyl ether and ethyl vinyl ether are about 36 °C and the reaction is exothermic.28 Acetal interchange from EE ethers to acetaldehyde acetals may happen during heating in acid according to a previous report;29 thus, TsOH must be removed completely by washing with saturated NaHCO3 solution before distillation. The successful Received: September 20, 2010 Revised: January 5, 2011 Published: February 09, 2011 518
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Scheme 1. Synthesis of the New Initiator, Trimethylolpropane Allyl (1-Ethoxyethoxy) Ether (TMPAEEE)
Scheme 2. Synthesis of Protected Heterobifunctional PEG, allyl(EE)-PEG-OH, and Its Derivatives: X(Y)-mPEG and X(Y)-PEGZa
a
X, Y, and Z are different functional groups.
Table 1. Results of Anionic Polymerization of Ethylene Oxide (EO) Using TMPAEEE as the Initiator 10-3 Mnb [EO]0/[TMPAEEE]0
time (h)
obsdc
yield (%)
1a
51
24
98
2.48
2a
222
36
97
9.94
calcdd 2.50 10.0
10-3 Mnd
Mw/Mnd
2.47
1.07
9.87
1.08
Solvent: THF/DMSO = 3/2, Temperature: 50 °C. b Mn denotes number average molecular weight. c Determined from the 1H NMR results. d Mw denotes weight average molecular weight. f Determined from the following equation: Mn(calcd) = Mw(EO)[EO]0/[TMPAEEE]0 þ Mw(TMPAEEE) = 44.05 [EO]0/[TMPAEEE]0 þ 246.18. d Determined from the SEC results. a
synthesis was confirmed by 1H NMR and elemental analysis (see Supporting Information). We used diphenylmethylpotassium (DPMK) as a base for generating alkoxide initiators.30 Lowering the ratio of DPMK to hydroxyl and using a DMSO-containing cosolvent system ensure the complete solubility of alkoxide and, more importantly, a reduced rate of polymerization. It is beneficial for the synthesis of PEG in reducing the polydispersity, especially for those of low molecular weight. The synthesis procedure is shown in Scheme 2. Polymerization was performed in a stainless steel kettle to
generate allyl(EE)-PEG-OH (3). The polymer was unimodal with an average molecular weight of 2.47 103 g/mol and a polydispersity of 1.07 according to SEC (Table 1). The low polydispersity with very high yield (98%) match the characteristics of anionic polymerization.31 The structure of allyl(EE)PEG-OH was confirmed by 1H NMR spectroscopy (Figure 1A). The signals of the protons of the 1-ethoxyethyl group are detected at δ (ppm) of 1.17 (t, CH3CH2O-), 1.30 (d, -OCH(CH3)-O-), and 4.68 (q, -O-CH(CH3)-O-). The number average molecular weight (Mn) of the polymer was 519
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determined by 1H NMR spectroscopy using end group analysis according to the following equation: Mn ¼ 44:05
This further confirmed that 1-ethoxyethyl protected PEG with allyl group (allyl(EE)-PEG-OH) (3) has been successfully synthesized using trimethylolpropane allyl (1-ethoxyethoxy) ether (TMPAEEE) as the initiator. We capped the terminal hydroxyl groups of allyl(EE)-PEG-OH with methyl iodine to synthesize allyl(EE)-mPEG (4). Figure 1A, B shows the 1H NMR spectra of PEG before and after methylation. The new peak corresponding to the methoxy end group at δ = 3.38 ppm was found to have the same peak area as the methyl group at δ = 0.84 ppm. This equimolar ratio indicates that the hydroxyl group was completely methylated. According to SEC, allyl(EE)-mPEG remains unimodal with a low polydispersity (PDI = 1.08). Following hydrolysis under acid conditions similar to a previously reported procedure,30 heterobifunctional PEG allyl(OH)-mPEG (5) was obtained. The structure was confirmed by 1H NMR (Figure 1C). The signals assigned to 1-ethoxyethyl group at δ (ppm) 1.17 (t, CH3CH2O-), 1.30 (d, -O-CH(CH3)O-), and 4.68 (t, -O-CH(CH3)-O-) disappeared completely, meaning that the hydroxyl group was completely recovered. To demonstrate that this synthesis approach is applicable for preparing heterotrifunctional PEG derivatives, we modified the
3AEO - 2Am = 3 þ 246:18 4Am
where AEO and Am are the peak areas of the sum of protons in the PEO main chain at δ = 3.45-3.80 ppm and methyl protons at δ = 1.17 ppm, respectively, and 246.18 and 44.05 are the molecular weight of TMPAEEE and EO, respectively. The Mn calculated by 1H NMR is 2.48 103 g/mol, which is in close agreement with the measurement by SEC (Mn = 2.47 103 g/mol) and by MALDI-TOF mass spectrum (Mn = 2.42 103 g/ mol, Figure 2). The results of MALDI-TOF corroborate with the results of SEC, showing that the polymer is unimodal and has a low polydispersity of 1.03. Signals corresponding to the side reaction product are absent. The major series of the molecular masses of the product can be calculated by the following equation: Mw ¼ 44:05nðEOÞ þ 246:18ðTMPAEEEÞ þ 39:01ðpotassiumÞ
Figure 1. 1H NMR spectra of allyl (EE)-(PEG-OH)2 (A), allyl(EE)-(mPEG)2 (B), and allyl(OH)-(mPEG)2 (C), respectively; (CDCl3 at 20 °C).
Figure 3. 1H NMR spectra of allyl(EE)-PEG-alkyne (A) and allyl(OH)-PEG-alkyne (B) (CDCl3 at 20 °C).
Figure 2. MALDI-TOF mass spectrum of allyl(EE)-(PEG-OH)2. 520
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allyl(EE)-PEG-OH with propargyl bromide using a method previously reported.32 The 1H NMR spectrum (Figure 3A) of allyl(EE)-PEG-alkyne (6) shows new signals at δ (ppm) 2.40 (s, -CCH) and 4.17 (s, -CH2-CCH) that correspond to the alkyne groups. Analysis of the peak areas indicates that the alkyne groups and the methyl groups (δ = 1.17 ppm) are present in an equimolar amount, meaning that the hydroxyl groups were completely modified to alkyne groups. Alkyne is a useful functional group for the “click chemistry” reaction, which involves a highly specific cycloaddition between alkyne and azido groups for forming a stable triazole under mild conditions.33 Following the hydrolysis of allyl(EE)-PEG-alkyne (6) under acidic conditions,30 allyl(OH)-PEG-alkyne (7) was obtained. The structure was confirmed by 1H NMR (Figure 3B). The signals assigned to 1-ethoxyethyl group at δ (ppm) 1.17 (t, CH3CH2O-), 1.30 (d, -O-CH(CH3)-O-), and 4.68 (t, -O-CH(CH3)-O-) disappeared completely, meaning that the hydroxyl group was completely recovered. It is worth pointing out that many heterobifunctional X(Y)mPEG and heterotrifuntional X(Y)-PEG-Z can be synthesized due to the versatility of hydroxyl groups4,32 and the ease of modifying allyl groups with thiol-containing compounds by radical addition reactions.23,34 In conclusion, heterobifunctional allyl(EE)-PEG-OH, which contains acetal as the protection group, was successfully synthesized by the anionic polymerization of ethylene oxide (EO) from a new initiator trimethylolpropane allyl (1-ethoxyethoxy) ether (TMPAEEE). After modification, heterobifunctional allyl(OH)mPEG and heterotrifunctional allyl(OH)-PEG-alkyne were obtained. This approach is applicable for synthesizing a wide variety of X(Y)-(EO)n-OCH3 type heterobifunctional and X(Y)-(EO)nZ type heterotrifunctional PEGs. These PEGs have potential applications in combination drug delivery and for preparing block copolymers with heterobifunctionalized terminal groups on PEG.
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’ ASSOCIATED CONTENT
bS
Supporting Information. Materials, methods, and experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Corresponding author. Tel.: þ852-2358 8935; Fax: þ852-2358 0054; E-mail address:
[email protected].
’ ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Hong Kong Research Grant Council (General Research Fund 600207). ’ REFERENCES (1) Harris, J. M. (1992) Introduction to biotechnical and biomedical applications of poly(ethylene glycol), In Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications (Harris, J. M., Eds.) Plenum Publishing Corporation, New York. (2) Zalipsky, S., and Harris, J. M. (1997) Introduction to chemistry and biological applicatons of poly(ethylene glycol), In Poly(ethylene glycol) Chemistry and Biological Applications (Harris, J. M., and Zalipsky, S., Eds.) American Chemical Society, Washington, DC. 521
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