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Bioconjugate Chem. 2004, 15, 954−959
ARTICLES Facile Synthesis of Chain-End Functionalized Glycopolymers for Site-Specific Bioconjugation Sijian Hou,† Xue-Long Sun,*,† Chang-Ming Dong,† and Elliot L. Chaikof*,†,‡ Laboratory for Biomolecular Materials Research, Departments of Surgery and Biomedical Engineering, Emory University School of Medicine, Atlanta, Georgia 30322, and School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30320. Received March 19, 2004; Revised Manuscript Received June 22, 2004
A series of derivatized arylamine initiators were used to generate chain-end functionalized glycopolymers by cyanoxyl-mediated free-radical polymerization. Significant features of this strategy include the capacity to produce polymers of low polydispersity (PDI < 1.5) under aqueous conditions using unprotected monomers bearing a wide range of functional groups. In addition, the presence of a phenyl ring simplifies calculation of polymer saccharide content and molar mass by 1H NMR. It is particularly noteworthy, however, that derivatized arylamine initiators in conjunction with the presence of a terminal cyanate group provide a convenient approach for synthesizing polymers with a variety of distinct functional groups at R and ω chain ends. In the process, the capacity to label glycopolymers or otherwise conjugate them to proteins or other molecules is greatly enhanced.
INTRODUCTION
Polymers containing carbohydrate moieties as pendant groups, referred to as “glycopolymers”, have potential applications in medicine and biotechnology. In particular, glycopolymers potentiate multivalent protein-carbohydrate interactions, which lead to biomolecular recognition events that are dramatically different from those elicited by monovalent interactions (1-3). In the past decade, considerable efforts have focused on the application of living/controlled polymerization strategies for the production of glycopolymers that exhibit low fluctuation in size and composition. Examples include cationic polymerization (4-7), ring-opening polymerization (8, 9), ringopening metatheses polymerization (10, 11), and nitroxidemediated radical polymerization (12-16). Likewise, recent reports have also demonstrated the efficacy of transition metal-catalyzed atom transfer radical polymerization (ATRP) for the synthesis of carbohydrate-bearing polymers (17-21). Recently, the potential utility of glycopolymers in bioand immunochemical assays (22-25), as biocapture reagents (26, 27), and as glycosurfaces (28, 29) has been investigated by the addition of functional anchoring groups either pendant to the backbone or at the chain end. In this regard, a chain-end functionalized glycopolymer facilitates site-specific immobilization required for generating a uniformly oriented multivalent array of carbohydrates (25). However, the desire to generate glycopolymers with oligosaccahride units of increasing * To whom correspondence should be addressed. Mailing address: 1639 Pierce Drive 5105 WMB, Emory University, Atlanta, GA 30322. Phone: (404) 727-8027. Fax: (404) 727-3660. E-mail addresses:
[email protected] or
[email protected]. † Emory University School of Medicine. ‡ Georgia Institute of Technology.
functional complexity poses a number of significant challenges, including a requirement for serial protectiondeprotection steps or further polymer derivatization after initial synthesis. We have previously reported that cyanoxyl (OCN)-mediated free radical polymerization of unprotected glycomonomers can be conducted in aqueous solution, is tolerant of a broad range of functional groups including -OH, -NH2, -COOH, and SO3- moieties, and can yield glycopolymers with low polydispersity (PDI < 1.5) (29-31). In the present study, we describe a straightforward approach to synthesize chain-end functionalized glycopolymers (Figure 1) using functionalized arylamine initiators in conjunction with cyanoxyl-mediated polymerization. As a model system, copolymerization of acrylamide with either lactose or sulfated lactose acrylamide monomer was performed. Notably, polyacrylamide-based neoglycoconjugates have been used as diagnostic reagents (32) or as solid-phase coatings in enzyme-linked immunosorbent assays (ELISA) (22, 33). EXPERIMENTAL PROCEDURES
Chemicals. p-Anisidine(4-methoxyaniline), 2-(4-aminophenyl) ethylamine, 4-aminobenzoic hydrazide, 4-aminophenylacetic acid, N-(9-fluorenymethoxycarbonyloxy) succinimide, piperidine, sodium nitrite, fluoroboronic acid (48% aqueous solution), sodium cyanate, tetrahydrofuran (THF), acrylamide (AM), peracetyl lactose, dimethylforamide (DMF), methanol, trifluoron boron in ether (BF3‚ OEt2), sodium azide, acryloyl chloride, platinum oxide, sodium methoxide, and triethylamine were used as received, unless otherwise specified. Lactose acrylamide monomer (2) and sulfated lactose acrylamide monomer (3) were synthesized as previously described (31). Cyanoxyl-Mediated Free-Radical Polymerization of Acylamide-Derived Glycomonomers with Acrylamide Initiated by RC6H4NtN+BF4-/NaOCN
10.1021/bc0499275 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/12/2004
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Figure 1. Schematic structure of chain-end functionalized glycopolymer. Scheme 1: Synthesis of Chain-End Functionalized Glycopolymers via Cyanoxyl-Mediated Free Radical Polymerization
(Scheme 1). In a three-neck flask, 6.03 × 10-5 mol of arylamine was reacted with 17 mg of HBF4 (48 wt % aqueous solution, 9.04 ×10-5 mol) in 2 mL of water at 0 °C and under an Ar atmosphere. The diazonium salt RC6H4N+tNBF4- was then generated by adding 5 mg (7.2 × 10-5 mol) of sodium nitrite (NaNO2) to the reaction medium. After 30 min, a degassed mixture of 225 mg (6.03 × 10-4 mol) of glycomonomer (2/3), 171 mg (2.41 × 10-3 mol) of acrylamide, and 4 mg (6.03 × 10-5 mol) of NaOCN dissolved in 1 mL of water were introduced into the flask containing the diazonium salt. The polymerization solution was then heated to 65 °C for 16 h to yield statistical copolymers (4/5) after quenching the reaction and dialysis for 2 days at room temperature to remove inorganic salt and impurities. The conversions were determined by weight for the resultant glycopolymers. Complex Formation of Biotin Chain-End Functionalized Glycopolymer with Streptavidin. The biotin chain-end functionalized glycopolymer 4c/5c (1.33 × 10-4 mmol) dissolved in PBS (0.2 mL) was incubated with streptavidin (1.67 × 10-5 mmol) at room temperature for 2 h. UV-vis spectroscopy was performed before and after adding 4′-hydroxyazo-benzene-2-carboxylic acid (HABA, 1.33 × 10-4 mmol). All spectra were acquired on a Cary 50 Bio UV-visible spectrophotometer (Varian). Conversion of Cyanate (OCN) of Glycopolymer 4b to a Hydroxyl End Group. Pyridine (0.5 mL) was added to a solution of glycopolymer 4b (16 mg, 2.1 × 10-3 mmol) in water (2 mL). The mixture was stirred at room temperature for 2 h, followed by dialysis against water at room temperature for 2 days to remove excess pyridine and glutaconic aldehyde. Infrared spectra of 4b and 6b were acquired using a Digilab/BioRad FTS-4000 Fourier transform infrared (FT-IR) spectrometer (Randolf, MA) equipped with a wide band MCT detector, collected with 512 backgrounds scans, triangular apodization, and 4 cm-1 resolutions. Synthesis of Fmoc-Protected Arylamine 1e. 2-(4Aminophenyl) ethylamine (1d) (220 mg, 1.615 mmol) was dissolved in a sodium carbonate aqueous solution (10%, 7 mL) and was stirred 30 min at room temperature. A
solution of N-(9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-NHS, 545 mg, 1.615 mmol) in 1,2-dimethoxyethane (7 mL) was slowly added, and the reaction mixture was stirred overnight at room temperature. A white solid in the reaction mixture was removed by filtration. The filtrate was evaporated to yield a residue, which was dissolved in ethyl acetate, washed three times with distilled water, and then concentrated. The resulting residue was purified by silica gel column chromatography using chloroform/methanol (95/5) as eluent to obtain 1e (265 mg, yield 46%). 1H NMR (CDCl3) δ ppm: 7.75 (2H, d, J ) 7.5 Hz), 7.6 (2H, d, J ) 7.2 Hz); 7.40 (2H, t, J ) 7.2 Hz), 7.30 (2H, t, J ) 4.8 Hz), 6.95 (2H, d, J ) 4.8 Hz), 6.60 (2H, d, J ) 8.1 Hz), 4.40 (2H, d, J ) 6.6 Hz), 4.2 (1H, t, J ) 6.9 Hz), 3.4 (2H, m), 2.70 (2H, t, J ) 6.9 Hz). MS/FAB, m/z: 358 (M + Li)+. Removal of Fmoc Protecting Group of Glycopolymer 5e. Fmoc-protected glycopolymer 5e (50 mg) was dissolved in 3 mL of DMF. Piperidine (3 mL) was dropped into the polymer solution, which was stirred overnight at room temperature. The reaction mixture was dialyzed using a cellulose membrane for 2 days and then extracted three times using chloroform. The solution was freeze-dried using a lyophilizer to give glycopolymer 5d (33 mg). RESULTS AND DISCUSSION
Previous investigations have demonstrated that cyanoxyl (OCN)-mediated free-radical polymerization of acrylamide glycomonomers could be conducted in aqueous solution to yield glycopolymers of low polydispersity. In this polymerization scheme, cyanoxyl radicals are generated by an electron-transfer reaction between cyanate anions (-OCtN) from a NaOCN aqueous solution and aryl-diazonium salts (RC6H4NtN+BF4-) prepared in situ through a diazotization reaction of arylamine in water. In addition to cyanoxyl persistent radicals, aryl-type active radicals were simultaneously produced, and only the latter species is capable of initiating chain growth. Therefore, we hypothesized that chain-end functionalized
956 Bioconjugate Chem., Vol. 15, No. 5, 2004
Hou et al.
Table 1. Free-radical Copolymerization of Nonsulfated and Sulfated Lactose Glycomonomers 2/3 (GM) with Acrylamide (AM)a
GM/AM (mol)
[M]0/[I]0
glycopolymer
yield (%)
polymer composition (x/y)b
Mnb (g/mol)
initiator
R
glycomonomer (GM)
1a 1b 1c
Cl MeO biotin-NHCH2
2 2 2
1/7 1/7 1/7
20 48 20
4a 4b 4c
71 78 75
1/6 1/9 1/7
9 000 7 500 11 000
1a 1b 1c 1d 1e 1f 1g
Cl MeO biotin-NHCH2 H2NCH2CH2 Fmoc-1d H2NHNCO HOOCCH2
3 3 3 3 3 3 3
1/1 1/8 1/7 1/5 1/7 1/5 1/1
10 25 20 12 28 22 10
5a 5b 5c 5d 5e 5f 5g
80 65 61 25 57 35 57
1/10 1/11 1/8 1/6 1/9 1/6 1/1
9 300 11 100 16 000 11 300 15 900 12 800 10 800
a Polymerizations were performed for 16 h at 65 °C. b Mass content of saccharide and molecular weight were determined by 1H NMR analysis (in D2O).
Figure 2.
1H
NMR spectrum of methoxyl phenyl chain-ended glycopolymer 4b in D2O.
glycopolymers could be obtained if functionalized arylamines could act as efficient initiators for polymerization. A series of 4-substituted phenylamines (1a-1g) were investigated as initiators of cyanoxyl-mediated copolymerization of nonsulfated and sulfated lactose acrylamide monomers (2/3) with acrylamide. Consequently, a series of chain-end functionalized glycopolymers of varying molecular weight were prepared by altering either monomer conversion or the initial ratio of monomer to initiator concentrations ([M]0/[I]0). As shown in Scheme 1 and summarized in Table 1, copolymerization of lactosyl acrylamide (2/3) and acrylamide generated a series of expected chain-end functionalized glycopolymers (4/5). Of note, carboxylic acid, amine, and hydrazine end groups will facilitate glycopolymer conjugation with amine-, acid-, and aldehyde-containing molecules, respectively, while the biotin end group facilitates glycopolymer binding to streptavidin (29), and the methoxyl phenyl end group provides an activated aromatic ring for radiohalogenation of the glycopolymer (34). The resultant glycopolymers were characterized by NMR spectroscopy. It is noteworthy that the phenyl group at the end of the glycopolymer provides a conve-
nient mechanism for calculating carbohydrate content and the molar mass through comparison of integrated phenyl proton peaks with those of carbohydrate and backbone protons, respectively. For example, as illustrated in Figure 2, comparison of the integrated signals produced from the chain-end phenyl protons (H2′,6′ and H3′,5′) with those due to the anomeric protons of lactose (H1′-Lac and H1-Lac) and the backbone protons indicated that the average composition of the methoxylphenyl chain-end functionalized glycopolymer 4b was seven lactose units and 63 acrylamide units with Mn of 7500. In addition, downfield shift of end phenyl protons H2′,6′ of resultant glycopolymers confirmed C-C bond formation between phenyl and the polymer backbone (Table 2). When compared to chlorine-, methoxyl-, carboxylic-, and biotin-containing arylamine initiators, amine- and hydrazine-derivatized arylamines (1d and 1f) afforded the corresponding glycopolymers (5d and 5f) in lower yield. This may be a result of quaternization of the amino group, limiting subsequent radical formation. Therefore, Fmoc-protected arylamine (1e) initiated polymerization was investigated providing an amine-protected chain-end
Chain-End Functionalized Glycopolymers
Bioconjugate Chem., Vol. 15, No. 5, 2004 957
Figure 3. Monitoring of streptavidin-biotin-glycopolymer (4c/5c) interactions by UV-vis spectroscopy: (A) streptavidin + 4a + HABA; (B) streptavidin + 5a + HABA; (C) streptavidin + HABA; (D) streptavidin + biotin + HABA; (E) streptavidin + 4c + HABA; (F) streptavidin + 5c + HABA. Scheme 2. Representitive Scheme of Glycopolymer-Protein Hybrid Fabrication
Table 2. Chemical Shifts of Phenyl Protons of Initiators and Polymers (ppm)a initiator
polymerb
initiator
ending group
H3′,5′
H2′,6′
H3′,5′
H2′,6′
polymer
1a 1b 1c
ClMeOBiotin-NHCH2-
7.10 6.70 7.13
6.60 6.60 6.78
7.10 6.80 7.08
7.20 7.00 7.06
4a 4b 4c
1d 1f 1e
H2NCH2CH2H2NHNCOHOOCCH2-
6.95 7.55 7.85
6.60 6.60 7.35
7.10 7.80 7.10
7.10 7.20 7.10
5d 5f 5g
a 1H NMR spectra were recorded in D O (4.65 ppm of DHO as 2 standard) at room temperature. b Broad, 2H, polymeric phenyl protons.
functionalized glycopolymer (5e) in high conversion yield. 1 H NMR comfirmed that piperidine completely deprotected the Fmoc groups producing an amine chain-end functionalized glycopolymer. High-affinity binding of biotin to streptavidin (ca. 1013-15 M-1) has led to the use of streptavidin as a molecular adapter in diverse applications (35). For example, polymers with biotinylated end groups have been found to be significant for a self-organizing proteinpolymer hybrid amphiphile that uses the molecular recognition between streptavidin and biotin (36). In the present study, glycopolymer-based noncovalent neoglycoproteins were produced by incubation of streptavidin with biotin chain-end functionalized polymers 4c and 5c (Scheme 2). The ability of biotin-glycopolymers to specifically bind streptavidin was accessed using a HABAstreptavidin assay (37). HABA ′ (λmax 350 nm) changes color from yellow to red (λmax 500 nm) upon binding to streptavidin (Figure 3, trial C). When HABA was added to a solution of streptavidin saturated with free biotin or biotin glycopolymers 4c and 5c, a red shift was not
observed (Figure 3, trials D, E, and F). In contrast, a color change was noted when HABA was added to a solution of streptavidin with non-biotin-containing glycopolymers 4a and 5a (Figure 3, trials A and B). These results demonstrate glycopolymer-protein hybrid formation through specific binding of chain-end biotin of glycopolymers 4c and 5c to streptavidin. In principle, streptavidin has four free biotin-binding sites. However, the HABA assay revealed an average occupancy of 3.0-3.6 glycopolymer chains per streptavidin molecule. Full occupancy may be limited as a result of steric factors. Another significant feature of cyanoxyl (OCN)-mediated polymerization is the presence of a terminal cyanate group at the opposing chain end (Scheme 1). Cyanatebased ligand immobilization via specific reaction with an amine group to form an isourea bond has proven to be a useful tool in bioconjugate chemistry (38, 39). As such, the OCN end group provides a second feasible reaction pathway for conjugation of glycopolymers with aminecontaining surfaces and molecules (40). Consequently, this scheme establishes a facile approach for producing R,ω asymmetric bifunctional chain-terminated polymers. Moreover, the potential to conjugate glycopolymers that have been selectively labeled with a chromophore, fluorophore, or radiotracer is apparent. It is also noteworthy that desired the cyanate group can be converted to a hydroxyl group (Scheme 3) by treatment with pyridine in water (41). IR spectroscopy demonstrated that the OCN absorption band at 2157 cm-1 disappeared after pyridine treatment of glycopolymer 4b to 6b thereby confirming complete hydrolysis of the cyanoxyl group. Further 1H NMR of 6b indicated that this hydrolysis reaction did not induce any further structural changes. In summary, a straightforward approach for the synthesis of a series of R,ω chain-end functionalized glyco-
958 Bioconjugate Chem., Vol. 15, No. 5, 2004 Scheme 3. Conversion of Terminal Glycopolymer Cyanate Group (OCN) to a Hydroxyl Moiety
polymers via cyanoxyl-mediated free-radical polymerization has been presented. Significantly, a variety of arylamine derivatives proved to be acceptable initiators for cyanoxyl-mediated free-radical polymerization yielding chain-end functionalized glycopolymers that can be successfully conjugated or otherwise linked to a variety of molecules or surfaces. ACKNOWLEDGMENT
This work was supported by grants from the NIH and NSF. The authors acknowledge the Emory University NMR and Mass Spectrometry Centers for use of their facilities and Dr. Keith Faucher for assistance with IR spectroscopy. LITERATURE CITED (1) Lee, Y., and Lee, R. T. (1995) Carbohydrate-protein interactions: basis of glycobiology. Acc. Chem. Res. 28, 321. (2) Strong, L. E., and Kiessling, L. L. (1999) A general synthetic route to defined, biologically active multivalent arrays. J. Am. Chem. Soc. 121, 6193. (3) Lundquist, J. J., and Toone, E. J. (2002) The cluster glycoside effect. Chem. Rev. 102, 555. (4) D’Agosto, F., Charreyye, M. T., Pichot, C., and Mandrand, B. (2002) Polymer of controlled chain length carrying hydrophilic galactose moieties for immobilization of DNA probes. Macromol. Chem. Phys. 203, 146. (5) D’Agosto, F., Charreyye, M. T., Delolme, F., Dessalces, G., Cramail, H., Deffieux, A., and Pichot, C. (2002) Kinetic study of the “living” cationic polymerization of a galactose carrying vinyl ether. MALDI-TOF MS analysis of the resulting glycopolymers. Macromolecules 35, 7911. (6) Yamada, K., Minoda, M., and Miyamoto, T. (1999) Controlled synthesis of amphiphilic block copolymers with pendant N-acetyl-D-glucosamine residues by living cationic polymerization and their Interaction with WGA lectin. Macromolecules 32, 3553. (7) Yamada, K., Minoda, M., and Miyamoto, T. (1997) Controlled synthesis of glycopolymers with pendant D-glucosamine residues by living cationic polymerization. J. Polym. Sci., Part A: Polym. Chem. 35, 751. (8) Tsutsumiuchi, K., Aoi, K., and Okada, M. (1997) Synthesis of polyoxazoline-(Glyco)peptide block copolymers by ringopening polymerization of (Sugar-Substituted) R-amino acid N-carboxyanhydrides with polyoxazoline macroinitiators. Macromolecules 30, 4013. (9) Aoi, K., Tsutsumiuchi, K., and Okada, M. (1994) Glycopeptide synthesis by an alpha-amino acid N-carboxyanhydride (NCA) method: ring-opening polymerization of a sugarsubstituted NCA. Macromolecules 27, 875. (10) Nomura, K., and Schrock, R. R. (1996) Preparation of “sugar-coated” homopolymers and multiblock ROMP copolymers. Macromolecules 29, 540. (11) Fraser, C., and Grubbs, R. U. (1995) Synthesis of glycopolymers of controlled molecular weight by ring-opening metathesis polymerization using well-defined functional group tolerant ruthenium carbene catalysts. Macromolecules 28, 7248. (12) Gotz, H., Horth, E., Schiller, S. M., Frank, C. W., Knoll, W., and Hawker, C. J. (2002) Synthesis of lipo-glycopolymer amphiphiles by nitroxide-mediated living free-radical polymerization. J. Polym. Sci., Part A: Polym. Chem. 40, 3379.
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