“Clickable” Agarose for Affinity Chromatography - Bioconjugate

In-column preparation of a brush-type chiral stationary phase using click chemistry and a silica monolith. Michael D. Slater , Jean M. J. Fréchet , F...
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Bioconjugate Chem. 2005, 16, 1536−1541

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“Clickable” Agarose for Affinity Chromatography Sreenivas Punna, Eiton Kaltgrad, and M. G. Finn* Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037. Received August 8, 2005; Revised Manuscript Received August 12, 2005

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynessthe premier example of “click chemistry”sis an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

INTRODUCTION

EXPERIMENTAL PROCEDURES

Biological processes depend on specific interactions between molecules, such as between antibodies and antigens, carbohydrates and lectins, and enzymes and inhibitors. Affinity chromatography (1) exploits these various interactions for the analysis and purification of mixtures of biomolecules. One of the members of the interacting pair is immobilized on an insoluble support and is used to “fish out” the complementary agent from a solution passed through or over the column. In many cases, affinity chromatography is a crucial step in the isolation of desired species, for example, providing the only practical means for the separation of tagged or functional proteins from cellular extracts. Beaded agarose bearing functional groups such as amine (-NH2), carboxylic acid (-COOH), aldehyde (-CHO), thiol (-SH), and hydroxyl (-OH) is the support of choice for most ligands of interest (2) due to its inexpensive and biocompatible nature. For selective immobilization, a ligand should possess a functional group for attachment to the support that is not essential to its binding property. Ligands can be immobilized in ways detrimental to their intended function when more than one of the attachment units is present in a ligand or when other reactive functional groups on the ligand compete in the immobilization chemistry. A matched pair of groups which is selective in reacting with each other while being unreactive to other functional groups in the molecule would therefore be highly useful in the preparation of functional agarose beads. Organic azides and terminal alkynes fit this description, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. While their uncatalyzed [3+2] dipolar cycloaddition is typically very slow at room temeperature (3), Cu(I) catalysts accelerate the reaction to a great degree (4, 5). Here we report the first use of azides, alkynes, and their “click reaction” (6) for the convenient immobilization of ligands on agarose for affinity chromatography and related purposes.

General. 1H and 13C NMR spectra were recorded on Bruker DRX-500, AMX-400, or Varian Mercury 200 spectrometers, using CDCl3 or CD3OD as solvents. Mass spectra were obtained on an Agilent 1100 LC/MS spectrometer (model G1946A). Melting points were measured in a Mel-Temp II capillary melting point apparatus and are uncorrected. Infrared spectra were recorded on a MIDAC EM200 instrument with horizontal attenuated total reflectance accessory from Pike Instruments or in KBr pellets. Column chromatography was performed on EM Science silica gel (40-63 µm mesh). TLC analysis was facilitated by the use of phosphomolybdic acid or KMnO4/H2O stains in addition to UV light with fluorescent-indicating plates. Anhydrous sodium sulfate was used for drying solutions. Carboxy Link coupling gel (50% aqueous slurry, 16 µmol of amine groups per milliliter), biotin-PEG-amine, and avidin-HABA complex were purchased from Pierce. All other commercially available reagents were purchased from Acros or Aldrich. Compounds 6 and 7, kindly provided by Mr. Warren G. Lewis, have been previously described (5). Reactions requiring anhydrous conditions were performed under nitrogen. 5-Azidopentanoic Acid 4-Nitrophenyl Ester (2). 1-(3-Dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDCI‚HCl, 703.8 mg, 3.67 mmol) was added to a solution of p-nitrophenol (510.5 mg, 3.67 mmol) and 5-azidopentanoic acid (500 mg, 3.49 mmol) in CH2Cl2 (25 mL) at room temperature and stirred overnight, followed by the addition of water and excess EtOAc. The separated organic phase was washed with H2O and brine solution, dried over Na2SO4, and evaporated. The crude product was purified by silica gel column chromatography (1:4 EtOAc/hexane) to give 2 (514 mg) as a pale yellow oil in 52% yield. 1H NMR (CDCl3) δ 1.62-1.92 (m, 4H), 2.60 (t, J ) 6.6 Hz, 2H, -CH2N3), 3.34 (t, J ) 6.6 Hz, 2H), 7.25 (d, J ) 9.2 Hz, 2H), 8.23 (d, J ) 8.8 Hz, 2H); 13C NMR (CDCl3) δ 22.1, 28.4, 33.9, 51.2, 60.6, 122.6, 125.4, 145.5, 155.5, 170.8; IR (thin film, cm-1): 2946, 2100 (azide), 1768, 1593, 1534, 1345, 1211, 1130, 910, 865, 748; ESMS m/z (relative intensity) 287 (M + Na)+ (75), 237 (100), 223 (70).

* To whom correspondence should be addressed. E-mail: [email protected].

10.1021/bc0501496 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/13/2005

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Scheme 1

Agarose Azide and Alkyne Beads (4 and 5). CarboxyLink gel (8 mL, maximum of 64 µmol of amine groups) was transferred to a disposable frit, allowed to settle for 30 min, and equilibrated with 5 column volumes of pH 4.7 MES buffer (0.1 M). To the gel was added p-nitrophenyl ester 2 (143.4 mg, 64 mmol, 16 µmol/mL) dissolved in DMF with 5% of the MES buffer (8.4 mL), and the capped fritted tube was rotated gently at room temperature for 2 days. Phosphate buffer (600 µL, pH 7, 1.5 M) was added to the reaction mixture, which was then drained into a clean scintillation vial and the absorbance at 412 nm was measured directly. An agarose sample lacking the amine group was used in a control experiment to correct for residual hydrolysis of 2, which was negligible under these conditions. The amount of attached azide was assumed to be equivalent to the amount of released p-nitrophenol, which was determined with the aid of an extinction coefficient measured by the addition of authentic p-nitrophenol to the same mixture of DMF, pH 4.7 MES, and pH 7 phosphate ( ) 432 M-1 cm-1). Agarose 5 was prepared and analyzed by the same procedure, using p-nitrophenyl ester 3. Fluorescein-alkyne (8). To fluorescein NHS ester (97 mg, 0.205 mmol) in THF (30 mL) was added propargylamine (32 µL, 0.451 mmol), and the reaction was stirred under nitrogen atmosphere at room temperature overnight. After the sample was filtered to remove a small amount of insoluble material, the solvent was evaporated and the residue was washed with CH2Cl2 and then Et2O to afford 8 as a yellow solid in quantitative yield (87 mg). ESMS m/z (relative intensity) 414 (M+, base peak). Typical Immobilization Procedure. Stock solutions (50 mM) of the following reagents were prepared: 2,6lutidine (DMF), 2,2′-bipyridine (DMF), CuBr (DMF), and sodium ascorbate (water). A slurry of agarose-azide/ alkyne beads (1.0 equiv) in DMF in a disposable frit was treated with the molecule to be attached bearing the complementary (alkyne or azide) group (4.0 equiv), followed by 2,6-lutidine (8.0 equiv), 2,2′-bipyridine (8.0 equiv), cuprous bromide (4.0 equiv), and finally sodium ascorbate (8.0 equiv). The resulting suspension was bubbled with a gentle flow of nitrogen for 1 min, capped, and rotated at room temperature for 12-18 h. The reaction mixture was drained and washed sequentially with approximately 5 column volumes each of DMF, H2O, MeOH, 0.1 M aq EDTA, H2O, and DMF. Biotin-PEG-alkyne (12). To a solution of biotin-PEGamine (11, 33 mg, 0.079 mmol) in CH2Cl2 (5 mL) was added 4-pentynoic acid (7.4 mg, 0.045 mmol) followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDCI‚HCl; 704 mg, 3.67 mmol). The mixture was stirred at room temperature overnight and was then partitioned between EtOAc (10 mL) and water. The organic layer was dried (Na2SO4) and evaporated to give

compound 12 (9 mg) as a colorless syrup. A further 11 mg was recovered from the aqueous layer by stirring in the presence of additional EtOAc and excess NaCl (overall yield 51%). 1H NMR (CDCl3) δ 1.49-1.55 (m, 2H), 1.86-1.88 (m, 4H), 2.25-2.85 (m, 2H), 2.36 (s, 1H), 2.492.60 (m, 6H), 2.75-2.85 (m, 2H), 2.92-3.02 (m, 2H), 3.25-3.30 (m, 1H), 3.40 (m, 2H), 3.41-3.50 (m, 4H), 3.58-3.64 (m, 4H), 3.67-3.70 (m, 2H), 4.35-4.40 (m, 1H), 4.55-4.60 (m, 1H), 8.20 (br s, 1H), 8.4 (br s, 1H);13C NMR (CDCl3) δ 14.7, 25.8, 28.5, 28.7, 34.9, 35.7, 40.0, 56.03, 60.64, 62.4, 68.9, 69.5, 70.2, 70.27, 70.6, 74.4, 82.5, 175.2, 177.7, 178.05; IR (film, cm-1): 3287 (CCH), 3084, 2864, 2114 (alkyne), 1629, 1553, 1265, 1140, 951, 879, 726; ESMS m/z (relative intensity) 521 (M+Na)+ (100), 499 (M+1)+ (25), 375 (60). HIV-Protease Peptide Alkyne (15). Standard Fmocbased solid phase peptide synthesis techniques were employed (7), starting with Wang resin bearing the C-terminal amino acid (Fmoc-Phe-OH, 0.77 mmol/g). Fmoc deprotection was accomplished with two treatments of 20% piperidine in DMF, and amino acid coupling was typically performed with 5.5 equiv of Fmocamino acid, 5.0 equiv of HBTU, and excess iPr2NEt. Fmoc-propargylglycine (CSPS Chemicals, San Diego) was installed as the final (N-terminal) amino acid, using DIC/ HOBt (5.0 equiv) in CH2Cl2. The crude peptide containing the N-terminal Fmoc protecting group was obtained by treating the resulting resin with 94/2.5/2.5/1 CF3CO2H/ H2O/1,2-ethanedithiol/SiH(iPr)3 mixture for 3 h at room temperature. The desired peptide was identified by MALDI-MS of fractions from analytical HPLC of the crude product, and then purified by preparative HPLC. The final purified product (peptide G*QCTLNF, where G* ) propargylglycine) was obtained in approximately 9% yield, calculated from the weight of the isolated material relative to the amount of amino acid on the starting resin. After attachment of 15 to 4 by the above procedure, the resulting beads (16) were treated with 20% piperidine (4 mL) at room temperature for 10 min. The solution was drained, and the absorbance at 300 nm was measured against a blank composed of 20% piperidine in DMF. Using the measured fluorene molar extinction coefficient of 7800 M-1 cm-1 determined under identical conditions, the attached peptide concentration was found to be 0.71 µmol/mL, representing approximately 39% conversion of the available azides on the resin (measured as above at 1.84 µmol/mL). Attachment of Fmoc-propargylglycine alone proceeded in 63% yield by the same assay. A contributing factor to the relatively poor attachment of the peptide is its poor solubility, even in DMF, causing it to aggregate and precipitate slowly during the reaction. 4-Azido-N-[4-(4-oxobutoxy)phenyl]butyramide (19, Scheme 1). To the N-hydroxysuccinimide (Su) ester of

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Scheme 2

4-azidobutyric acid (2.2 g, 18.32 mmol) in THF (20 mL) was added p-aminophenol (1.0 g, 18.32 mmol), and the mixture was stirred overnight at room temperature. The solvent was evaporated and the resulting residue was partitioned between equal volumes of 1 M HCl and EtOAc. The aqueous layer was extracted twice more with EtOAc and the combined organic layers were washed with 1 N HCl, H2O (2×), brine, dried, and evaporated. The crude product (designated as compound A) was purified by column chromatography (1:3 EtOAc/hexanes) to give a brown solid (1.19 g, 57% yield). mp 67-69 °C; 1 H NMR (CDCl3) δ 1.50-1.57 (m, 2H), 1.63-1.71 (m, 2H), 2.26 (t, J ) 7.5 Hz, 2H), 6.68 (d, J ) 8.0 Hz, 2H), 7.19 (dd, J ) 8.5, 3.5 Hz), 8.37 (s, 1H); 13C NMR (CD3OD) δ 24.3, 29.6, 37.2, 52.3, 116.3, 123.6, 131.7, 155.5, 173.9; IR (KBr, cm-1): 2959, 2465, 2096 (azide), 1665, 1449, 1238, 839; ESMS m/z (relative intensity) 257 (M + Na)+ (22), 235 (M+H)+ (100). To a solution of 4-pentene-1-ol (3.0 g, 35 mmol), Et3N (14.5 mL, 105 mmol), and DMAP (425 mg, 3.5 mmol) in THF (50 mL) was added p-TsCl (6.64 g, 34.88 mmol), and the reaction was stirred for 6 h at room temperature. The resulting mixture was filtered through a sintered funnel, the solid was washed with additional THF, and the combined solution was evaporated. The crude product was purified by column chromatography (silica gel, 1:4 EtOAc/hexanes) to obtain the desired tosylate B (Scheme 1) as a colorless liquid (6.6 g, 79% yield). 1H NMR (CDCl3) δ 1.58-1.72 (m, 2H), 1.92-2.08 (m, 2H), 2.36 (s, 3H), 3.96 (t, J ) 6.4 Hz, 2H), 4.80-4.94 (m, 2H), 5.50-5.73 (m, 1H), 7.27 (d, J ) 8.0 Hz, 2H), 7.70 (d, J ) 7.8 Hz); 13C NMR (CDCl3) δ 13.9, 21.3, 27.7, 29.1, 69.6, 115.5, 127.6, 132.8, 136.4, 144.6; IR (thin film, cm-1): 3075 (olefin), 2981, 1926, 1643, 1598, 1449, 1346, 1185, 920, 825, 749; ESMS m/z (relative intensity) 241 (M + H)+ (10).) This material (720 mg, 3.00 mmol) was combined with compound A (702 mg, 3.00 mmol) and K2CO3 (1.24 g, 9.0 mmol) in DMF (5.0 mL), and the mixture was stirred at 80 °C for 12 h. Saturated aqueous NH4Cl solution (25 mL) and EtOAc (25 mL) were added, and the organic layer was separated and washed with additional NH4Cl solution, water, and brine. The solution was dried and evaporated, and the residue was purified by column chromatography (silica gel, 1:4 EtOAc/hexanes) to give the ether-amide intermediate C (627 mg, 69% yield) as a red solid. mp 60-62 °C; 1H NMR (CDCl3) δ 1.63-1.69 (m, 2H), 1.75-1.82 (m, 2H), 1.82-1.89 (m, 2H), 2.22 (q, J ) 7.0 Hz, 2H), 2.35 (t, J ) 7 Hz, 2H), 3.30 (t, J ) 6.5 Hz, 2H, -CH2N3), 3.93 (t, J ) 6 Hz), 4.98-5.08 (m, 2H), 5.84 (m, 1H), 6.83 (d, J ) 10.0 Hz), 7.37 (d, J ) 8.5 Hz, 2H), 7.41 (br s, 1H); 13C NMR (CDCl3) δ 22.3, 27.9, 29.6, 36.2, 50.7, 66.9, 114.3, 115.0, 121.4, 130.3, 137.3, 155.4, 170.15; IR (KBr, cm-1): 2950, 2100 (azide), 1650, 1310, 1250, 820, 760; ESMS m/z (relative intensity) 303 (M + H)+ (100). To a cooled (ice bath) solution of olefin C (500 mg, 1.65 mmol) in 2:1 acetone/water (6 mL) was added a small crystal of OsO4. After 5 min, N-methylmorpholine Noxide (193.3 mg, 1.65 mmol) was added, and the mixture

was stirred at room temperature for 2 days. EtOAc and brine were added, and the aqueous layer was further extracted with EtOAc (2 × 25 mL). The crude product obtained from the combined organic layers was purified by column chromatography (silica gel, EtOAc) to give the desired diol (D, Scheme 1, 248 mg, 51% yield) as a white solid. Approximately 65 mg of the starting material was also recovered. mp 82-84 °C; 1H NMR (CDCl3) δ 1.501.90 (m, 8H), 2.34 (t, J ) 7.0 Hz, 2H), 2.70 (br s, -OH), 3.29 (t, J ) 5.5 Hz, 2H, -CH2N3), 3.40-3.44 (m, 1H), 3.60-3.85 (m, 2H), 3.92 (s, 2H), 6.79 (d, J ) 9.5 Hz), 7.36 (dd, t, J ) 7.0, 5.0 Hz, 2H), 8.32 (br s, 1H); 13C NMR (CDCl3) δ 23.9, 26.5, 29.3, 30.8, 36.9, 52.0, 67.2, 69.0, 72.8, 115.4, 122.9, 132.6, 157.1, 173.6; IR (thin film, cm-1): 2945, 2869, 2132 (azide), 1661, 1490, 1301, 1238, 1018, 830; ESMS m/z (relative intensity) 337 (M + H)+ (100). To a solution of diol D (100 mg, 0.297 mmol) in CH2Cl2 (5 mL + 2 drops of MeOH) was added saturated aqueous NaHCO3 (0.5 mL) followed by NaIO4 (127 mg, 0.60 mmol). After the sample was stirred at room temperature for 4 h, anhydrous Na2SO4 (approximately 1 g) and CH2Cl2 (25 mL) were added and the mixture was stirred for a further 30 min. Filtration, washing of the solid with CH2Cl2, and evaporation of the combined organic solutions provided compound 19 (67 mg, 74% yield) as a thick colorless syrup. 1H NMR (CDCl3) δ 1.601.70 (m, 2H), 1.74-1.85 (m, 2H), 2.08-2.15 (m, 2H), 2.34 (t, J ) 6.4 Hz, 2H), 2.65 (t, J ) 5.8 Hz, 2H), 3.28 (t, J ) 5.6 Hz, 2H, -CH2N3), 3.95 (t, J ) 5.4 Hz, 2H), 6.8 (d, J ) 10.8 Hz, 2H, ArH), 7.38 (d, J ) 10.8 Hz, 2H, ArH), 7.74 (s, 1H), 9.8 (s, 1H, -CHO); 13C NMR (CDCl3) δ 21.3, 22.0, 27.6, 35.8, 39.9, 50.4, 66.2, 113.9, 121.2, 130.6, 154.7, 170.3, 201.4; IR (film, cm-1): 2949, 2877, 2096 (azide), 1727, 1664 (CdO), 1301, 1233, 959, 833, 743; ESMS m/z (relative intensity) 305 (M+1)+ (85). Test of Affinity Chromatography Function with Aldehyde 19. Functionalized agarose 20, prepared in the standard manner from 19, was poured into a column (Amersham Tricom, 5 mm × 50 mm; 1 mL volume; cat#18-1163-09). At pH 7, the column was found to retain the catalytic antibody 84G3 originally obtained by Barbas and co-workers (8, 9) and not an unrelated antibody; details can be found in Figure 5. RESULTS AND DISCUSSION

Commercially available Carboxy-Link agarose (1), which bears addressable amine groups, was treated with the p-nitrophenyl ester of 5-azidopentanoic acid (2) and 5-pentynoic acid (3) to give azide- and alkyne-functionalized beads 4 and 5, respectively (Scheme 2). The degree of acylation was determined by measurement of the concentration of released p-nitrophenol. Loadings of between 3.3 and 4.6 µmol/mL of gel were typical, representing approximately 20-30% of the available amine groups according to the commercial specifications of the gel. However, the standard Kaiser (ninhydrin) test (10) gave a positive signal for 1 and negative signals for 4 and 5, suggesting that most or all of the accessible amine groups of 1 were acylated by 2 and 3.

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Figure 1. CuAAC coupling of dye reagents to agarose azide and alkyne; a, standard Cu‚bipy-mediated coupling conditions (see Experimental Procedures), followed by a wash procedure including 0.l M EDTA to remove Cu ions.

Figure 2. Biotinylation of agarose azide; a, standard Cu‚bipy-mediated coupling conditions (see Experimental Procedures), followed by a wash procedure including 0.l M EDTA to remove Cu ions.

To test the feasibility of resin attachment by the Cumediated azide-alkyne cycloaddition (CuAAC) reaction, agarose-azide beads 4 were treated with a dabsyl-alkyne derivative (6) and the corresponding dye with an n-propyl group in place of the alkyne (7), in the presence of cuprous bromide, 2,2′-bipyridine as accelerating ligand (5), sodium ascorbate to maintain a reducing environment, and 2,6-lutidine as mild base (Figure 1). The dye was successfully attached with 6, as indicated by the production of an intense and permanent red color in the resulting beads (9). Use of the control 7 or omission of Cu(I) from the reaction of 6 gave colorless beads. In addition, no dye could be washed from 9 even upon extensive treatment with DMF; these observations show that the dye does not adsorb to the resin. Similarly, 4 was treated with fluorescein alkyne 8 under CuAAC conditions to give yellow beads that were strongly fluorescent under ultraviolet light. The same successful results were obtained with agarose-alkyne 5 and the complementary azide-substituted dabsyl compound (data not shown). Biotinylated supports are a mainstay of protein immobilization and therefore of experimental biochemistry. The CuAAC reaction provides a convenient addition to the many methods already in use for constructing such materials. Biotin alkyne 12 was prepared from commercially available biotin-PEG-amine 12 and 4-pentynoic acid (Figure 2). Immobilization on agarose azide beads gave 13, which was treated with a red solution of avidinHABA (HABA ) 2-(4′-hydroxyphenyl)azobenzoic acid) complex. Tight avidin-biotin interaction gave the result-

ing beads a persistent red color (14), whereas a mixture of avidin-HABA with agarose-azide 4 resulted in no such binding. The immobilization of peptides is a common application of activated agarose supports. To demonstrate the ease and convenience of clickable agarose in this endeavor, we prepared a derivative of the C-terminal sequence of HIV protease (QCTLNF), which contains primary amide, carboxylic acid, thiol, and alcohol functional groups which would otherwise complicate the choice of attachment stragtegy. An extra alkyne-containing amino acid was used to end the standard solid-phase peptide synthesis sequence, and the final oligopeptide was cleaved from the resin retaining its N-terminal Fmoc protecting group (compound 15, Figure 3). Purification by reversed-phase chromatography was followed by reaction with agarose azide 4 by the method described above. Fmoc deprotection of 16 with piperidine revealed the amine-terminated immobilized peptide 17 and showed by quantitation of the released fluorene chromophore a capture of 39% of the azide groups installed on the agarose support. The resulting peptide concentration is more than enough to be useful for applications such as aptamer selection and binding studies, which are currently underway. Affinity purification of enzymes and antibodies by reversible interaction with a binding site or reactive tag is enabled by attachment of the complementary group to the solid support with a chemically compatible linker. An example is shown in Figure 4. Certain monoclonal antibodies generated against diketone 20 (Figure 5) have been shown to possess crucial active-site lysine residues

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Figure 3. Peptide immobilization on agarose azide; a, standard Cu‚bipy-mediated coupling conditions (see Experimental Procedures), followed by a wash procedure including 0.l M EDTA to remove Cu ions; b, 20% piperidine in DMF, RT, 10 min.

Figure 4. Aliphatic aldehyde immobilization on agarose azide; a, standard Cu‚bipy-mediated coupling conditions (see Experimental Procedures), followed by a wash procedure including 0.l M EDTA to remove Cu ions.

Figure 5. Elution profiles on a column of agarose 19 of (A) antibody 84G3 and (B) IgY isolated from chicken eggs obtained prior to an immunization. A greater wash volume at pH 7.4 was used in A, but the patterns were reproducible. The antibody isolated from chromatograph A was found to be catalytically active in a standard retro-aldol reaction. The structure of the hapten used to generate 84G3 (20) is shown.

which catalyze asymmetric aldol and retro-aldol reactions by the formation of enamine intermediates from aldehyde substrates (8, 11). It was envisioned that an immobilized aldehyde could be used to purify such antibodies by virtue of reversible imine formation. The attachment of such an aldehyde to agarose by standard amide or ether bondforming methods would usually require protection/deprotection steps, which are avoided by the use of the CuAAC process. Thus, azidoaldehyde 18 was ligated without difficulty to agarose-alkyne 5 by the standard protocol to give 19. The presence of the aldehyde group was confirmed by treatment of agarose 19 with p-nitrophenylhydrazine to give a yellow color consistent with the formation of the corresponding hydrazone. As shown in

Figure 5, agarose 19 did indeed bind an antibody raised against 20, but not an unrelated polyclonal antibody. The results described here illustrate the convenient use of a single pair of functionalized agarose materials, azide 4 and alkyne 5, for the attachment of a chemically diverse set of functional molecules. While we produced 4 and 5 for this study from a commercial “specialty” agaroseamine, it should be possible to access similar materials from agarose itself and much less expensive reagents; such large-scale production of clickable agarose materials is underway in our laboratory. Given the established applicability of the CuAAC reaction to a widely diverse set of building blocks in organic, materials, and biological chemistry, we believe that there are few limitations to

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the nature of structures that can be married to soluble or insoluble supports in this manner for a wide variety of applications. ACKNOWLEDGMENT

We thank the Skaggs Institute for Chemical Biology and the National Institutes of Health (GM48870) for support of this work, Dr. Sayam Sen Gupta for helpful discussions, and Dr. Diane Kubitz for a sample of antibody 84G3. LITERATURE CITED (1) Wilchek, M., Miron, T., and Kohn, J. (1984) Affinitychromatography. Methods Enzymol. 104, 3-55, Part C.; Scouten, W. H. (1981) Affinity Chromatography, John Wiley & Sons, New York. (2) Janson, J.-C., and Ryden, L. (1998) in Protein Purification, pp 375-442, John Wiley & Sons, New York. (3) Huisgen, R. (1984) 1,3-Dipolar cycloaddition - introduction, survey, mechanism, in 1,3-Dipolar Cycloaddition Chemistry (Padwa, A., Ed.) pp 1-176, Wiley, New York. (4) (a) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective ligation of azides and terminal alkynes. Angew. Chem., Int. Ed. 41, 2596-2599. (b) Tornøe, C. W., Christensen, C., and Meldal, M. (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057-3062. (c) Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless, K. B., and Finn, M. G. (2003) Bioconjugation by copper(I)-catalyzed

azide-alkyne [3+2] cycloaddition. J. Am. Chem. Soc. 125, 3192-3193. (d) Rodionov, V. O., Fokin, V. V., and Finn, M. G. (2005) Mechanism of the ligand-free CuI-catalyzed azidealkyne cycloaddition reaction. Angew. Chem., Int. Ed. 44, 2210-2215. (5) Lewis, W. G., Magallon, F. G., Fokin, V. V., and Finn, M. G. (2004) Discovery and characterization of catalysts for azide-alkyne cycloaddition by fluorescence quenching. J. Am. Chem. Soc. 126, 9152-9153. (6) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem., Int. Ed. 40, 2004-2021. (7) Chan, W. C. W., and White, P. D. (2000) Fmoc Solid-Phase Peptide Synthesis: A Practical Approach, Oxford University Press, New York. (8) Zhong, G., Lerner, R. A., and Barbas, C. F. I. (1999) Broadening the aldolase catalytic antibody repertoire by combining reactive immunization and transition state theory: new enantio- and diastereoselectivities. Angew. Chem., Int. Ed. 38, 3738-3741. (9) Turner, J. M., Bui, T., Lerner, R. A., Barbas, C. F. I., and List, B. (2000) An efficient benchtop system for multigramscale kinetic resolutions using aldolase antibodies. Chem. Eur. J. 6, 2772-2774. (10) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598. (11) Wagner, J., Lerner, R. A., and Barbas, C. F. I. (1995) Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Science 270, 1797-1800.

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