Azide Amidation

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Protein C-Terminal Modification through Thioacid/Azide Amidation Xiaohong Zhang, Fupeng Li, Xiao-Wei Lu, and Chuan-Fa Liu* Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551. Received November 10, 2008; Revised Manuscript Received January 6, 2009

The preparation of protein bioconjugates has been largely dependent on the development of selective chemistries that are orthogonal to the diverse functionalities present in a protein. Here, we report a new method for C-terminusdirected modification of recombinant proteins. The method is based on the thioacid/azide amidation reaction. Essentially, hydrothiolytic cleavage of the thioester intermediate in protein splicing yields a recombinant protein with a unique thioacid group at the C-terminus, which is then chemoselectively amidated with an electron-poor organic azide carrying a biofunctional tag. The small ubiquitin protein was used as a model system to demonstrate the utility of this new bioconjugation method. C-terminal PEGylation or biotinylation of ubiquitin was readily achieved through amidation of ubiquitin thioacid with a sulfonazide-functionalized PEG or biotin derivative. Our data validate that thioacid/azide amidation is a mechanistically novel and practically useful method for siteselective protein modification.

Site-selective protein modification techniques are important tools for protein structure-function study and drug discovery (1-3). For instance, such techniques can be used to label proteins with a special biophysical probe for structural and biochemical studies (4) or with a biocompatible polymer for the development of protein-based therapeutic agents (5). They are also useful as immobilizing techniques to tether proteins to solid supports for the fabrication of protein chips (6). Traditional methods for protein modification rely mainly on classic acylation or alkylation chemistries involving the side chain functional groups of Lys, Asp/Glu, and Cys residues (1). However, the use of these methods is often limited by the fact that proteins typically carry multiple copies of the targeted residue, making it difficult to control the degree and site of modification. This commonly results in product mixtures with considerable structural heterogeneity. Nontraditional methods have also been developed recently that target the electron-rich side chain aromatic rings of Trp or Tyr residues (7). Because these aromatic amino acids are found less frequently on the protein surface, it may be possible to achieve single-site modification if one such residue is solvent-exposed (8). However, these new techniques also have limitations because of the low likelihood of having a single solvent-exposed aromatic residue in most proteins. Molecular biology methods that make use of the cell’s protein synthesis machinery offer powerful alternatives to chemically based techniques for protein modification. Recent excitement is seen with the methodology developed by Schultz’s group and others (9). These methods rely on the use of an amber stop codon and a corresponding suppressor tRNA to incorporate into a protein a desired unnatural amino acid which may already carry the biophysical probe of interest in place or contain a unique chemical/photochemical reactivity for later use (9). Compared to modifying the internal residues, protein Cterminal modification offers some distinct advantages. The C-terminal tail of a protein is usually flexible and solvent* To whom correspondence should be addressed. Chuan-Fa Liu, Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551. E-mail: [email protected]. Tel: +65-6316-2867. Fax: +65-6791-3856.

exposed, which makes it easily accessible by the tagging reagent. Modifying the C-terminus is also less likely to adversely affect a protein’s biological activity as the active spot of a protein is often distant from the terminal end. Moreover, since there is only one C-terminus in a protein, in principle it should be possible to distinguish it from the internal residues for singlesite modification. There exist several enzymatic and biosynthetic methods to introduce a C-terminal modification onto a protein. For instance, C-terminal specific labeling has been carried out by catalytic transacylation reactions using carboxypeptidase Y (10, 11). Puromycin-based techniques have also been developed that allow the incorporation of various C-terminal tags (12, 13). Exploitation of intein-mediated protein splicing (14) has generated very useful approaches to protein C-terminal modification (15-17), which are mostly based on the thioestermediated ligation chemistry (18). In our efforts to develop new chemoselective methods for protein synthesis and modification, we have paid much attention to a unique functional group, the thioacid. For instance, we have made use of the soft and powerful nucleophilicity of thioacids in the development of a highly effective thioacid capture peptide ligation method (19). The synthetic utility of the thioacid group is also experiencing renewed interest in the thioacid/azide amidation reaction (20) for the preparation of complex amides (21). Thorough mechanistic studies by Williams et al. suggest that this reaction proceeds through the formation of a thiatriazoline intermediate which subsequently decomposes to yield the amide product (21, 22). The reaction is especially fast with electron-deficient azides such as sulfonazides, owing to a highly efficient prior capture mechanism (22). Although previous studies have only demonstrated thioacid/azide coupling with small thioacids such as thiobenzoic acid and N-protected amino thioacids (21, 23-25), its unique reaction features imply that it should have the required chemical orthogonality to work in a chemoselective manner with large and complex thioacid compounds such as peptides and proteins. In our recent work, clean and near-quantitative formation of the amidation products was shown between unprotected thioacid peptides and tosyl azide in highly dilute solutions (26). In the present study, we extend the application of thioacid/azide amidation to C-terminal modification of recombinant proteins (Figure 1).

10.1021/bc800488n CCC: $40.75  2009 American Chemical Society Published on Web 01/13/2009

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Figure 1. General scheme of protein C-terminal modification through thioacid/azide amidation.

Figure 2. Purification and characterization of ubiquitin thioacid. (A) HPLC profile of C8 semiprep purification of the ubiquitin thioacid eluate from chitin beads after hydrothiolysis. The ubiquitin thioacid was eluted by a linear gradient of buffer B in buffer A from 0% to 60% in 60 min. (B) MALDI-TOF MS of purified ubiquitin thioacid. m/z [M + H]+ found: 8580.8, MW calcd: 8580.9 (average)/8576.3 (isotopic). The higher MW species in the spectrum were due to adducts of the sinapic acid matrix.

Figure 3. Sulfonazides useful for C-terminal PEGylation (1) or biotinylation (2) of recombinant thioacid proteins.

We chose the small ubiquitin protein as a model system to test this new protein C-terminal modification method. A crucial step is to obtain the thioacid protein in a sufficient amount. We referred to the method previously proposed by Begley et al. for the production of recombinant proteins with a C-terminal thioacid (27), which is based on the mechanism of capturing and cleaving the thioester intermediate of protein splicing with hydrosulfide ions. Although the potential utility of this method was discussed by the authors (27), no application examples have been reported since its publication. Therefore, in our work, we cloned the ubiquitin gene into the pTYB1 vector (New England Biolabs), which contains the VMA intein from S. cereVisiae. The ubiquitin-intein-CBD fusion protein was overexpressed in E. coli and affinity-purified by binding to chitin beads. The cleavage of ubiquitin from the fusion protein was performed by incubation at 37 °C of the bound fusion protein in the hydrothiolytic cleavage buffer (0.1 M Na2S, 0.25 M HEPES, 0.5 M NaCl, 1 mM EDTA, pH 8.0) overnight. These conditions were similar to those used for hydrothiolysis of synthetic peptide thioesters (26, 28). In the previous work by Begley et al. of preparing a 66-residue protein thioacid ThiS-COSH, the cleavage was done with 30 mM ammonium sulfide at 4 °C (27). The cleaved ubiquitin thioacid was eluted from chitin beads and purified by RP-HPLC (Figure 2A). High-resolution MS analysis confirmed the presence of the thioacid group (Figure 2B). The yield of the ubiquitin thioacid was typically 4-5 mg per liter of cell culture. Two benzenesulfonazide-functionalized tagging agents 1 and 2 (Figure 3) were prepared for introducing a PEG or biotin moiety, respectively, onto the C-terminus of the recombinant ubiquitin (see Supporting Information for the synthesis of these two compounds).

The PEGylation reagent or PEG-sulfonazide 1 (Figure 3) contains a PEG component with an average MW of ca. 5000 Da. It was synthesized to test thioacid amidation with large sulfonyl azides. The amidation reaction of the ubiquitin thioacid with the PEGylation reagent 1 was performed in an aqueous solution: 0.3 mM ubiquitin thioacid, 2 mM PEG sulfonazide 1 in 6 M Gdn-HCl containing 3 mM 2,6-lutidine. The reaction took about 10 h to complete at r.t. and was accompanied by some hydrolysis (Figure 4). 2,6-Lutidine is the recommended base catalyst from previous studies with small thioacids (21, 22). We observed that, if the reaction was conducted in a HEPESbuffered solution (pH 7 or 8) instead, more hydrolysis occurred and the hydrolyzed ubiquitin-COOH appeared immediately after the ubiquitin thioacid was mixed with the azide (data not shown). Therefore, the use of 2,6-lutidine helped minimize the hydrolysis of the ubiquitin thioacid. On the basis of HPLC analysis, the yield of the PEGylation reaction was about 65%. Despite an excess amount of PEGylation agent used, only the mono-PEGylation product (Figure 4A, peak 3 from trace b) was formed in the reaction, which was also confirmed by MS analysis (Figure 4B), indicating that tagging PEG to the protein was highly chemoselective and site-specific. The relatively lengthy reaction time and some degree of hydrolysis may be a result of the large molecular weight and steric hindrance of the PEGylation reagent 1 in which the relatively hydrophobic sulfonylazide may be made less accessible by a surrounding long and hydrophilic PEG chain. The small Gly residue at the C-terminus of ubiquitin may also have contributed to the hydrolysis of the thioacid. The biotinylation reagent or biotinyl-Lys(N3SO2Bz)-Lys-NH2 2 (Figure 3) was prepared by solid-phase synthesis. In this compound, the biotin and sulfonyl azide moieties are linked to

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Figure 4. PEGylation of ubiquitin thioacid. (A) Analytical RP-HPLC (Vadyc C4 column) monitoring of the amidation of ubiquitin thioacid with the PEGylation reagent. Trace a, purified ubiquitin-COSH (peak 1). Trace b, reaction at 10 h. Peak 2, the hydrolyzed product ubiquitin-COOH; peak 3, the PEGylated ubiquitin; peak 4, excessive PEGylation reagent 1. HPLC gradient: 0-60% of buffer B in buffer A for 60 min. (B) MALDITOF of the PEGylated product, average m/z [M + H]+ found ∼13 350, average MW calcd ∼13 500).

Figure 5. Reaction of ubiquitin-COSH with biotin-Lys(N3SO2Bz)-Lys-NH2. (A) Analytical RP-HPLC monitoring of amidation of ubiquitin thioacid with biotinyl-Lys(N3SO2Bz)-Lys-NH2 2 on a Vadyc C4 column. HPLC gradient: 0-40% of buffer B in buffer A for 40 min. Trace a, peak 1: ubiquitin thioacid. Trace b, the reaction mixture at 4 h. Peak 2, biotinyl-Lys(N3SO2Bz)-Lys-NH2 2; peak 3, the biotinylation product containing some minor amount of hydrolyzed ubiquitin in the front. (B) ESI-MS spectrum profile of the last three quarters of peak 3 which contained the biotinylated ubiquitin. MW found 9232.3, MW calcd 9228.5. (C) Western blot analysis of the biotinylated ubiquitin using an avidin-HRP conjugate (Bio-Rad Laboratories, USA).

the respective R- and ε-amino groups of a Lys residue. A second Lys residue is included to increase its water solubility. In the thioacid/azide coupling reaction, 0.3 mM ubiquitin thioacid in 6 M Gdn-HCl containing 3 mM 2,6-lutidine was reacted with ca. 10 equiv of biotinyl-Lys(N3SO2Bz)-Lys-NH2 2 at r.t. for about 4 h to give the biotinylated ubiquitin product (Figure 5). The ligation product eluted a little earlier than the thioacid and almost coeluted with (only slightly behind) the hydrolysis product. In fact, according to MS analysis, the first quarter of fractions of the product peak (peak 3 of trace b, Figure 5A) contained about 30% ubiquitin-COOH (see Supporting Information), and the last three quarters of the peak contained only the ligation product (Figure 5B). No remaining ubiquitin-COSH was detected. The desired amidation product had the expected molecular weight as analyzed by ESI-MS (Figure 5B). As it is much smaller than the PEGylation reagent, the biotinylation reagent reacted faster with the ubiquitin thioacid. Again, except for a minor amount of hydrolysis, no other side reactions were detected. The amidation yield was estimated to be at least 85% by HPLC analysis. The biotinylated ubiquitin was able to bind to avidin as shown by Western blot analysis (Figure 5C).

PEG has been used as a biofunctional group for protein modification since 1977 (29). Covalent attachment of PEG to therapeutic proteins has some desirable functions such as increasing their in vivo stability and solubility (5). It is also very common to covalently tag biotin to a biomolecule such as a protein for use in biochemical assays (http://en.wikipedia.org/ wiki/Biotinylation). Traditional PEGylation and biotinylation methods are nonspecific and introduce PEG or biotin nonspecifically onto nucleophilic amino acids in proteins, giving a mixture of product isomers (30). Thioacid/azide amidation represents a novel method for bioconjugation with a distinctly different mechanism from the intermolecular acylation reactions of conventional amidation. The amidation of thioacids with electron-poor azides involves a prior capture step followed by efficient intramolecular addition to give a thiatriazoline intermediate which spontaneously collapses to form the amide bond (22). These unique features determine the highly chemoselective nature of this reaction, as demonstrated herein by the Cterminus-specific PEGylation or biotinylation of ubiquitin. The reaction can accommodate a broad range of solvents from organic to aqueous (21-26). Notably, in this study the amidation

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of ubiquitin with both the PEG and biotin azide compounds was conducted in a denaturing solution containing 6 M GdnHCl, which will be useful for minimally soluble biomolecules such as membrane proteins. In conclusion, we have demonstrated herein that thioacid/azide amidation is a simple and efficient method for specific C-terminal modification of recombinant proteins. Amidation of ubiquitin thioacid with excess PEG and biotin sulfonazides shows that this reaction is highly specific despite the presence of a large number of functional groups in the protein and remains effective even with large azide compounds. Since protein and peptide thioacids can be easily prepared through hydrothiolysis of the corresponding thioesters (26-28), the thioacid/azide amidation reaction has the potential to become one of the most useful methods for C-terminal modification of bioactive peptides and proteins.

ACKNOWLEDGMENT The authors thank the Ministry of Education (MOE) of Singapore for financial support, as well as Nanyang Technological University. Supporting Information Available: Experimental procedures and supplementary data. This material is available free of charge via the Internet at http://pubs.acs.org.

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