Selective Coupling of Click Anchors to Proteins via Trypsiligase

Dec 15, 2015 - Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has bee...
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Selective Coupling of Click Anchors to Proteins via Trypsiligase Christoph Meyer,† Sandra Liebscher,† and Frank Bordusa* Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany S Supporting Information *

ABSTRACT: The combination of pure chemical methods with enzymatic approaches offers a kit system with maximum flexibility for site-specifically tagging proteins with a broad variety of artificial structures. Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has been used to introduce click anchors into the human protein cyclophilin 18 and the antibody Fab fragments anti-TNFα and anti-Her2. The subsequent click reactions with tetrazine or norbornene moieties lead to quantitative conversions to the corresponding dihydropyridazine products, thereby forming a stable covalent linkage between the label and the protein of interest. With this technology, cyclophilin 18 has been efficiently modified with the fluorescent dansyl moiety and the pharmaceutically relevant polymer PEG exclusively at its N-terminus. With the same methodology, the Fab fragments of anti-TNFα and anti-Her2 were derivatized exclusively at their C-terminal ends with PEG and the fluorescent dye carboxyfluorescein in the case of anti-TNFα or with the cytotoxic payload DM1 in the case of antiHer2, to form a homogeneous antibody−drug conjugate (ADC).

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INTRODUCTION The introduction of click chemistry by Sharpless in 20011−3 has led to applications not only in the field of bioconjugation but also in drug discovery, material sciences, and organic chemistry in general.4 A clear disadvantage of Sharpless’ original clickchemical approach between azides and terminal alkynes is the need of copper(I) species as catalysts. Since biomolecules have the tendency to aggregate upon contact with those exogenous metal species,5,6 copper-free alternatives were developed.7 In this context, the so-called “strain-promoted” 1,3-dipolar cycloaddition reactions between alkynes and azides were introduced specifically for protein labeling in living systems.8 High biocompatibility was also shown for the inverse Diels− Alder reaction between 1,2,4,5-tetrazine derivatives9,10 and electron-rich alkenes (e.g., styrenes, norbornenes, or cyclooctenes) forming stable dihydropyridazine ligation products11,12 without the need of potentially explosive azides. For inverse Diels−Alder reactions, ambient temperatures and aqueous environments suffice and therefore are ideally suited for the chemical modification of sensitive biomolecules. In addition, the electronic structure of tetrazines and the associated absorption characteristics above 500 nm allow for a direct photometric control where a consumption of the tetrazine moiety provides a means to monitor the reaction progress. Furthermore, superior electron-rich dienophiles, such as transcyclooctenes, are readily available. However, the poor water solubility and bulky structure limit their applications, especially in protein chemistry. The drawbacks with bulky transcyclooctenes are partially circumvented by the use of norbornenes. Not only are the norbornenes less sterically demanding, but they are also readily available in large amounts at lesser costs. Furthermore, product formation with norbornenes as dienophiles is in the range of minutes, making © XXXX American Chemical Society

the velocity of such reactions comparable to those with transcyclooctenes.13 The combination of chemical and enzymatic approaches has obvious advantages, namely, the inherent regio- and stereospecificity of enzymes on the one hand and the broad versatility of click-chemical reactions on the other. Consequently, the combination of both characteristics should result in an approach with maximum flexibility of site-specific protein modifications not reachable with either method alone. Currently, three of the most commonly used enzymes for bioconjugation are formylglycine-generating enzyme (FGE),14 sortase (sortagging),15 and the recently published trypsiligase approach.16 The trypsiligase reaction combines the mechanism of proteases with those of ligases by specifically improving the protease’s reverse reaction, the ligase activity. The respective shift in activity has been achieved by a unique substrate-assisted activation mechanism of the engineered biocatalyst obtaining its enzymatic activity exclusively in the presence of the tripeptide sequence YRH. The introduction of this recognition sequence at the N- or C-terminal region of appropriate target proteins via standard mutagenesis enables highly selective enzymatic couplings of click anchor molecules (Figure 1). N-terminal modification of proteins proceeds via the concept of substrate mimetics (such as peptidyl 4-guanidinophenyl esters OGp), whose ester leaving groups mimic enzyme-specific amino acid side chains, allowing the coupling of diverse and even nonpeptidic acyl moieties to the target protein (Figure 1a).16 C-terminal modification reactions are mechanistically comparable to transpeptidation reactions between the introReceived: November 13, 2015 Revised: December 14, 2015

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Figure 1. Reaction schematics of the trypsiligase-catalyzed coupling of click anchors to proteins of interest (POI). (a) N-terminal protein modification. POI is equipped with an N-terminal (purification) tag and the recognition sequence YRH. Trypsiligase-catalyzed tag cleavage is followed by the introduction of the click anchor. Acyl transfer is initiated by the addition of acyl 4-guanidinophenyl esters (OGp). Addition of the click counterpart leads to the final protein product. (b) C-terminal protein modification. POI’s C-terminus is elongated by the recognition sequence YRH and an optional (purification) tag. Trypsiligase-catalyzed modification reaction occurs via a transpeptidation mechanism. Cleavage of the tag initially results in a transient acyl−enzyme intermediate. In the presence of Arg-His-containing acceptor peptides bearing the appropriate click anchor, the desired product is formed. Addition of the second click reactant leads to the final protein product.

Figure 2. Inverse electron-demand Diels−Alder cycloaddition of tetrazine and norbornene. (a) Tetrazines and norbornenes react under formation of dihydropyridazines. (b) Trypsiligase substrates conjugated with tetrazine or norbornene. RHAAC(Tet) for C-terminal protein modification with tetrazine and Norb-OGp for N-terminal introduction of norbornene. Trypsiligase recognition site and click moieties are highlighted in red and blue, respectively. (c) Time course of the reaction of RHAAC(Tet) (1 mM) with 5-norbornene-2-carboxylic acid (2 mM) in 100 mM HEPES buffer, pH 7.8, recorded at 513 nm, proving the complete consumption of the tetrazine peptide within 20 min.

duced YRH-sequence and a nucleophilic acceptor peptide (RHX) (Figure 1b). The enzyme’s high ligation activity and reduced proteolysis rate enabled the use of this approach for selective C-terminal modifications even for proteins of higher complexity, such as antibody fragments.17 The specificity toward proteolytic cleavage of YRH sequence allows for the introduction of detection tags prior to the subsequent labeling of the N- or C-terminus of the target protein in a one-pot reaction. In this study, we demonstrate the ability of the trypsiligase approach to catalyze the site-directed coupling of click anchors to the N- and C-terminus of selected proteins. The use of those intermediate proteins as substrates for subsequent click reactions finally broadens the usability of the trypsiligase approach to virtually any tag moiety, even with moieties that

are poorly soluble and/or toxic to the enzyme activity or stability.

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RESULTS AND DISCUSSION For click reactions, derivatives of norbornenes, the electronpoor p-1,2,4,5-tetrazinylbenzoic acid and tetrazinylphenol were used (Figure 2a). To mediate enzymatic recognition of the anchor moieties by trypsiligase, click reactants need to be provided as acyl-4-guanidinophenyl (OGp) ester (N-terminal modification) or as a part of a specific peptide sequence with a N-terminal Arg-His motif (C-terminal modification). Accordingly, the peptide RHAAC(Tet) for the C-terminal introduction of tetrazine (Tet) and the ester Norb-OGp for the Nterminal protein modification with norbornene (Norb) moieties were synthesized (Figure 2b). As mentioned above, B

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Figure 3. (a) Trypsiligase-catalyzed acyl transfer of Norb-OGp toward the N-terminus of the 5,6-carboxyfluorescein (CF)-conjugated tetrapeptide RHAK(CF). HPLC analysis of the reaction mixture at t = 0 min (red curve), t = 30 min (green curve), and t = 1 h (black curve) and ESI mass spectrometric analysis of the product peak Norb-RHAK(CF): m/zcalc = 1101, m/zfound = 1101.5 ± 1 (M + H+), 551 ± 1 ((M + 2H+)/2). Reaction conditions: 1 mM RHAK(CF)-OH, 1.5 mM Norb-OGp, 20 μM trypsiligase, 100 mM HEPES buffer, pH 7.8, 100 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2, T = 30 °C. HPLC conditions: Lichrospher 100 RP18 column (125 mm × 4 mm), 10−80% ACN in 15 min. (b) UPLC analysis of trypsiligase-catalyzed transpeptidation reaction between Bz-AAYRHAAG and RHAAC(Tet) at t = 0 min and t = 20 min under formation of the desired product Bz-AAYRHAAC(Tet) and the side product Bz-AAY. Product peak was identified via ESI mass spectrometric analysis: m/zcalc = 1290 Da, m/zfound = 646 ± 1 ((M + 2H+)/2). Reaction conditions: 1 mM Bz-AAYRHAAG, 2 mM RHAAC(Tet), 20 μM trypsiligase, 100 mM HEPES buffer, pH 7.8, 100 mM NaCl, 10 mM CaCl2, 0.1 mM ZnCl2. UPLC conditions: Acquity BEH130 C18 column (2.1 mm × 100 mm), 10−90% ACN in 5 min.

at other positions of Cyp18 could be detected. In the second step, the enzymatic acyl transfer reaction was initiated by addition of Norb-OGp to the truncated Cyp18. Finally, further conversion of the formed Norb-RH-Cyp18 took place without prior purification by adding the click counterparts dansyltetrazine (Dns-Tet; Figure 4c) or PEG-tetrazine (PEG-Tet; Figure 4e) again in a one-pot reaction. Clicking was completed within 20 min, resulting again in a quantitative conversion that could be confirmed via hydrophobic interaction chromatography (HIC) for the dansylated protein (Figure 4c) and SDS− PAGE analysis for the PEGylated Cyp18 derivative (Figure 4e) indicating that even the trypsiligase-catalyzed acyl transfer proceeded with quantitative product yields. Identity of the dansylated protein product was confirmed via mass spectrometry, while no remaining unclicked protein species could be detected (Figure 4d). Also, PPIase activity measurements of the dansylated Cyp18 showed full enzymatic activity when compared with unmodified protein (Figure S2). In addition, similar results were shown in reactions with human Pin1, suggesting that the current method is independent of the individual nature of the protein of interest (cf. Figure S3). Due to our particular interest in antibody drug conjugates (ADCs), we extended our studies using pharmaceutically relevant Fab fragments. Fab fragments generally consist of two disulfide-bridged peptide chains, whose N-terminal regions are involved in antigen binding making those regions unsuitable for covalent modification. For our studies we chose Fab antiHer2, derived from the FDA-approved therapeutic antibody trastuzumab (Herceptin) clinically used for the treatment of Her2-positive breast cancers.19 The recombinant Fab fragment was extended at the C-terminus of its heavy chain by the recognition sequence YRH fused to a myc-tag.20 General acceptance of those YRH-bearing Fab fragments by trypsiligase was demonstrated recently.17 The modified Fab fragment was expressed in the periplasm of Escherichia coli BL21 cells and

the absorption characteristics of the tetrazine substrates allow for the photometric control of the click reaction at 513 nm showing complete consumption of the tetrazine moiety within 20 min (Figure 2c). General acceptance of the synthesized substrates by the biocatalyst was confirmed by initial model reactions. In the case of Norb-OGp its general substrate behavior was proven by acyl transfer experiments using the carboxyfluorescein (CF)containing peptide RHAK(CF) as model acceptor peptide (Figure 3a). The desired product formation was confirmed via ESI mass spectrometric analysis. Most importantly, a quantitative formation of the norbornene modified peptide Norb-RHAK(CF) could be detected via UPLC analysis within 1 h reaction time, suggesting that the trypsiligase reaction occurs with quantitative product yield. In case of the trypsiligase-mediated C-terminal modification reaction, the model donor peptide Bz-AAYRHAAG was converted with the acceptor peptide RHAAC(Tet) (Figure 3b). Enzymatic product formation was again recorded via UPLC analysis. Besides the desired main product BzAAYRHAAC(Tet), small amounts of the hydrolysis product Bz-AAY and the educt Bz-AAYRHAAG were observed. To further test the applicability of our method to N-terminal derivatization of functional proteins, we evaluated the human peptidylprolyl cis/trans isomerase (PPIase) cyclophilin 18 (Cyp18) as the protein of interest (Figure 4a). In common with most proteins, native Cyp18 contains several nucleophilic amino acid side chain residues besides the N-terminal amino function (e.g., lysine, arginine, or cysteine; cf. Scheme S1) that make the use of conventional chemical labeling procedures to form homogeneous products for obvious reasons impossible. In the first step, the N-terminal Strep-tag II18-YRH fusion of Cyp18 was quantitatively clipped by trypsiligase specifically at its recognition sequence to produce selectively truncated Cyp18 with the enzyme specific N-terminal Arg-His motif (RH-Cyp18; Figure 4b). Importantly, no undesired proteolysis C

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Figure 4. N-terminal modification of human cyclophilin 18 (Cyp18) via the combined trypsiligase/click chemistry approach. (a) Schematic course of the combined labeling approach. (b) Time course of trypsiligase-catalyzed cleavage of the Strep-tag II fusion monitored by SDS−PAGE. M: molecular weight marker. Tl: trypsiligase. Reaction conditions: 100 μM StrepII-YRH-cyclophilin 18, 10 μM trypsiligase, 50 μM ZnCl2, 100 mM HEPES buffer, pH 7.8, 100 mM NaCl, 10 mM CaCl2. (c) Trypsiligase-catalyzed acyl transfer of norbornene and subsequent click reaction with DnsTet. Acyl transfer was initiated by the addition of 0.2 mM Norb-OGp and click reaction by the addition of 0.5 mM Dns-Tet derivative. Click reaction was followed via HIC analysis: (blue curve) click reaction t = 0 min (λ = 280 nm), (red curve) t = 30 min (λ = 280 nm), (gray curve) t = 30 min (λ = 320 nm). HIC conditions: TSK-gel butyl-NPR, A = 1.5 M ammonium sulfate/0.05 M HEPES buffer, pH 7.5, B = 0.05 M HEPES buffer, pH 7.5; 0− 2 min 100% A, 2−15 min 100% A to 100% B. (d) ESI-MS analysis of the reaction mixture prior (blue curve) and after (red curve) click reaction. Norb-RH-Cyp18: Mcalc = 18 478 Da, Mfound = 18 480 ± 1 Da. Dns-RH-Cyp: Mcalc = 18 857 Da, Mfound = 18 859 ± 1 Da. Mass spectra are deconvoluted. For original data see Figure S1. (e) Click reaction of PEG-Tet to Norb-RH-Cyp18 followed via SDS−PAGE and Coomassie or iodine/Coomassie staining. M: molecular weight marker. R: fusion protein (StrepII-YRH-cyclophilin 18) as reference.

Fab-anti-Her2-YRH-Tet results in rapid formation of specific DM1-conjugated Fab-anti-Her2-YRH-DM1 with nearly quantitative product yields within 30 min reaction time (Figure 5b). Product was again isolated via HIC, and its identity was confirmed by mass spectrometry (Mcalc = 49 658 Da, Mfound = 49 659 Da). Finally, full functionality of the isolated Fab-antiHer2-YRH-DM1 was proven by ELISA experiments confirming unaltered KD values of the Fab fragments prior and after the modification reactions and therefore high bioorthogonality of the applied conjugation procedure (Figure 4d). To demonstrate the universality of the combined trypsiligase/click approach, we confirmed our results for the selective modification of Fab anti-TNFα which corresponds to the approved antibody fragment certolizumab pegol (CDP870, CIMZIA) clinically used for the treatment of Crohn’s disease and rheumatoid arthritis.23,24 Trypsiligase-catalyzed introduction of tetrazine as click anchor proceeds similar to the efficiency observed with anti-Her2 Fab fragment (cf. Figure S5a). We further broadened the click chemistry portfolio with additional synthetic substrates, i.e., Norb-CF (Figure S5b) and PEG-conjugated norbornene (Norb-PEG, Figure S6b). Consistently, with all substrates the desired and specifically modified Fab products could be obtained in high efficiencies

purified to homogeneity by affinity chromatography (for details see Supporting Information). In the presence of the tetrazine-bearing RH-peptide, trypsiligase rapidly converted the Fab fragment to the desired primary product Fab-anti-Her2-YRH-Tet. Reaction analysis, quantification, and product isolation were performed via hydrophobic interaction chromatography (HIC). Besides the remaining educt Fab-anti-Her2-YRH-myc, two additional product peaks were observed in the HIC profile representing the desired main product, tetrazine-modified Fab-anti-Her2YRH-Tet and hydrolyzed starting Fab fragment (Fab-antiHer2-Y) (Figure 5a). A maximum yield of about 70% after 20 min reaction time for the enzymatic reaction was determined. Identity of the desired Fab-anti-Her2-YRH-Tet was confirmed via mass spectrometric analysis (Mcalc = 48 688 Da, Mfound = 48 690 Da). For subsequent conjugation via the inverse Diels− Alder click reaction we synthesized Norb-DM1 (Figure 5c). DM1 is a potent cytotoxin that inhibits cell proliferation with IC50 values in the nanomolar range and is approved for treatment of breast cancer when conjugated to anti-Her2 antibody (trastuzumab emtansine, KADCYLA).21,22 Prior to subjecting to click chemistry, the HIC-isolated Fab-anti-Her2YRH-Tet was desalted. Addition of Norb-DM1 to the desalted D

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Figure 5. C-terminal modification of anti-Her2 Fab fragment via the combined trypsiligase/click chemistry approach. (a) HIC and MS analyses of the enzymatic modification reaction of Fab-YRH-myc with RHAC(Tet) to the corresponding product Fab-YRH-Tet at t = 20 min, which was identified via ESI mass spectrometry (Mcalc = 48 688 Da, Mfound = 48 690 ± 1 Da, Mfound = 48 786 ± 1 Da (M + SO42−)). Reaction conditions: 100 μM Fab-YRH-StrepII, 1 mM RHAC(Tet), 10 μM trypsiligase, 100 μM ZnCl2, 100 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.8. (b) HIC and MS analyses of the chemical modification reaction of Fab-YRH-Tet to Fab-YRH-DM1 with Norb-DM1 at t = 0 min (black curve) and t = 30 min (red curve). HIC conditions: TSK-gel butyl-NPR, A = 1.5 M ammonium sulfate/0.05 M HEPES buffer, pH 7.5, B = 0.05 M HEPES buffer, pH 7.5; 0−2 min 100% A, 2−15 min 100% A to 100% B. ESI mass spectrometry confirms identity of Fab-YRH-DM1 (Mcalc = 49 658 Da, Mfound = 49 659 ± 1 Da, Mfound = 49 755 (M + SO42−)). Mass spectra are deconvoluted. For original data see Figure S4. Reaction conditions: 80 μM Fab-YRH-Tet, 160 μM Norb-DM1, 50 mM HEPES buffer, pH 7.0. (c) Chemical structure of Norb-DM1. (d) ELISA test of Fab-anti-Her2-YRH-myc (●) in comparison with Fab-anti-Her2-YRH-DM1 (▲). Labeling procedure does not affect binding affinity of Fab-anti-Her2-YRH-DM1 to its antigen.

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CONCLUSION This work proves the feasibility of combining the trypsiligase approach with click chemistry to provide a toolbox for the efficient and site-specific covalent coupling of relevant molecules to proteins not only for scientific objectives but also for pharmaceutical purposes. The proof of concept was shown for the N-terminal labeling of the human enzymes cyclophilin 18 and Pin1 as well as the C-terminal derivatization of the therapeutically relevant Fab fragments anti-Her2 and anti-TNFα. Fab-anti-Her2 was selectively modified with DM1 to form a homogeneous ADC derivative, whereas Fab antiTNFα was labeled with a fluorescent moiety on the one hand and polyethylene glycol on the other. From the course of all reactions, the universality of the current approach can be expected. In all individual cases, click anchors could be introduced enzymatically with full regiospecificity under conditions compatible with protein stability and functionality. The subsequent click reactions occurred without the formation

and without any unexpected additional byproducts (Figures S5 and S6). Thus, the trypsiligase-based method of introducing an anchor sequence into biomolecules in combination with a subsequent click reaction offers a modular tool for being flexible in terms of substrate choice and site-selective, especially when the production of homogeneous antibody−drug conjugates (ADC) is the major focal point. Furthermore, the use of excessive nucleophilic peptide functionalities usually required in purely enzymatic transpeptidation reactions often leads to high costs for chemical synthesis which causes a significant need for investment toward the recycling of unreacted material. Effectiveness of the introduced click anchors provides the possibility to introduce rather inexpensive click anchors for the introduction of more expensive drugs as, for example, maytansines in economical amounts as shown. E

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(3) 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. (4) Lutz, J. F., and Zarafshani, Z. (2008) Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using azide-alkyne "click" chemistry. Adv. Drug Delivery Rev. 60, 958−70. (5) Gaetke, L. M., and Chow, C. K. (2003) Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology 189, 147−63. (6) Capanni, C., Taddei, N., Gabrielli, S., Messori, L., Orioli, P., Chiti, F., Stefani, M., and Ramponi, G. (2004) Investigation of the effects of copper ions on protein aggregation using a model system. Cell. Mol. Life Sci. 61, 982−991. (7) Jewett, J. C., and Bertozzi, C. R. (2010) Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39, 1272−9. (8) Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A StrainPromoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 126, 15046−15047. (9) Sauer, J., Mielert, A., Lang, D., and Peter, D. (1965) Umsetzungen von 1,2,4,5-Tetrazinen mit Olefinen. Zur Struktur von Dihydropyridazinen. Chem. Ber. 98, 1435. (10) Devaraj, N. K., Weissleder, R., and Hilderbrand, S. A. (2008) Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging. Bioconjugate Chem. 19, 2297−2299. (11) Bluestone, H., Bimber, R., Berkey, R., and Mandel, Z. (1961) Chlorinated Derivatives of Butadiene Sulfone and Diels-Alder Reactions of 3,4-Dichlorothiophene 1,1-Dioxide. J. Org. Chem. 26, 346−351. (12) Sauer, J., Heldmann, D. K., Hetzenegger, J., Krauthan, J., Sichert, H., and Schuster, J. (1998) 1,2,4,5-Tetrazine: Synthesis and Reactivity in [4+2] Cycloadditions. Eur. J. Org. Chem. 1998, 2885−2896. (13) Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity. J. Am. Chem. Soc. 130, 13518−13519. (14) Hudak, J. E., Barfield, R. M., de Hart, G. W., Grob, P., Nogales, E., Bertozzi, C. R., and Rabuka, D. (2012) Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag. Angew. Chem., Int. Ed. 51, 4161−5. (15) Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E., and Ploegh, H. L. (2007) Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3, 707−708. (16) Liebscher, S., Schoepfel, M., Aumueller, T., Sharkhuukhen, A., Pech, A., Hoess, E., Parthier, C., Jahreis, G., Stubbs, M. T., and Bordusa, F. (2014) N-Terminal Protein Modification by SubstrateActivated Reverse Proteolysis. Angew. Chem., Int. Ed. 53, 3024−3028. (17) Liebscher, S., Kornberger, P., Fink, G., Trost-Gross, E.-M., Hoess, E., Skerra, A., and Bordusa, F. (2014) Derivatization of Antibody Fab Fragments: A Designer Enzyme for Native Protein Modification. ChemBioChem 15, 1096−1100. (18) Schmidt, T. G., and Skerra, A. (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat. Protoc. 2, 1528−35. (19) Hudis, C. A. (2007) Trastuzumab-mechanism of action and use in clinical practice. N. Engl. J. Med. 357, 39−51. (20) Hilpert, K., Hansen, G., Wessner, H., Kuttner, G., Welfle, K., Seifert, M., and Hohne, W. (2001) Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose. Protein Eng., Des. Sel. 14, 803−6. (21) Niculescu-Duvaz, I. (2010) Trastuzumab emtansine, an antibody-drug conjugate for the treatment of HER2+ metastatic breast cancer. Curr. Opin. Mol. Ther. 12, 350−60. (22) Widdison, W. C., Wilhelm, S. D., Cavanagh, E. E., Whiteman, K. R., Leece, B. A., Kovtun, Y., Goldmacher, V. S., Xie, H., Steeves, R. M., Lutz, R. J., Zhao, R., Wang, L., Blattler, W. A., and Chari, R. V. (2006) Semisynthetic maytansine analogues for the targeted treatment of cancer. J. Med. Chem. 49, 4392−408. (23) Goel, N., and Stephens, S. (2010) Certolizumab pegol. mAbs 2, 137−47.

of undesired byproducts in quantitative product yields under bioorthogonal reaction conditions resulting in fully active modified protein species. Thus, while the chemical click reaction benefits from the regioselectivity of the enzyme, the enzymatic approach profits from the flexibility and the efficiency of the click reaction. Furthermore, the combined approach also allows for the introduction of functionalities that are otherwise incompatible with enzymatic reaction conditions or even toxic to the biocatalyst. These characteristics represent a significant progress compared to the purely chemical or enzymatic modification of proteins based on processes where synthetic functionalities are directly introduced, thus leading to heterogeneity in drug-to-protein compositions, unsatisfying yields, noneconomic reactant stoichiometries, restrictions toward the nature of the protein target or functionalities that can be introduced.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00618. Synthesis protocols, experimental details, and additional characterization data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

C.M. and S.L. contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the DFG priority program SPP1623 “Chemoselective reactions for the synthesis and application of functional proteins”. The authors are grateful for the professional technical assistance of C. Langer and S. Kaufmann, the help of Katja Gölldner with ELISA testing, and Dr. C. Wiedemann for NMR measurements.

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ABBREVIATIONS CF, carboxyfluorescein; Bz, benzoyl; FDA, U.S. Food and Drug Administration; DIPEA, N,N-diisopropylethylamine; DM1, N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)maytansine or mertansine; HIC, hydrophobic interaction chromatography; Norb, norbornene; OGp, 4-guanidinophenyl ester; POI, protein of interest; PEG, polyethylene glycol; PPIase, peptidylprolyl (cis/trans) isomerase; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; Tet, tetrazine; TFA, trifluoroacetic acid

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REFERENCES

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