A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins

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Citation Cohen, Daniel T. et al. "A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins." Nature Chemistry 11, 1 (November 2018): 78–85 © 2018, The Author(s), under exclusive licence to Springer Nature Limited

As Published http://dx.doi.org/10.1038/s41557-018-0154-0

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Author ManuscriptAuthor Manuscript Author Nat Chem Manuscript Author . Author manuscript; Manuscript Author available in PMC 2020 January 01. Published in final edited form as: Nat Chem. 2019 January ; 11(1): 78–85. doi:10.1038/s41557-018-0154-0.

A Chemoselective Strategy for Late Stage Functionalization of Complex Small Molecules with Polypeptides and Proteins

Daniel T. Cohen*,#, Chi Zhang^, Colin M. Fadzen^, Alexander J. Mijalis, Liana Hie‡, Kenneth D. Johnson†, Zachary Shriver†, Obadiah Plante†, Scott J. Miller‡, Stephen L. Buchwald, and Bradley L. Pentelute* Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA ‡Department of Chemistry, Yale University, 225 Prospect Street, New Haven, Connecticut 06520, USA †Visterra Inc., 1 Kendall Square, Cambridge, Massachusetts 02139, USA

Abstract Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products has presented a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide-vancomycin conjugates without prefunctionalization of vancomycin. These conjugates had enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we showed that a 6 kDa affibody protein and a 150 kDa IgG antibody could be modified without diminishing bioactivity.

Table of Contents Summary The preparation of conjugates between proteins and small molecules is often challenging and requires several synthetic steps to functionalize each component for conjugation. Herein, a conjugation methodology is described that leverages an electrophilic Se-S bond of selenocysteine to create bioconjugates between polypeptides and complex small molecules.

*Correspondence to: [email protected], [email protected]. #Current address: AbbVie, Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, USA ^These authors contributed equally Contributions D.T.C and B.L.P. conceived the work and designed the experiments. D.T.C, C.Z., and C.M.F. performed the peptide and protein labeling experiments. D.T.C., C.M.F., and A.J.M. developed the seleno-affibody synthesis. L.H. and S.J.M performed the NMR characterization to assign regioselectivity for the selenocysteine conjugation. K.D.J., Z.S. and O.P. carried out the cytotoxicity and hemolysis assays on 23v analogues. D.T.C., C.Z., C.M.F., and B.L.P. wrote the manuscript, with input from all other authors. Competing interests K.D.J., Z.S. and O.P. are employees of Visterra Inc. D.T.C., C.Z., S.L.B., and B.L.P. are inventors on a patent filed by MIT-TLO to cover this work (US patent application no. 15187169). Cohen et al. Page 2

Synthesizing covalent conjugates of a peptide or protein and a complex small molecule is Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author often challenging with available chemical tools. However, such conjugates may have clinical value as they can direct small molecule toxins to certain tissues, widen therapeutic windows, and tune pharmacokinetic and pharmacodynamic properties often beyond the capabilities of each component alone.1 For example, a conjugate of the highly cytotoxic agent emtansine (DM1) with trastuzumab, a HER2 selective antibody, is used clinically to treat late stage breast cancer.2

Early antibody-drug conjugates (ADCs) were prepared in a heterogeneous fashion, usually by linking exposed lysine residues on the antibody with N-hydroxysuccinimide ester derivatives of the small molecule payload.3–5 Alternatively, cysteine conjugation is facilitated by reduction of the four interchain disulfide bonds, followed by reaction with a maleimide-linked payload.6–8 Given the varying stability and efficacy of heterogeneous ADCs, the ADC field has prioritized the development of new methods to create homogenous ADCs.9 Current methods to manufacture homogenous ADCs include leveraging the reactivity of cysteine and lysine, incorporating unnatural amino acids into the antibody, and utilizing conserved glycans on the antibody surface.9 Many of these strategies require modification of both the antibody and the small molecule in order to enable homogeneous linkage, which is frequently laborious.

The direct and chemoselective conjugation of complex small molecules to peptides or proteins is a formidable task due to the presence of multiple nucleophilic and electrophilic functional groups. To achieve this conjugation with high selectivity, the reactivity of the small molecule must be complementary with the reactivity of the coupling functional group in the biopolymer. In particular, a technique for the replacement of an aromatic C–H bond of a small molecule with a biopolymer would be an especially straightforward and appealing means of coupling two complex fragments. There are several examples of using both metal- free and copper-catalyzed reactions to connect aryl thiols to arenes.10–12 Additionally, Fang et al. have demonstrated C-S and C-Se bond formation with indoles using iron-catalyzed chemistry with diaryldisulfides and diarylselenides.13 Unfortunately, these techniques often require bioincompatible solvents such as DMF and are performed between two small molecules. However, these types of reactions suggest that an electrophilic disulfide, diselenide, or mixed Se-S bond in a biopolymer may be able to be used for direct, aromatic C-H bond replacement if the conditions were milder.

We recently reported the selective formation of carbon-selenium bonds in unprotected peptides containing an oxidized, electrophilic selenocysteine residue.14 This approach exploited an electrophilic selenium-sulfur (Se–S) bond in combination with a copper reagent and a boronic acid to selectively arylate the selenium of selenocysteine. Despite the elaborate scope and mild conditions, this method required the use of a boronic acid functional group to facilitate the C–Se bond formation. The need for the boronic acid hampers the overall efficiency for conjugating complex small molecules to biopolymers because additional synthetic steps are needed to install the prerequisite boronic acid.15 Having demonstrated that we can access electrophilic selenocysteine under mild biocompatible conditions, we questioned whether our electrophilic selenium reagent in combination with electron-donating arenes could be used to achieve the direct conjugation

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of unmodified small molecules to peptides and proteins (Fig. 1). If effective, this would be a Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author general route for the conjugation of natural products and pharmaceuticals with electron-rich units to peptides and proteins. Herein we report an approach to creating peptide and protein- small molecule conjugates by matching the inherent reactivity of a small molecule with an oxidized selenocysteine residue (Sec). This strategy exploits the unique nature of electrophilic Sec in combination with the electron-rich nature of arene-containing small molecules to provide the respective conjugate (Fig. 1).

Results and Discussion We began our investigations with the conjugation of vancomycin to antimicrobial peptides containing an oxidized selenocysteine residue. Vancomycin provided a good candidate for the small molecule component, as the embedded electron-rich resorcinol ring of vancomycin serves as a site for conjugation. Additionally, vancomycin is a potent antibiotic often used as first-line treatment for methicillin-resistant S. aureus.16 We hypothesized that conjugation to antimicrobial peptides may be beneficial as vancomycin is unable to penetrate the outer membrane of Gram-negative bacteria.17 Also compared with larger proteins, small antimicrobial peptides would be a lower complexity starting point for the development of our chemistry. Upon incorporation of an oxidized selenocysteine residue into various antimicrobial peptides, the conjugation reaction described herein provides a rapid and convenient single step approach to prepare a small library of antibacterial peptide- vancomycin conjugates in multi-milligram quantities (Fig. 2A).

The reactions were run in aqueous, pH 8 media at 37 °C for 3–5 hours. The corresponding conjugates were formed in 40–90% yield based on liquid chromatography-mass spectrometry (LC-MS) of the unpurified reaction mixture. After purification by reverse- phase high-performance liquid chromatography (RP-HPLC), the corresponding vancomycin-antimicrobial conjugates were isolated in milligram quantities (Supplementary Section 13). To ascertain the regioselectivity for the conjugation of vancomycin to these antibacterial peptides, we prepared a vancomycin-peptide complex lacking any aromatic residues (13i, Fig. 2B) and fully characterized it using detailed 2D NMR experiments (Supplementary Section 10). The ROESY and HMBC correlations that were observed indicated that selenation had occurred on “carbon d” between the two-hydroxyl groups of the resorcinol ring of vancomycin (Fig. 2B).

We treated several strains of Gram-positive and Gram-negative bacteria with a small library of antibacterial peptide-vancomycin conjugates generated through our conjugation chemistry. This library served two key purposes: 1) to determine if the antibacterial peptide- vancomycin conjugates could overcome resistance mechanisms in known vancomycin- resistant Gram-positive bacterial strains and 2) to see if our conjugates are effective against Gram-negative bacteria, as the antibacterial peptide may improve delivery of vancomycin across the outer membrane. From the library of peptide-vancomycin compounds we discovered two conjugates that were more active than either the parent peptide or vancomycin.

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With regards to treating Gram-positive strain E. faecalis VRE (VanB), the K4-S4 (1–13a) Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author dermaseptin-vancomycin conjugate18 (19v) was more potent than vancomycin alone, any of the parent peptides tested (19p and 19ep), and the unconjugated mixtures of vancomycin with the parent peptides (van+19p and van+19ep). On a molar basis, 19v was over five times more potent than vancomycin and 20 times more potent than the parent peptide (Fig. 2C). For Gram-negative bacteria, the kinocidin congener Peptide RP-1-vancomycin conjugate (23v)19 was active against the Gram-negative bacterium A. baumannii with a minimum inhibitory concentration (MIC) of 16 μg/mL (4 μM, Fig. 2D). This result is surprising as separately, vancomycin and the parent peptide (23p) are both ineffective for treating A. baumannii (Fig. 2D). Effective therapies for A. baumannii would be highly valuable, as this organism can become multidrug resistant easily and is a source of nosocomial (hospital-acquired) infection, especially in immune-compromised patients.20–22 Future work will be necessary to determine the mechanism of these conjugates and whether or not the vancomycin component is functionally consequential. However, these initial studies validate the chemistry and suggest that it may enable the discovery of antibiotic conjugates with efficacy against resistant pathogens.

One other consideration for the in vivo application of this chemistry is the metabolism of the conjugates. While 23v shows no toxicity against eukaryotic cells in vitro (Supplementary Section 20), selenium metabolism in vivo is more complicated and toxicity could be difficult to predict. For example, one described pathway of metabolism for selenocysteine Se- conjugates in mice is β-elimination in the kidney to generate selenol metabolites.23,24 Selenols can be strong nucleophiles and strong reducing agents, which are advantageous properties that enable scavenging of toxic electrophilic metabolites and provide protection against radicals. However, selenol species can also auto-oxidize to diselenides in the presence of molecular oxygen, producing superoxide anion radicals and hydrogen peroxide as byproducts. Reduction back to the selenol by intracellular glutathione and subsequent oxidation can lead to a self-propagating cascade that leads to toxic levels of reactive oxygen species.25 Since the properties of the selenol species depend on the substituent on selenocysteine, the metabolites of selenocysteine-conjugated vancomycin-antimicrobial peptide conjugates could have beneficial or toxic effects. Therefore, in vivo metabolism will be the subject of future investigation.

The conjugation reaction proceeded smoothly for the attachment of 1–3kDa peptides to vancomycin, so we aimed to expand the scope of our chemistry by attaching small proteins onto vancomycin. We chemically synthesized a Sec-containing variant of the trihelical affibody protein (25) frequently used in protein engineering and designed to target proteins such as the HER2 receptor.26 Conjugating small molecule drugs to specific affibody variants has enabled localization of these small molecules to specific targets.27 The p-methoxybenzyl (PMB) protected selenocysteine affibody derivative (25) folded into an alpha-helical structure that had a circular dichroism (CD) spectrum nearly identical to the wild type affibody (Fig. 3C), indicating that the addition of a glycine-selenocysteine dipeptide at the N-terminus did not significantly change the affibody structure. The activated selenium-sulfur (Se-S) bond was installed in one step and the resulting crude material was directly conjugated to vancomycin to afford the corresponding conjugate (27) in 23% isolated yield

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after purification by RP-HPLC (Fig. 3A and 3B). The folded affibody-vancomycin Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author conjugate (27) also retained the alpha-helical structure, as indicated by its characteristic CD spectrum (Fig. 3C). Furthermore, the conjugate (27) readily bound to trastuzumab with a dissociation constant (KD) of 2.6±0.2 nM, a value comparable to that of the wild type affibody (KD = 2.0±0.2 nM, Supplementary Section 25) (Fig. 3D). Collectively, these results demonstrate that there was minimal perturbation of the affibody structure and function after conjugation to vancomycin.

Another potential application of our chemistry is to the generation of homogeneous ADCs. As a first step, we prepared functional antibody conjugates using sortase-mediated ligation of a selenocysteine-containing peptide (Fig. 4).28 We first conjugated unmodified genistein29 onto a polyglycine sortase A substrate peptide 28 providing peptide conjugate 28a in 72% yield after RP-HPLC purification (Fig. 4A). We then reacted the loaded polyglycine peptide 28a with a HER2-targeted IgG (150kDa) bearing a sortase A LPSTG motif on the C- terminus of each heavy chain. Following deglycosylation with PNGase F, analysis by LC- MS of the final trastuzumab-genistein conjugate (30) showed complete and specific drug conjugation on the heavy chains of the antibody (Fig. 4B). Conjugate 30 retained binding to HER2 (Fig. 4C). Additionally, this experiment indicates that the Se–aryl linkage does not inhibit sortase A.

Our experiments highlight both the promises and challenges of using this chemistry for protein-small molecule conjugates. While this methodology successfully yielded a homogenous ADC that retained binding, the reaction itself was performed on a peptide and an additional sortase ligation was necessary to generate the final construct. In the future, our conjugation chemistry could be used in conjunction with techniques to site-specifically incorporate selenocysteine into large proteins, obviating the need for sortase ligation.30,31 However, selenocysteine incorporation into proteins has its own challenges, and the oxidation step to generate an electrophilic selenocysteine may affect other cysteine residues in the protein. Finally, it is critical that the small molecule retains activity after substitution at its electron-rich arene. As long as these parameters are considered in the molecular design of conjugates, we believe this chemistry is a powerful tool, especially in cases of complex small molecules that are difficult to functionalize for conjugation.

Given that the use of complex small molecules is one of the most appealing aspects of this conjugation technique, we wanted to determine the breadth of the small molecule substrate scope. This study began by investigating the reaction between molecules possessing electron-rich arene groups and a model peptide containing an activated selenocysteine (peptide 8, sequence NH2-Gly-Sec*-Ala-Asn-Ser-Leu-Arg-Phe-Tyr-His-Asp-Lys-CONH2; Sec* is the activated selenocysteine, Fig. 5). We found arenes with two electron-donating substituents at the 1- and 3-positions are necessary for efficient conjugation (9a–9e). Phenol (9f) was unreactive under standard conditions, although some product was observed when CuSO4 and a ligand (4,4’-di-tert-butyl-2,2’-dipyridyl, L1) were added. Lastly, the reaction of 1,3,5-trimethoxybenzene (9g) with 8 yielded no desired conjugate. These results suggest that phenolic substrates are more reactive in their deprotonated forms. Next we surveyed nucleophilic heteroaromatic substrates (10a–10q, Fig. 5). Electron-rich 5-membered heterocycles such as indoles, pyrroles, and aminoisoxazoles provided the respective

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conjugates in moderate yield to good yields, although the addition of CuSO4/L1 was Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author sometimes needed to the improve the overall efficiency of the conjugation (Supplementary Section 8). Lastly, electron-rich 6-membered heterocycles, such as 4-hydroxycoumarin (10m), 2,4-quinolinediol (10n), and 2,6-diaminopyridine (10o), were converted to the corresponding conjugates in good to excellent yields.

Having established the scope of aromatic substrates that conjugate effectively to peptide 8, we turned our attention to complex small molecules (e.g., natural products and pharmaceuticals) that have the intrinsic reactivity we imagined to be compatible with electrophilic selenocysteine-based conjugation. Natural products and pharmaceuticals with 32 an embedded resorcinol ring, such as β2 adrenoreceptor agonist 11a (fenoterol), dehydrogenase inhibitor 11c (gimeracil),33 and metabotropic glutamate receptor agonist 11e ((S)-3,5-dihydroxyphenylglycine),34 were all readily linked to peptide 8 in good to excellent yields. In general, these reactions were highly chemoselective, providing one regioisomer as indicated by NMR spectroscopy of the respective small molecule-peptide conjugate (Supplementary Section 10). Additionally, 2-Carbazolol (11g) and β-carboline harmol (11h) gave the corresponding conjugates in excellent yields, although a copper reagent was needed to improve the yield for the former. Phentolamine,35 an anti-hypertensive drug (11i), also reacted efficiently. Lastly, indolebased molecules such as pindolol (11j, a β-blocker)36 and serotonin (11k, a neurotransmitter)37 provided the corresponding products in high yields in the presence of the copper reagent.

In summary, we have described a reaction that enables the conjugation of peptides and proteins containing a selenocysteine residue to native, unfunctionalized small molecules. This strategy has been demonstrated with over 30 different small molecules and 20 different polypeptide/protein sequences. We have done proof-of-concept experiments in two different areas of biological and clinical interest that show the utility of this conjugation strategy. First, we used our reaction to rapidly prepare conjugates between antimicrobial peptides and vancomycin. We screened our conjugates against several pathogens and identified conjugates with activity against vancomycin-resistant Enterococcus and A. baumannii. Future research will focus on the role of both selenocysteine and vancomycin in the efficacy of the reported conjugates. However, our results suggest our conjugation chemistry may facilitate the design and discovery of new conjugates aimed at antibiotic-resistant infections. Secondly, we showed that for a small protein with a defined fold (affibody), our conjugation strategy does not disrupt the structure or binding affinity. We also utilized sortase ligation to generate an IgG antibody containing the C-Se linkage to a small molecule and showed that the antibody retains its binding affinity. These experiments were a first step towards using this chemistry to create homogeneous ADCs. We envision combining this conjugation chemistry with techniques to incorporate selenocysteine into antibodies in the future to have a one-step route to homogeneous ADCs. Given these results, we believe our chemistry will be a valuable bioconjugation technique for the community and may lead to the generation of therapeutic conjugate molecules.

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Author ManuscriptAuthor Methods Manuscript Author Manuscript Author Manuscript Author Detailed experimental methods, synthetic procedures and characterization of the compounds are described in the Supplementary Information.

Procedure for Copper-Mediated Selenocysteine Conjugation A microcentrifuge tube was charged with 32.5 μL of deionized water, 5 μL of 1.0 M Tris Buffer (pH = 8.0), and 10 μL of a 1 mM stock solution of peptide (containing an activated electrophilic selenocysteine – Supplementary Section 3) in water. A separate microcentrifuge tube was charged with copper (20, 40, or 80 μmol), ligand (20, 40, or 80 μmol), the small molecule (containing a (hetero)aryl nucleophile) (40 or 80 μmol) and 0.5 mL or 0.25 mL of 200 proof EtOH (making a 40 or 80 mM stock solution, respectively). The small molecule solution was subjected to sonication for 1 minute, vortexed for 30 seconds, and 2.5 μL of the solution was added to the peptide solution. The resulting reaction mixture was capped, vortexed for 30 seconds, and placed in a 37 °C water bath for 3 hours. The reaction mixture was quenched with 5 μL of EDTA (200 mM in water), 45 μL of water, and 100 μL of acetonitrile containing 0.2% trifluoroacetic acid. The quenched reaction mixture was subjected to immediate analysis by liquid chromatography-mass spectrometry.

Procedure for Copper-Free Selenocysteine Conjugation A microcentrifuge tube was charged with 32.5 μL of deionized water, 5 μL of 1.0 M Tris Buffer (pH = 8.0), and 10 μL of a 1 mM stock solution of peptide (containing an activated electrophilic selenocysteine – Supplementary Section 3) in water. A separate microcentrifuge tube was charged with the small molecule (containing a (hetero)aryl nucleophile) (40 or 80 μmol) and 0.5 mL or 0.25 mL of 200 proof EtOH (making a 40 or 80 mM stock solution, respectively). The small molecule solution was subjected to sonication for 1 minute, vortexed for 30 seconds, and 2.5 μL of the solution was added to the peptide solution. The resulting reaction mixture was capped, vortexed for 30 seconds, and placed in a 37 °C water bath for 3 hours. The reaction mixture was quenched with 50 μL of water and 100 μL of acetonitrile containing 0.2% trifluoroacetic acid. The quenched reaction mixture was subjected to immediate analysis by liquid chromatography-mass spectrometry.

Data Availability All data generated or analyzed during this study are included in this published article (and in the supplementary information files). Further details are available from the corresponding authors upon request (D.T.C.: [email protected]; B.L.P.: [email protected]).

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

Financial support was provided by NIH Awards GM46059 (S.L.B.), F32GM108294 (D.T.C.), F32GM122204 (L.H.), F30HD093358 (C.M.F.) and GM110535 (B.L.P.) and also by MIT startup funds, a Damon Runyon Cancer Research Foundation Award, and a Sontag Distinguished Scientist Award for B.L.P. C.Z. is a recipient of a George Büchi Summer Research Fellowship, a Koch Graduate Fellowship in Cancer Research, and a Bristol-Myers Squibb

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Fellowship in Synthetic Organic Chemistry. A.J.M. is a National Science Foundation Graduate Research Fellow.

Author ManuscriptAuthor Manuscript Author The authors Manuscript Author acknowledge the Manuscript Author Biological Instrument Facility of MIT for providing the Octet BioLayer Interferometry System (NIH S10 OD016326) and circular dichroism spectrometer (NSF-0070319). We thank Dr. Yi-Ming Wang and Dr. Michael Pirnot (MIT) for help in preparing this article. We also acknowledge Samreen Bano (Merck) for Cu ICP-MS analysis of purified peptides 13c, 13d, 13e, and 13f. MIT has filed a U.S. patent application (US15/187169) and international application (PCT/US16/38372) on the current work. Correspondence and requests for materials should be addressed to D.T.C.: [email protected] and B.L.P.: [email protected].

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Figure 1. The conjugation of oxidized selenocysteine to electron-rich small molecules is a one-pot chemoselective reaction for the covalent attachment of natural products and pharmaceuticals to polypeptides and proteins. (a) Site-selective conjugation of vancomycin to polypeptides and proteins. (b) Examples of bioconjugates prepared through our conjugation reaction.

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Figure 2. Selenocysteine-based conjugation of vancomycin to antibacterial peptides enables the discovery of potent Gram-positive and Gram-negative antibacterial agents. (a) Conjugation of antimicrobial-peptides to vancomycin. (b) ROESY and HMBC correlations for vancomycin in peptide-vancomycin conjugate 13i. (c and d) Two vancomycin-antibacterial peptide conjugates (19v and 23v) showed lower minimum inhibition concentration (MIC) compared to the controls including vancomycin only (van), antibacterial peptide only (19p and 23p), antibacterial peptide with a p-methoxybenzyl protected selenocysteine and a glycine on the N-termini (19ep and 23ep), and the mixtures

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of vancomycin and antibacterial peptides. MIC values are from a single activity screening Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author experiment (Supplementary Section 14).

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Figure 3. Conjugation of vancomycin to affibody generates conjugates with retained structure and binding affinity. (a) Conjugation of affibody to vancomycin. The affibody variant 27 containing activated selenocysteine was generated in situ from variant 25 (Supplementary Section 23). (b) Deconvolved mass spectrum of purified affibody-vancomycin conjugate. (c) Far-UV Circular dichroism (CD) spectrum of affibody, affibody-selenocysteine variant 25, and affibody-vancomycin conjugate 27. The CD spectrum of 27 was obtained by subtracting the measured spectra with that of vancomycin only (Supplementary Section 24). (d) Sensorgrams from BioLayer interferometry analysis of affibody-vancomycin conjugate binding to trastuzumab antibody. Retained binding was observed compared to the native affibody (KD = 2.0 ± 0.2 nM; the final KD is the average of the KD obtained from three different concentrations, the error is standard error.)

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Figure 4. Conjugation of genistain to antibody leads to conjugates with retained target binding affinity. (a) A two-step strategy of conjugating an antibody to genistein. Genistein was first modified with a polyglycine peptide through the selenocysteine-mediated conjugation (step 1); the obtained product was then conjugated to antibody through sortase-mediated ligation reaction (step 2). (b) Deconvolved mass spectra of the reduced trastuzumab-genistein conjugate 30. The N-linked glycans on antibody heavy chains were removed by treatment with PNGase F. (c) Sensorgrams from BioLayer interferometry analysis of trastuzumab-genistein conjugate 30 binding to recombinant HER2 protein. Similar binding affinity was observed compared to native trastuzumab (dissociation constant KD = 0.3 ± 0.2 nM; the final KD is the average of the KD obtained from three different concentrations, the error is standard error.)

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Figure 5. Selenocysteine-based conjugation of peptides to (hetero)arenes, natural products, and pharmaceuticals proceeded in moderate to high yields. Amino acids are shown in one-letter code, U – selenocysteine; Yields were determined by integration of the total ion currents (TICs) from LC−MS analyses of the crude reaction mixtures (Supplementary Section 8). *Yield in parentheses run with 1 mM CuSO4, 1 mM fl § 4,4’-di-tert-butyl-2,2’-dipyridyl (L1). 100 μM 8 and 1 mM small molecule. 4 mM CuSO4, ‡ # 4 mM L1, 4 mM small molecule. 4 mM small molecule. 2 mM CuSO4, 2 mM L1.

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