Enzymatic and Site-Specific Ligation of Minimal Size Tetrazines and

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Enzymatic and Site-Specific Ligation of Minimal Size Tetrazines and Triazines to Proteins for Bioconjugation and Live-Cell Imaging Mathis Baalmann, Michael J. Ziegler, Philipp Werther, Jonas Wilhelm, and Richard Wombacher Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00157 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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Bioconjugate Chemistry

Enzymatic and site-specific ligation of minimal size tetrazines and triazines to proteins for bioconjugation and live-cell imaging Mathis Baalmann, Michael J. Ziegler, Philipp Werther, Jonas Wilhelm, and Richard Wombacher* Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany ABSTRACT: Diels-Alder reactions with inverse electron demand (DA inv) have emerged as an indispensable tool for bioorthogonal labeling and manipulation of biomolecules. In this context, reactions between tetrazines and strained dienophiles have received attention due to high reaction rates. Current methods for DA inv mediated functionalization of proteins suffer from either slow reactivity, impaired stability, isomerization or elimination of the incorporated strained dienophiles. We here report a versatile platform for the posttranslational, highly selective and quantitative modification of proteins with stable dienes. A new synthetic access to minimal size tetrazine and triazine derivatives enabled us to synthesize tailored diene substrates for the lipoic acid protein ligase A (LplA) from Escherichia coli which we employ for rapid, mild and quantitative bioconjugation of proteins by DA inv. The presented method benefits from the minimal tag size for LplA recognition and can be applied to proteins from any source organism. We demonstrate its broad suitability by site-specific in vitro protein labeling and live cell labeling for fluorescence microscopy. With this work we expand the scope of DAinv bioorthogonal chemistry for site-specific protein labeling, providing additional experimental flexibility to prepare well-defined bioconjugates or to address biological questions in complex biological environments.

group9 and our work10, gives virtually full control over the labeling method and features a high flexibility. The Diels-Alder cycloaddition with inverse electron demand (DAinv) has become one of the most popular bioorthogonal reactions for facile modification or labeling of macromolecules due to its high biocompatibility, non-toxic reagents, superior reaction rates and commercial availability of DA inv “click chemistry” reagents. It is thus not surprising that the DAinv has emerged as the bioorthogonal reaction of choice for many applications. The fastest DA inv reactions proceed between electron-deficient dienes (e.g. tetrazine moieties) and strained cycloalkenes (e.g. trans-cyclooctenes, TCO)11 or ringstrained alkynes (e.g. bicyclononynes, BCN)12 as dienophiles with second order rate constants up to 10 5 M-1s-1 in aqueous systems.13 Due to their impressively fast kinetics, TCOs have been regarded as the ne plus ultra-dienophiles for DAinv in the first years of emergence. However, today it is well known that TCOs suffer from isomerization to the respective non-reactive cis-cyclooctene scaffolds in presence of thiols.10, 14 An improved version of trans-cyclooctene (TCO*) has been shown to be less prone to isomerization but suffers from elimination upon cycloaddition resulting in bond cleavage.15-18 Less strain (as exemplified by comparison with other cyclic dienophiles such as norbornenes, cyclopentenes or linear terminal alkenes) usually translates to more stable but less reactive dienophiles.19 On the side of the diene reaction partner, the stability of the employed 1,2,4,5-tetrazines under physiological conditions depends on the steric and electronic properties of the substituents in positions 3 and 6 of the tetrazine scaffold. Degradation of tetrazines can occur through two different processes: (1) the reversible reduction by a postulated radical or a non-radical

INTRODUCTION In the past years, the lipoic acid protein ligase A (LplA) from Escherichia coli (E. coli) and specific mutants have been established as a platform to site-specifically ligate a variety of functional handles to a 13 amino acid peptide tag attached to proteins of interest. Various functional handles have been reported to date including alkyne-1, azide-1, 2, 4-iodophenyl-3 and even coumarin-4 or resorufin derivatives.5 While small non-natural lipoic acid derivatives are readily accepted by the wildtype enzyme, a gatekeeper mutation (Trp37Val, W37V) in the lipoic acid binding pocket enables ligation of substrates with a higher steric demand.4 Labeling with LplA belongs to the posttranslational labeling methods, resulting in high functionalization rates due to the irreversible nature of the ligation reaction. The peptide tag is small in size and, contrary to self-modifying protein tags (e.g. SNAP-, Halo-tag), the labeling efficiency does not require the correct fold of the protein tag. LplA-catalyzed protein modification is independent of the protein expression system or sample complexity exemplifying its enormous potential as a tool for life science and bioconjugation. The 13 amino acid tag recognized by the LplA can be fused genetically to the protein of interest and even inserted into internal loops to yield functional proteins.6-8 This offers flexibility and precise control about the location of the desired modification which is often crucial for functionality or application of the modified protein.7 Aside from its excellent performance in extracellular settings or in vitro labeling approaches, the LplAW37V-mutant is also capable of ligating functional handles to peptide-tagged proteins in living cells.8 Using the LplAW37V in combination with state-of-the art bioorthogonal reactions (two-step labeling methods), as demonstrated by the pioneering work of the Ting -1-

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mechanism or (2) nucleophilic addition followed by tetrazine destruction.20-22 Notably, the reactivity of tetrazines also seems to be inversely correlated with the stability under physiological conditions, implying an inherent reactivity-stability balance of both the dienophiles and dienes. The selection of diene-dienophile pair is thus dictated by the experimental demands, such as incubation times, sample complexity (single purified protein, lysate, ex vivo, in vivo) and the presence of molecules or moieties that cause degradation (such as thiols). A current popular approach for site-specific protein labeling using DAinv is to modify the protein of interest (POI) with the dienophile first by using posttranslational attachment strategies9, 10 or incorporation of noncanonical amino acids (Figure 1, strategies I and II).23-25 The cycloadduct is then formed with a probe bearing the diene moiety. Generally, a fast bioorthogonal reaction is favored for any labeling situation, to drive the reaction of low abundant molecules (proteins) to completion even at low probe concentrations. Further, these low concentrations minimize unwanted by-product formation due to side reactions. Also long incubation times can hamper labeling efficiencies in complex biological settings as isomerization of the dienophiles attached to the biomolecule to be targeted progresses leading to low modification yields. Extended incubation times are, however, usually necessary for the sitespecific installation of bioorthogonal handles, as exemplified by introduction of dienophiles or dienes by genetic code expansion.

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Here we present a two-step protein labeling strategy utilizing LplA mediated ligation with stable and highly reactive dienes for bioconjugation by DA inv (Figure 1, strategy IV). For that we investigated various 6-methyl-1,2,4,5-tetrazines, 1,2,4,5-tetrazines and 1,2,4-triazines as potential diene substrates for an LplAW37V mutant. The strategy fully utilizes the advantage of fast reacting dienophiles like TCO and BCN while circumventing their stability issues by reversing the order of DAinv reaction partners in two-step labeling settings. The reversed order allows rapid protein labeling with DA inv, while keeping incubation times with TCO- and BCN-probes short. We demonstrate the versatility of the two-step labeling approach by highly efficient labeling of purified LAP tagged proteins as well as specific and efficient labeling of LAP tagged proteins in complex biological mixtures like cell lysates and living cells. While previous work using genetic code expansion with tetrazine amino acids has been demonstrated for protein labeling in E.coli, our rapid, straightforward and robust method can be applied to various expression systems. The herein reported novel diene substrates for LplA are a valuable addition to the toolbox of bioorthogonal chemistry, providing a robust, efficient and highly flexible protein labeling method for in vitro and live cell applications. RESULTS AND DISCUSSION General considerations and synthesis of the substrate library Currently, methyl-substituted 1,2,4,5-tetrazines are the most commonly used dienes in bioorthogonal reactions because of their superior stability in aqueous systems. 30 As a prerequisite for tetrazine dienes as substrates for LplA mediated labeling, we focussed on establishing a synthesis of minimally sized and sufficiently stable tetrazine headgroups that can be attached to linkers bearing a carboxyl function to serve as substrates for the LplA. Besides being small, the tetrazine substrates need to be stable under physiological conditions and exhibit maximal reactivity with regard to the optimal reactivity-stability balance. Derivatives with carboxylic acids directly attached to the tetrazine scaffold would provide ideal functionality to be converted to LplA-substrates. While these electron poor tetrazines show high reactivity in DA inv, they appear to be highly instable in aqueous solution and are not suited for application under physiological conditions. We thus envisioned the synthesis of 3-hydroxymethyl-6-methyl tetrazine (MeTzCH2OH, 14) which, when converted to the respective carbamates, results in less electron poor tetrazine LplA substrate derivatives with good stability under physiological conditions. A naïve consideration for a synthesis strategy would be to use hydroxyacetonitrile (glycolonitrile) as a precursor but its basecatalyzed tendency toward rapid explosive decomposition 31 makes protective groups for the hydroxyl group of the nitrile a necessity. The synthesis of the small methyltetrazine 10 was accomplished by Pinner-type tetrazine synthesis using acetonitrile, methoxyacetonitrile and nickel triflate as catalyst to obtain MeTzCH2OMe (9a). 32 Subsequent methoxydeprotection with BBr3 yielded the desired MeTzCH2OH (10) (Scheme 1). As observed previously,1-3, 10 unnatural substrates for LplA and mutants thereof (LplAW37V) show maximal ligation efficiency with optimized spacer lengths between the carboxylic acid function and the head group of the substrate to be ligated. Therefore, we synthesized a panel of putative LplAW37V sub-

Figure 1. Schematic overview about current two-step protein labeling techniques using site-specific protein labeling with small tags and bioorthogonal DAinv. Strategies I and II (dienophiles attached to the protein, labeling with diene-molecules) have been commonly applied in DAinv-based protein labeling. Diene attachment using genetic code expansion in E.coli has been reported previously (strategy III). In our work, we report the first generalizable introduction of dienes by post-translational, tag-based ligation to proteins (strategy IV).

To minimize the inherent isomerization or elimination problem of TCOs and at the same time benefit from its outstanding reaction speed, a reversed labeling strategy can be advantageous where the diene is incorporated into a POI and the conjugation is accomplished with dienophile-modified probes.20, 26-28 The Mehl group used genetic code expansion to sitespecifically introduce triazine and tetrazine amino acids into proteins in E. coli (Figure 1, strategy III).20, 26, 28, 29 -2-


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strates. We employed carbamate formation between the novel MeTzCH2OH moiety (10) and five different linear aliphatic amino acids using the activated carbonates of the diene alcohol as 4-nitrophenyl carbonates (Figure 2A, 2C). Next, we attempted to synthesize the respective 3hydroxymethyl tetrazine (HTzCH2OH) building block via the same route using formamidine acetate and acetonitrile with different metal catalysts. ZnI2 was found to be a superior catalyst over Zn- or Ni-triflates and gave us access to the HTzCH2OMe precursor (11), but BBr3-mediated methoxy deprotection failed to deliver the desired product (Scheme 1). Also, TMS-iodide- or TMS-chloride/NaI-mediated cleavage did not provide detectable product formation. To have a homologous HTz substrate in hand, we synthesized a HTz carbamate closely matching the steric dimensions of wellaccepted and so far reported LplA W37V substrates using a HTz(CH2)3OH scaffold and formed the carbamate with an amino acid to closely resemble the MeTz substrate with n = 3 (HTz)(Figure 2A).

Chart 1. Structures of the diene-fluorophore conjugates as probes and reaction partners for tetrazines

Scheme 1. Synthesis of the novel minimal size tetrazine scaffold

Substrate acceptance by the LplAW37V and wildtype LplA Next, we evaluated the various diene substrates (Trz1 and Trz2, MeTz1-MeTz5, HTz) in LplA-mediated labeling in a peptide-based enzyme assay. We analyzed the formation of ligation product for each substrate in presence of Mg2+ and LplA (wildtype or W37V mutant) after 45 min by HPLC, a timescale in which none of the reactions shows full conversion and ligation yields among the individual substrates can be compared. All enzymatic ligation reactions proceed to completion until reactant limitation is reached. The reaction was stopped by the addition of EDTA removing the essential cofactor Mg2+. Due to an expected change of the extinction coefficient of the lipoate acceptor peptide (LAP) conjugated with the aromatic tetrazine or triazine substrates, the yields of the ligations were calculated from the HPLC chromatograms by the change in intensity of the unmodified LAP peptide peak. The natural substrate α–lipoic acid is accepted by both the wildtype and mutant enzyme. The enzyme acceptance of MeTz substrates by the LplAW37V was found to be strongly dependent on the linker length and displays an optimal linker length characteristic that is comparable to what has been observed previously for other non-natural LplAW37V-substrates.2-4, 10 Among the methyl tetrazines we tested, MeTz3 was by far the best-accepted LplAW37V substrate without detectable acceptance by wildtype LplA providing another layer of orthogonality (Figure 1). For the triazines, Trz1 showed the highest ligation yield with nearly full conversion within the 45 min reaction time using LplAW37V while being poorly accepted by the wildtype LplA. We could confirm the identity of all ligation products by mass spectrometry (see Figure S2 and Figure S3). HTz displayed no acceptance at all which was surprising at first glance since it should have the perfect dimensions to fit into the active site of the mutant enzyme just like the wellaccepted MeTz3. The carbamate function in HTz, however, is closer to the carboxyl function that might impede binding within the active site. These findings show that the development of non-natural substrates for enzymes requires rational design, careful optimization and tailoring of the steric and electronic properties. To demonstrate that the ligated tetrazine handle is fully functional in DAinv, we further analyzed the derivatization of the peptide with BCN-Pip-TAMRA by HPLC and mass spec-

For the homologous triazine-substrates, we selected 1,2,4triazine carboxylic acid for straightforward amide conjugation with the linear amino acids.33 We first synthesized the triazine amide carboxylic acid substrates as tert-butyl esters. The corresponding triazine amides with two different linker lengths were prepared by Brønsted-acidic tert-butyl ester-deprotection resulting in the desired substrate candidates Trz1 and Trz2. Design and synthesis of DAinv reaction partners Although the DAinv is widely used for biomolecule labeling, fluorescent probes linked to dienophiles still lack commercial availability, while tetrazine-bearing fluorophores are available from multiple suppliers. This also reflects the lack of strategies and approaches to introduce dienes to biomolecules. To examine the performances of TAMRA-dienophile fluorophores with different polarities, we linked the fast dienophiles BCN and TCO to the fluorophore 4’-carboxytetramethylrhodamine (5-TAMRA), a standard fluorophore for live cell imaging. Together with the commercially available TCO-Prop-sulfoCy5, BCN-Pip-TAMRA, BCN-PEG-TAMRA and TCOProp-TAMRA were synthesized as a broad probe selection for this study (Chart 1).27 -3-



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after 30 min reaction time with EDTA and labelled with BCNPip-TAMRA (1.25 eq. related to the MeTz3) and analyzed by RP-HPLC. The asterisk corresponds to an impurity of the BCNPip-TAMRA fluorophore sample. Peak 1 corresponds to unmodified LAP, Peak 2 to the MeTz3-BCN-Pip-TAMRA DAinv cycloadduct, Peak 3 to LAP(MeTz3), Peak 4 to LAP(MeTz3)BCN-Pip-TAMRA DAinv cycloadduct. For uncropped chromatograms, see Figure S1.

trometry (Figure 1D, Figure S1/S2). After enzymatic modification of the LAP peptide for 30 min with MeTz3 at 37 °C, the reaction was stopped and BCN-Pip-TAMRA was added to the reaction mixture (1.25 eq. over MeTz3), incubated for 5 min at 37 °C and the reaction mixture was analyzed by RPHPLC. After addition of the dienophile-fluorophore, the LAP(MeTz3) peak (Figure 2D, Peak 3) vanished entirely and yielded the LAP(MeTz3)-BCN-Pip-TAMRA DAinv cycloadduct (Figure 2D, Peak 4). The unmodified LAP peak (Figure 2D, Peak 1) did not show any change upon treatment with BCN-Pip-TAMRA. As expected, the excess of MeTz3 substrate from the ligation reaction resulted in the largest peak (MeTz3-BCN-Pip-TAMRA, Figure 2D, Peak 2, Figure S1).

Tetrazine derivatives are often regarded as unstable moieties.9, 34 However, stability studies of different tetrazine compounds do only support this claim for dipyridyl- and pyrimidyl-substituted tetrazines.30, 35 Methyl-substituted tetrazines have been identified as the most stable derivatives with optimal stability to reactivity balance. Thus, it is not surprising that many of the commercially available tetrazine compounds are methyl-substituted tetrazines. A methyltetrazinylphenylalanine derivative introduced recently by the Mehl group for genetic code expansion also proved to be perfectly stable under physiological conditions.20 We measured the stability of MeTz3 in comparison with the frequently employed tetrazine scaffold MeTzBnNH2•HCl at 37 °C (Figure S4). The data indicate comparable high stability in phosphate buffer and reduced but sufficient stability in media or serum. MeTz3 alone was found to be perfectly stable in neat form or as a DMSO stock solution for several months at -20 °C and for multiple days at RT (data not shown). Protein modification with tetrazine moieties Next, we were interested in the ability to site-specifically introduce these diene moieties onto proteins. We choose a LAP-tagged maltose binding-protein (MBP-LAP-HA) (Figure 3A) for diene functionalization which we can express and purify in high yield using E. coli.36 Molar ratios of 10:1 to 5:1 (protein:LplAW37V) were found to be sufficient for almost quantitative protein ligation at 37 °C within 1 h (Figure 3B, 3C). For the biorthogonal DAinv, we chose BCN-Pip-TAMRA for facile detection of fluorescence after the two-step modification procedure. Protein labeling was analyzed by SDS-Page and in gel-fluorescence (Figure 3D). Omitting any key component of the two-step labeling reaction (LplAW37V, ATP. MeTz3, BCN-Pip-TAMRA) did not lead to fluorescent modification of MBP-LAP-HA. Likewise, the wildtype enzyme showed no detectable MeTz3 ligation either. We also subjected all protein species of the two-step workflow (non-modified MBP-LAP-HA, MBP-LAP(Trz1)-HA, MBP-LAP(MeTz3)HA, BCN-Pip-TAMRA-DAinv cycloadduct with MBPLAP(MeTz3)-HA) to whole protein mass determination in order to detect possible byproducts and to follow the individual reaction steps (Figure 2C). The spectra confirm each expected modified protein species as the major peak in agreement with the calculated molecular weights. Most importantly, this also indicates the quantitative modification in both, the enzymatic ligation step and the DAinv conjugation. We did not observe any peaks corresponding to species from the previous modifications steps in the individual samples (Figure 2C). When MeTz3 is attached to the model protein, snap-freezing of the buffered protein solution in liquid nitrogen and storage at -20 °C is possible for at least two months without considerable loss of reactivity for DAinv (data not shown). The triazine substrate Trz1 was as well ligated quantitatively to MBPLAP-HA as evident from the mass spectra (Figure 2C, yellow trace). Because triazines require high equivalents of corresponding dienes for DAinv29and are better suited for proximity-

Figure 2. Evaluation of diene ligation using a peptide based LplA assay. (A) Substrate structures of the diene substrate library. (B) Ligation scheme. LAP = Lipoate acceptor peptide. (C) Enzymatic conversion of the different substrate candidates using the mutant and wildtype lipoic acid ligase. 250 µM of the lipoic acid acceptor peptide (LAP) was treated with 1 µM of LplA (mutant or wildtype), 5 mM ATP, 5 mM of Mg2+ and the respective substrate (500 µM) in sodium phosphate buffer (pH 7.0) at 37 °C for 45 min and quenched with EDTA (250 mM) to remove the Mg2+ cofactor. The reaction mixtures were analyzed by RP-HPLC. (n.c. = no conversion) (D) A separate ligation mixture was quenched

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Figure 3. High yield in vitro labeling of proteins with dienes using LplA W37V (A) The model protein is a maltose-binding protein with a C-terminal LAP tag. Protein structure modified according to Quiocho et al. (PDB: 1ANF).37 (B) Schematic for complete protein labeling with tetrazine or triazine substrates (C) Mass spectrometry data of samples from the functionalization reactions without prior separation or enrichment of certain species. The predicted molecular weight and molar differences of the two -step functionalization species are shown on the left side. The deconvoluted ESI-MS spectra show nearly quantitative ligation of the MBP-LAP-HA model protein with Trz1 (yellow trace), MeTz3 (pink trace) and the Diels-Alder cycloadduct of the MBP-LAPHA protein functionalized with MeTz3 substrate and reacted with BCN-Pip-TAMRA (blue trace). The unmodified protein is depicted as the dark grey trace. For full ESI spectra, see Figure S5-S8. (D) Labeling of MBP-LAP with MeTz3 and subsequent functionalization with DAinv-reactive BCN-Pip-TAMRA. 10 µM of MBP-LAP were treated with 1 µM of LplA W37V, 1 mM of MeTz3, 5 mM of Mg(OAc)2 and 5 mM ATP in sodium phosphate buffer (pH 7.0) for 1 h at 37 °C. The reaction was quenched with 250 mM of EDTA, washed and the MBP-LAP(MeTz3)-HA was labelled in PBS (pH 7.4) with BCN-Pip-TAMRA (50 µM) for 15 min. The samples were analyzed by SDS-PAGE with in-gel fluorescence measurement and Coomassie BrilliantBlue staining.

based biorthogonal chemistry38, we focused on MeTz3 for further ligation and bioconjugation experiments. The reaction rate between the novel MeTz moiety and endo-BCN was determined to occur with a bimolecular rate constant of 89.4 ± 1.2 M-1s-1 in PBS at 30 °C (Figure S9). We independently assessed the rapidness of the DA inv between BCN and MeTz3-functionalized MBP-LAP-HA at the protein level (Figure S10): 5 min of incubation with 5 eq. of BCNPip-TAMRA are sufficient for full fluorophore functionalization at low micromolar concentrations (Figure S10D) at 37 °C. By expanding the reaction time to more than 5 min, even substochiometric concentrations of the BCN-probe result in efficient labeling of the model protein (Figure S10B, S10D) making this combination (MeTz3-functionalized proteins and BCN-probes) an ideal way to achieve a maximum of protein functionalization and a defined and homogenously labeled protein population.

With these stable diene substrates for LplA-mediated labeling, we have a facile, robust and quantitative labeling technology for the modification and derivatization of functional proteins in hand. Nanobody labeling and immunostaining Modification and functionalization of proteins recognizing structures or targeting other macromolecules is a common procedure in life sciences and diagnostics and has many applications in the field of antibody-derived therapeutics.39 Socalled antibody-drug conjugates (ADCs) are comprised of (1) a macromolecule (usually an antibody or a fragment thereof) recognizing target structures or biomarkers overexpressed on diseased cells and (2) one or multiple payloads that are often cytotoxic drugs.40 When applied in cancer therapy, ADCs minimize the adverse effects of classical chemotherapy by a higher selectivity toward the tumor cells. For the payload attachment to the antibodies, biorthogonal reactions are -5-



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Figure 4. Two-step functionalization with enzymatic MeTz3-ligation and fluorescent probe conjugation by DAinv can be used to generate functional nanobody (Nb) probes for immunostaining procedures. (A) Single-domain Nbs can be generated from camelid heavy chain immunoglobulin G (hcIgG). The anti-GFP-Nb reported by Kubala et al.41 (PDB: 3OGO) was tagged C-terminally with the LAP. (B) Schematic of the LifeAct-EGFP-HaloTag protein bound to the actin cytoskeleton and the EGFP domain targeted by the TAMRA-labelled anti-GFP-Nb. (C) Structure of the SiR-Halo probe that was used as a positive control for the immunostaining. (D) Wide field microscopy analysis of fixed NIH/3T3 cells transfected with LifeAct-GFP-HaloTag. Fluorescence images show results from post-fixation immunostaining with TAMRA-labelled anti-GFP-Nb (2 µg/mL, 1% BSA in PBS for 20 min at RT) and labelling of LifeAct Halo tag with SiR-Halo (0.1 µM in medium, 15 min at 37ᵒC). Merged images of the GFP-, TAMRA and DAPI fluorescence are shown in the lower panel. The procedure for the LplAW37V mediated labeling of anti-GFP-Nb is depicted in the box on the left. (E) Confocal microscopy analysis of fixed cell immunostaining demonstrating colocalization of the actin-localized EGFP signal and TAMRA-modified anti-GFP-Nb. R = Pearson’s correlation coefficient. All scale bars = 25 µm.

preferred leading to a homogenous and well-defined ADC population contrary to the classical rather non-selective lysine or cysteine conjugations that were used in the past. 42 While a full-length human immunoglobulin G (IgG) has a size of around 150 kDa, smaller functional antibody fragments like single-domain antibodies have been introduced. These singledomain antibodies (also known as nanobodies, Nbs) are derived from heavy chain antibodies from camelidae (Figure 4A), cartilaginous fishes or protein engineering. 43, 44 Nanobodies are much smaller (12-15 kDa) than their cognate IgGs, greatly simplifying heterologous expression and purification while maintaining high specificity for the target antigen. 45, 46 We reasoned that the novel MeTz3 substrate for the LplAW37V can serve as a powerful tool to perform labeling for immunostaining procedures. For this, we cloned, expressed and purified the anti-GFP-Nb as a LAP fusion (Figure 4A).41 The anti-GFP-Nb-LAP was subjected to LplAW37V-mediated ligation of MeTz3 and subsequent covalent bond formation with BCN-Pip-TAMRA mediated by DAinv (Figure 4C). We selected a construct expressing a genetic fusion between the actin marker LifeAct, EGFP and HaloTag for facile detection of the interaction between GFP:anti-GFP-Nb by colocalization at the cellular structure of the cytoskeleton of mammalian cells(Figure 4B).47

NIH/3T3 cells transiently expressing LifeAct-EGFPHaloTag were incubated with SiR-Halo (Figure 4D), fixed and incubated with the TAMRA-labeled anti-GFP-Nb. In transfected cells, strong co-localization of the TAMRA signal with the GFP signal was observed (Figure 4D and 4E, R = 0.92). The intensity of both the TAMRA- and SiR-signal correlated with the GFP expression/signal intensity demonstrating the target selectivity of the fluorophore-labelled nanobody. As expected, in cells that do not express LifeAct-GFP-HaloTag (see DAPI channel, Figure 4D), no TAMRA-signal could be detected. The method is thus particularly useful for the precise and specific generation of functional and biologically active bioconjugates. By using the robust conjugation with our MeTz3 substrate, we were able to utilize the DA inv for fast and mild bioconjugation with low concentration of the reactants. Notably, Gray et al. have developed a cytotoxic HER2-nanobody that was modified with a LplAW37I-based two-step labeling procedure using hydrazine-aldehyde chemistry.48 Our highyielding protein derivatization with the fast MeTz3 substrate is compatible with proteins from any expression organism given that they possess an accessible LAP tag. With the method presented here, we expect leverage for the construction of novel ADCs for therapeutic use. -6-


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Specific labeling of low abundant proteins in lysates A challenge for protein labeling strategies is selectivity and efficiency for low abundant proteins. We thus directed our experiments to assess the selectivity of the two-step labeling method for a low abundant protein in a complex cell lysate. E. coli BL21 Star (DE3) cells carrying the anti-GFP-NbLAP expression plasmid were induced with a low concentration of IPTG and grown to mimic the presence of a low abundant LAP-tagged protein. Bacterial cells were lysed in presence of protease inhibitors and subjected to LplA W37Vmediated MeTz3-ligation. We were especially intrigued by the performance of the TCO and BCN moieties in combination with fluorophores having different polarities. We thus applied four different fluorophores (BCN-Pip-TAMRA, BCN-PEGTAMRA, TCO-Prop-TAMRA and TCO-Prop-sulfo-Cy5) to the tetrazine-modified protein lysates. In all MeTz3-treated samples, LAP-tagged proteins showed well-detectable fluorescence with an exceptional signal-to-background ratio considering the low abundance that is evident by the corresponding Coomassie Brilliant Blue staining (Figure 5). Two other faint bands result from expression artifacts and get labeled due to the presence of an LAP tag. These fluorescent bands were completely absent in the negative control (Figure S11). Because no labeling method occurs without background or byreactions, we analyzed for non-specific signal in the upper part of the gel, where the proteins are present in much higher concentrations than the low abundant target protein. We display these minimal side products by showing the upper part of the gel with increased contrast (Figure 5, bottom). For both BCN fluorophores (BCN-Pip-TAMRA, BCN-PEG-TAMRA), unspecific labeling of other proteins in the lysate was observed while TCO-fluorophores showed significantly less unspecific labeling. This also demonstrates that the background labeling is dependent on the nature of the dienophile which is in agreement with data from Murrey et al. showing TCOfluorophores much less prone to non-specific protein labeling than BCN-fluorophores.27 It has been described previously that strained alkynes like BCN are susceptible toward thiol-yne addition reactions.49 Another factor that can contribute to background labeling is the polarity of the fluorophores. Non-specific binding of the fluorophore to hydrophobic patches or pockets on proteins might amplify non-specific labeling reactions due to a proximity effect.50 Notably, the most polar fluorophore, TCO-Propsulfo-Cy5 completely abolished background labeling supporting this hypothesis. Introduction of highly polar moieties on probes (e.g. sulfonic acid groups, PEGs) thus represents an effective way to reduce background when working in vitro or on the outside of living cells. For cell-permeable fluorophores, however, there are restrictions on the polarity since sulfonic acid groups usually render any molecule cell-impermeable when there is no active transport mechanism involved. Altogether, the two-step method was found to be an excellent method for addressing, detecting and modifying proteins in complex samples. An additional important observation is that the type of the dienophile as well as the polarity of the fluorophores is crucial when dealing with background labeling issues.

Figure 5. Highly specific functionalization of low-abundant LAPtagged proteins in a bacterial cell lysate. E. coli BL21 Star (DE3) cells transformed with a plasmid coding for anti-GFP-LAP-Nb (Protein size: 15.8 kDa) were induced with 50 µM of IPTG, grown and harvested. Cell lysis was performed in PBS with protease inhibitors. The clarified lysates (1 mg/mL of total protein) were subjected to MeTz3 ligation using 5 µM of LplAW37V, washed by centrifugal filtration and incubated with a panel of different diene fluorophores (10 µM) and analyzed by SDSPAGE, followed by in-gel fluorescence measurements and Coomassie Brilliant Blue staining. For the upper part of the in-gel fluorescence scan, contrast was increased (linear histogram equalization from 0-255  0-27) to evaluate by-reactions (bottom). For the negative control (lysate w/o LAP-tagged proteins) see Figure S11.

Live-cell labelling We next wanted to explore if the diene ligation is suitable for protein labeling with organic fluorophores for live cell imaging. HEK293 cells were transfected with a plasmid encoding for extracellularly presented LAP-tagged cyan fluorescent protein anchored in the cell membrane (pDisplay-LAP2-CFP-TM). LplAW37V -mediated MeTz3-ligation was performed in Hepesbuffered artificial Cerebrospinal fluid (Hbf-aCSF) with 1 mM ATP and 2.2 mM Mg2+. After washing, the cells were incubated with TCO-Prop-sulfo-Cy5 for 5 min, a Cy5 derivative with no cell-permeability. The cells were washed and imaged live under an epifluorescence microscope. -7-



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Seeing LplAW37V-mediated tetrazine ligation as a general framework, we benefit from the site-specificity of enzymatic catalysis, irreversible ligation mechanism and high flexibility of the tag position within the POI. Further, the method is independent from the expression system, enabling quick and facile modification of proteins of bacterial or mammalian origin. Additionally, we expanded the scope of the LplA W37V to the ligation of triazines, which are less reactive in the DAinv but exhibit very high stability under biological conditions. Further, triazines have the potential to be used in proximity-driven chemistry. The expansion of the substrate scope of the lipoic acid protein ligase makes it an entire and modular platform for protein modification with state-of-the art probes and chemical handles – all with only one system. The posttranslational nature of the lipoic acid ligase shifts the modification with the chemical handle to a later stage allowing to implement quick adaptations for the type of modification and to screen for the appropriate chemistry required for particular experimental demands. This avoids dead-end situations, enables small-scale modification reactions and eliminates the need for extensive usage of valuable diene substrates like it is necessary when using unnatural amino acids in genetic code expansion. The LplA and their mutants might convey the impression that the enzyme is a somewhat promiscuous enzyme, but the development of novel substrates requires multiple cycles of rational design and acceptance experiments and only a certain size range of substrates is accepted. The tetrazine substrate ligation was demonstrated to be equally good even when the POI is present in low concentration within a highly complex environment. We noted that the fluorophore probe for the bioorthogonal reaction targeting the POI needs to be tailored carefully to the experimental needs. The polarity of the probes plays an important role for the downstream application. For the DA inv, the type of dienophile predefines the scope of by-reactions, but the polarity has a certain impact on the amount of unspecificity of the probe. Fluorophore design is a crucial step for more advanced applications, especially for intracellular applications and we hope that our conclusions provide a rationale for improved probes. Like other previous work on the “reversed” approach by Mehl et al.20, 28, the usage of sufficiently stable tetrazine moieties can be combined with the more DA inv-reactive dienophiles like the outstandingly rapid sTCO or dTCO moieties or other moieties that will be developed in the future.51, 52 The stability issues that have been reported for these highly strained scaffolds can be tackled by this reversed DA inv labeling approach since the MeTz3 substrate exploits the stability-reactivity balance for quantitative conversion in DAinv within reasonable timescales (1 -15 min) even at low concentrations of the dienophile reactants. We successfully used the diene ligation for extracellular protein labeling with living cells and demonstrate its potential for fluorescence microscopy. The herein presented diene ligase and DA inv labeling approach is an attractive addition to the chemical toolbox for protein labeling and manipulation, offering small tag size, high specificity, robustness and straightforward handling to be easily adapted in any laboratory. MATERIALS AND METHODS A detailed description of the synthesis procedures. experimental methods and supplementary figures can be found in the Supporting Information.

Positively transfected cells displayed distinct cell surface labeling with Cy5, indicated by colocalization of CFP and Cy5 signals at the cytoplasm membrane (Figure 6A, B). As expected, the internalized CFP pool showed no significant colocalization with Cy5 due to the lack of cell permeability of the sulfo-Cy5 fluorophore. Non-transfected cells did not show surface labeling.

Figure 6. Extracellular live-cell labeling using MeTz3-ligation and DAinv with TCO-Prop-sulfo-Cy5. HEK293 cells were transfected with pDisplay-LAP-CFP-TM. (A) Scheme of the procedure for live cell surface labeling with MeTz3 (100 µM) in Hbf-aCSF (pH 7.4) supplemented with 1 mM of Mg(OAc)2, 1 mM of ATP and 5 µM of LplAW37V. Subsequent DAinv was performed with TCO-Prop-sulfo-Cy5 (1 µM) in Hbf-aCSF. (B) Fluorescence images of extracellular live-cell labeled membrane proteins; Cy5 (upper left), CFP (upper right), merged (lower left) and merged with brightfield (lower right). White arrows in all images indicate non transfected cells that serve as internal negative controls. Scale bar = 25 µm. For images with a larger field of view and further controls, see Figure S12.

CONCLUSIONS In summary, we synthesized a novel minimal-size tetrazine moiety providing access to tetrazine substrates that are readily accepted by a mutant posttranslational enzyme for the sitespecific enzymatic attachment to proteins with high yield and selectivity. The new tetrazine motif is tailored to exhibit optimal reactivity-stability balance for easy and mild further derivatization, high stability in biological environment and reasonably fast kinetics in DAinv. -8-


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Supporting Information. Additional figures and experiments; full materials and methods including detailed and complete experimental procedures; synthesis procedures and characterization of fluorophores, substrate candidates and reagents. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions All authors have given approval to the final version of the manuscript

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT R. W. acknowledges funding from the Deutsche Forschungsgemeinschaft DFG (SPP1623, WO 1888/1-2). M. B. acknowledges funding from the Landesgraduiertenförderung BadenWürttemberg (LGF BW). M. J. Z. acknowledges funding from the Carl-Zeiss-Stiftung. We thank Martin Wolfring, Marcel Best, Sandra Suhm, Verena Straub, Stefanie Kühn, Luca Blicker, Anna Degen, Niklas Müller-Bötticher, Heiko Rudy and Tobias Timmermann for experimental support. We thank the ZMBH Core facility for mass spectrometry and proteomics (Heidelberg) for whole protein mass determinations. We are grateful to Jacob Piehler for providing the pSems-Lifeact-mEGFP-HaloTag7 and Dirk-Peter Herten for SiR-Halo as well as for access to their confocal fluorescence microscope.

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Enzymatic and Site-Specific Ligation of Minimal Size Tetrazines and

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