Tyrosine-Specific Chemical Modification with in Situ Hemin-Activated

Sep 10, 2015 - We succeeded in the functionalization of several proteins using azide-conjugated compound 18 using alkyne-conjugated probes by copper(I...
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Tyrosine-Specific Chemical Modification with in Situ Hemin-Activated Luminol Derivatives Shinichi Sato, Kosuke Nakamura, and Hiroyuki Nakamura* Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan S Supporting Information *

ABSTRACT: Tyrosine-specific chemical modification was achieved using in situ hemin-activated luminol derivatives. Tyrosine residues in peptide and protein were modified effectively with N-methylated luminol derivatives under oxidative conditions in the presence of hemin and H2O2. Both single and double modifications of the tyrosine residue occurred in the reaction of angiotensin II with N-methylated luminol derivative 9. Tyrosine-specific chemical modification of the model protein bovine serum albumin (BSA) revealed that the surface-exposed tyrosine residues were selectively modified with 9. We succeeded in the functionalization of several proteins using azide-conjugated compound 18 using alkyne-conjugated probes by copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC) or dibenzocyclooctyne (DBCO)-mediated copper-free click chemistry. This tyrosinespecific modification was orthogonal to conventional lysine modification by N-hydroxysuccinimide (NHS) ester, and dual functionalization by fluorescence modification of tyrosine residues and PEG modification of lysine residues was achieved without affecting the modification efficiency.

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proteins, however, depends on nucleophilic reagents, such as Nhydroxysuccinimide (NHS) ester and maleimide, and relies on the modification of lysine and cysteine residues. While those methods have significantly contributed to native protein modification, alternative methods that can modify different amino acid residues are still required. A tyrosine residue contains a polar phenolic hydroxyl group that is often exposed on a protein’s surface; thus, it is expected to be a good target for protein functionalization.11 Recently, remarkable attention has been given to tyrosine modification, including Pd-catalyzed phenol alkylation,12,13 the threecomponent Mannich reaction,14−16 the use of diazonium salt,17−20 and oxidative coupling reactions using Ni(II) catalyst,21 Ce(IV) ammonium molybdate,22 and Ru(bpy)3 photocatalyst.23,24 Barbas and co-workers reported a click-like ene-type reaction of tyrosine with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD, 1a, Figure 1).25,26 Following the publication of the first report, various PTAD derivatives were synthesized and used for the functionalization of peptides for radioisotope labeling,27

he chemical modification of proteins with synthetic probes is an important technique in chemical biology, protein-based therapy, and material science. For example, it is an essential tool for the development of antibody−drug conjugates1,2 and protein-based drug delivery agents,3,4 both of which have received immense attention recently. To achieve reliable bioconjugation in the chemical modification of proteins, the following requirements should be met: (1) stable covalent bond formation, (2) chemoselectivity to a specific moiety in a protein structure, (3) rapid reaction in aqueous solution, and (4) high conversion under physiological pH and mild temperature. A combination of the click reaction and the genetic introduction of unnatural amino acids is often used to append a desired function to a protein of interest.5 Several click reactions that meet the above requirements have been established, such as the azide−alkyne click reaction, the Staudinger−Bertozzi ligation, and oxime/hydrazine formation.6,7 Those techniques, however, require gene manipulation to introduce an unnatural amino acid residue into a protein of interest and are thus not applicable to native proteins. In the attempt to modify native proteins, various bioorthogonal chemical reactions that enable irreversible bond formation in any of the natural amino acids have been developed.8−10 Much of the chemical modification of native © XXXX American Chemical Society

Received: June 9, 2015 Accepted: September 10, 2015

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DOI: 10.1021/acschembio.5b00440 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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hemin (an iron−protoporphyrin IX complex), an ironcontaining component of hemoglobin (10 mol %), and H2O2 (10 equiv) in 100 mM phosphate buffer (pH 7.4) for 1 h. The results are summarized in Table 1. The adducts of compounds 2a, 6a−g, 7a, and 10 to angiotensin II were detected as singular Table 1. Reactivity of Luminol Derivatives toward Angiotensin II Modificationa

Figure 1. Concept of this work.

DNAs, 28 carbohydrates, 29,30 and bioactive small compounds.25,26 Although PTAD derivatives (1) are very promising for protein functionalization, they are unstable under physiological conditions. Therefore, it is necessary to prepare them immediately before use by oxidizing the PTAD precursor (2) with N-bromosuccinimide (NBS) or 1,3-dibromo-5,5dimethylhydantoin (DBH) and to use them immediately without purification.25−30 Even during peptide/protein modification, PTAD derivatives gradually decompose into isocyanate byproducts (3). Unfortunately, the generated isocyanate byproducts are highly electrophilic and react with amino groups, such as lysine residues and N-terminal amino groups (refs 24 and 28 and Figure 2). The relatively low selectivity for a tyrosine residue is caused by those side reactions. We thought that a reactive diazodicarboxyamide, which does not generate a electrophilic byproduct, would enable us to modify the tyrosine residue selectively. We focused on the intermediate of the luminol reaction (5) as a reactive diazodicarboxyamide. In this study, we succeeded in the tyrosine-specific modification of peptides and proteins using in situ hemin-activated luminol derivatives.

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RESULTS AND DISCUSSION Structure−Activity Relationship of Luminol Derivatives as a Tyrosine Modifier. The luminol reaction is a chemiluminescence reaction that is used to detect trace amounts of blood in criminal investigations. Iron in hemoglobin catalyzes the oxidation of luminol (6a) in the presence of hydrogen peroxide (H2O2) as an oxidant to generate an oxidized luminol intermediate (5), which has a cyclic diazodicarboxyamide structure common to PTAD.31,32 Luminol-dependent chemiluminescence is caused by emission of the excited 3-aminophthalate generated from 5. We focused on the oxidative activation of the cyclic hydrazide structure of luminol and speculated that oxidized luminol intermediate 5 would become an alternative tyrosine modifier to PTAD. Therefore, we examined the reactivity of activated luminol and its derivatives toward tyrosine-selective modification using angiotensin II (NH2-DRVYIHPF-COOH) as the tyrosinecontaining model peptide. Angiotensin II was treated with various luminol derivatives (10 equiv), a catalytic amount of

Reaction conditions: angiotensin II (100 μM), hemin (10 μM), H2O2 (1 mM), and compound (1 mM) in 100 mM Na-phosphate buffer (pH 7.4) for 1 h at RT. All reactions were quenched with DTT (10 mM) and analyzed by MALDI-TOF MS. Each reaction was repeated several times. bPTAD modification was performed using commercially available PTAD (Sigma-Aldrich). Angiotensin II (100 μM) was incubated with PTAD (1 mM) for 1 h at RT. cNot detected. a

B

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ACS Chemical Biology products by MALDI-TOF-MS (molecular weight: + compound − H2). In the case of N-methylated derivatives (compounds 9, 11a, and 11b in Table 1), a small amount of double-modified angiotensin II was detected along with a single-molecule adduct as the major product. The modification yields were calculated from the consumption of angiotensin II based on the intensity of the internal standard, angiotensin I (NH2-DRVYIHPFHLCOOH), by MALDI-MS analysis (see Figure S1, Supporting Information). PTAD (1a) reacted with angiotensin II (100 μM) in the presence of hemin (10 μM) and H2O2 (1 mM) to afford the adduct in 54% yield. PTAD precursor 2a was converted into PTAD 1a in the presence of hemin and H2O2, and comparable modification efficiency to that of 1a was observed (48%) (Figure S2). The result suggests that oxidative activation using hemin and H2O2 converts the cyclic hydrazide (CO−NH− NH−CO) into a cyclic diazodicarboxyamide (CO−NN− CO). Luminol 6a gave a low yield (21%), whereas phthalic hydrazide 6b (R = H) showed better efficiency (61%). Maleic hydrazide 7a reacted minimally with angiotensin II, whereas saturated succinic hydrazide 7b and acyclic hydrazide 8 did not react with angiotensin II at all. The substituents on the aromatic ring of phthalic hydrazides (6c−g) did not affect the reactivity drastically (34−61%). In contrast, N-substituted hydrazides (9, 10, 11a, and 11b) exhibited excellent efficiency and the modification yields were 90 to >95%. LC-ESI-TOF mass analysis revealed that both single and double modifications of angiotensin II occurred in the reaction between angiotensin II and compound 9 (Figure S3). The effects of the amount of hemin and H2O2 and the reaction time on the modification of a tyrosine residue were investigated using 100 μM angiotensin II and 1 mM compound 9 in 100 mM phosphate buffer (pH 7.4) (Figures S4 and S5). Against 1.0 equiv of angiotensin II having a single tyrosine residue, 10 mol % hemin and 10 equiv of H2O2 were required for the complete consumption of angiotensin II. Using 10 equiv of 9, the reaction was completed within 15 min. A reaction rate of 8.55 M−1 s−1 was determined (Figure S6).33 Despite the strong chemiluminescence property of the luminol, N-methylated derivatives 9 and 18 did not show any chemiluminescence property (see Figure S7). Tyrosine Selectivity of Compound 9. Angiotensin II variants in which Tyr is replaced by Ala, Cys, Lys, Met, Ser, or Trp and bradykinin fragment 1−7 (NH2-RPPGFSP-COOH) were also examined as substrates without a tyrosine residue. Those variant peptides were not modified under the optimum reaction conditions, suggesting that the modification is completely selective for a tyrosine residue among the natural amino acid residues. Although this reaction needed 10 equiv of H2O2 as the driving force for the oxidation reaction, only the oxidation of a cysteine residue was observed (approximately 40%) as a side reaction in the case of the Y4C variant (NH2DRVCIHPF-COOH) that contains SH-free cysteine instead of tyrosine (Figures S8 and S9). To estimate the binding mode between compound 9 and a tyrosine residue, cresol 12 was used for the modification. Cresol 12 reacted with 9 in the presence of 1 mol % hemin in DMSO, and compound 13 was identified as an adduct by NMR (Scheme 1). A C−N bond between the carbon at the position ortho to the phenolic hydroxyl group in the cresol structure and the nitrogen atom in 9 was formed. Furthermore, a doublemodified product at the two positions ortho to the phenolic hydroxyl group (14) was obtained under reaction conditions using 10 mol % hemin. MS/MS analysis of 9-modified

Scheme 1. Cresol Modification with Compound 9

angiotensin II also suggested that the single and double modifications proceeded only at a tyrosine residue (Figures S10 and S11). To compare the selectivity of the current modification between in situ activated luminol derivative 9 and PTAD 1a, the reactions were carried out in a 1:1 mixture of angiotensin II and angiotensin II Y4K variant (NH2-DRVKIHPF-COOH). The results are shown in Figure S12. In the case of 1a, the 1a adduct from angiotensin II (m/z = 1221) as well as other adducts from the Y4K variant (m/z = 1130) and angiotensin II (m/z = 1165) was detected, and those adducts were presumably a result of the reaction of the corresponding peptides with phenylisocyanate in 10 mM Tris buffer (Figure S12). MS/MS analysis of the modified peptides suggested that the lysine residue and the Nterminal amine moiety of angiotensin II and its Y4K variant were conjugated with phenylisocyanate generated from the decomposition of 1a (Figures S13−S15). Although it was reported that conjugation with phenylisocyanate is suppressed in highly concentrated Tris buffer (100 mM),26,30 the reactions between 1a and the amine groups of angiotensin II Y4K variant were still observed even in 100 mM Tris buffer (Figures S16 and S17). On the other hand, in the case of compound 9, the tyrosine residue in angiotensin II was modified predominantly without affecting the angiotensin II Y4K variant. These results indicate that in situ activated luminol derivative 9 is more potent for tyrosine-selective modification than PTAD 1a. We applied this methodology to the modification of a protein. Bovine serum albumin (BSA), which was selected as the model protein, was reacted with 9 (100 equiv) in the presence of hemin (1 equiv) and H2O2 (100 equiv) at RT. The increase in the molecular weight observed by MALDI-TOF-MS analysis corresponds to the conjugation mainly of six molecules of compound 9 to BSA. Under conditions without H2O2, the molecular weight of BSA did not change, suggesting that H2O2 is necessary for the modification of BSA with compound 9. Treatment with 1a (100 equiv) caused an increase in the molecular weight of 617, indicating that in situ activated compound 9 is more effective for the modification of BSA than 1a (Figure S18). The modified BSA was digested by trypsin or endoproteinase Glu-C, and the modified amino acid residues were determined by MS/MS analysis (Figures 2 and S19−S31). Interestingly, all modifications with 9 occurred at tyrosine residues, whereas lysine modification with phenyl isocyanate was observed in addition to the tyrosine modification with 1a (Figure S32−S34). As the modification sites of compound 9, eight tyrosine residues were detected among the total of 20 tyrosine residues in BSA’s amino acid sequence. Indeed, those tyrosine residues are exposed on BSA’s surface according to Xray crystallography (PDB: 4F5S).34 It must be noted that the C

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Figure 2. Tyrosine residues modified by compound 9 in the 3D structure of BSA. (A) The protein’s surface is shown in green. Modified tyrosine residues are shown by red sticks, and red areas on the protein’s surface indicate exposed parts of the modified tyrosine residues. (B, C) Snapshots taken from different angles.

Figure 3. Modification of BSA with 18 or azide-conjugated PTAD 19. BSA (10 μM) was treated with 18 or 19 (1 mM), hemin (10 μM), and H2O2 (1 mM) in 100 mM phosphate buffer (pH 7.4) for 1 h at RT. After the modification, BSA was visualized with alkyne-conjugated Alexa Fluor 488 by CuAAC. Fluorescence intensities (0.5 s exposure) were measured by image analysis using ImageJ software.35

double modification was observed only at Y400, which is the most exposed tyrosine residue among the tyrosine residues on the surface of BSA (Figure 2). The surface-exposed tyrosine residues are more likely to undergo modification by in situ activated compound 9 compared to the inner tyrosine residues. (Table S1). Functionalization of Proteins Using Azide-Conjugated Compound 18. As an application of the current tyrosine-selective modification to protein functionalization, we designed azide-conjugated probe 18 based on the structure of compound 9 in Table 1 to introduce a functional group into a protein by copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC). Compound 18 was easily synthesized from dimethyl 4-hydroxyphthalate (15) in four steps in a total yield of 78% (Scheme 2). After modification of BSA with 18 or azide-

modification reaction with 18 and alkyne-conjugated biotin (Figure S36). Approximately 40% of free cysteine residue was oxidized under the modification conditions using 10 μM hemin and 1 mM H2O2. We demonstrated the generality of this method using other proteins, ovalbumin (OVA, total 10 tyrosines), streptavidin (SAv, total 6 tyrosines), and bovine carbonic anhydrase (CA, total 8 tyrosines). All tested proteins underwent modification with 18 in the presence of hemin and H2O2, suggesting that this method is widely applicable to proteins that have a tyrosine residue on their surface (Figure 4; also see Figure S40). It is

Scheme 2. Synthesis of Azide-Conjugated Probe 18

Figure 4. Modification of proteins with 18 or 19. OVA, SAv, and CA (10 μM) were modified using the same method as that described in Figure 3. The SDS-PAGE samples (8 μM protein, 5 μL) were loaded in all lanes. Exposure times for fluorescence images were different among proteins (9 s for OVA and 2.5 s for SAv and CA; see Figure S40).

noted that the modification of OVA is less efficient than that of other proteins because it is known that OVA has some saccharide modification, causing low tyrosine accessibility of 18 to the surface of OVA. MALDI-TOF MS analysis suggested that an average 1 molecule of compound 18 per CA molecule was modified, whereas azide-conjugated PTAD 19 did not modify CA. The activity of CA was hardly affected by modification with 18 (Figure S37). To apply the in situ activated luminol derivative to the dual functionalization of BSA, its compatibility with lysine modification was tested (Figure 5). Lysine modification was carried out using 5 kDa PEG-conjugated NHS ester. As shown in lane 1, a molecular weight shift of BSA was observed in SDSPAGE. After the PEG modification of BSA, fluorescence modification was performed using the same method as that described in Figure 3. As shown in lane 2, the fluorescence of

conjugated PTAD 19 was carried out, azide moieties in the modified BSA were reacted with alkyne-conjugated Alexa Fluor 488 by CuAAC for visualization (Figure 3). In the presence of hemin and H2O2, BSA was effectively modified with compound 18 without inducing protein degradation (lane 5; also see Figure S40), and the modification efficiency was 2.1-fold higher than that by azide-conjugated PTAD 19 (lane 6). Needless to say, BSA modification was not observed in the absence of hemin or/and H2O2 (lanes 2−4 in Figure 3). To optimize the protein modification conditions, the concentrations of hemin and H2O2 were investigated. As shown in Figure S35, 10 μM hemin and 1 mM H2O2 are necessary to effectively modify BSA. In order to estimate the oxidation of a cysteine residue, a free SH group at Cys34 in BSA was modified with maleimideconjugated fluorescent dye (TAMRA-maleimide) after the D

DOI: 10.1021/acschembio.5b00440 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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antibody and FITC-conjugated secondary antibody. Furthermore, we applied the current method to trypsin immobilization on magnetic beads (Figure S39). The protease activity of trypsin modified with 18 and DBCO-biotin was observed (Figure S39A,B), and the protease activity was still retained in the immobilized trypsin on SAv-coated magnetic beads (Figure S39C). Indeed, BSA was digested by the immobilized trypsin and identified successfully from the resulting digested fragments by MS/MS analysis (Figure S39D). Conclusions. We have developed a tyrosine modification method that uses luminol derivatives based on the in situ generation of reactive cyclic diazodicarboxyamide. Hemin catalyzed the formation of a covalent bond between a tyrosine residue and a luminol derivative in the presence of a stoichiometric amount of H2O2. The reactions using Nmethyl-substituted phthalic hydrazide derivatives showed higher efficiencies and tyrosine selectivities than those of reactions using PTAD. We evaluated the reactivity of compound 9 relative to that of PTAD (1a) for protein modification using BSA as the model protein. PTAD (1a) modified not only the tyrosine residues but also the lysine residues in BSA, whereas compound 9 modified only the tyrosine residues without affecting other amino acid residues in BSA under the reaction conditions that used hemin and H2O2. It is also noteworthy that the surface-exposed tyrosine residues underwent modification with compound 9 more efficiently than did internal tyrosine residues. This phenomenon is considered to be a good indication for the further development of sitespecific tyrosine modifications by adjusting modifier structures sterically and electronically. We also demonstrated protein modification using azide-conjugated compound 18 and visualized the modified proteins using alkyne-conjugated probes by CuAAC or DBCO-mediated copper-free click chemistry. This method was orthogonal to conventional lysine modification using NHS ester, and dual modification via fluorescence modification of tyrosine and PEG modification of lysine was realized without affecting the modification efficiency. Finally, we succeeded in the modification of an antibody and trypsin. The recognition of the antigen by the antibody and the enzymatic activity of trypsin were retained after the modification, although the proteins’ structures were possibly damaged by oxidation with H2O2 under the current reaction conditions. The current tyrosine-selective and highefficiency reaction provides an attractive strategy for the modification of peptides and proteins and a new methodology for protein modification and immobilization.

Figure 5. Dual functionalization of BSA with lysine and tyrosine modifications. After lysine modification with 5 kDa PEG-NHS, tyrosine modification was performed using compound 18 or 19. Azide moieties were reacted with alkyne-conjugated Alexa Fluor 488 by CuAAC using the same method as that described in Figure 3. Fluorescence intensities (2.0 s exposure) and the intensity of CBBstained bands were measured by image analysis using ImageJ software.35

both BSA and PEG-modified BSA was detected. Furthermore, the fluorescence bands obtained from the tyrosine modification with PTAD derivative 19 were weaker in intensity than those obtained from the modification with 18, revealing that our tyrosine modification method using 18 is more efficient than the tyrosine-click method using 19 (lane 2 vs 3). According to the quantification of fluorescence and CBB-stained bands, modification with 19 was inhibited by PEG modification, whereas modification with 18 proceeded without affecting the efficiency by PEG modification. We applied our current method to antibody modification. An anti-tubulin antibody was chosen as the model antibody. Biotin modification of the anti-tubulin antibody was carried out using 18. The azide moiety of 18 was linked with biotin by copperfree click chemistry using biotin-conjugated dibenzocyclooctyne (DBCO-biotin), as shown in Figure S38. Although the antibody’s antigen selectivity was slightly decreased after modification using 10 μM 18, 10 μM hemin, 1 mM H2O2, and DBCO-biotin, probably due to oxidative damage of the antibody’s structure, tubulin in HeLa cell lysate was recognized by the modified antibody. Next, we examined immunofluorescent imaging of microtubule formation by the antibody modified with 18 and DBCO-fluor 488 (Figure 6). Microtubule formation was detected by the modified antibody in a similar manner to that using the combination of an anti-tubulin

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METHODS

General Method for Peptide Modification. Angiotensin II and bradykinin fragment 1−7 were purchased from Sigma-Adlrich, and angiotensin II variants were customized by GeneScript Inc. To a solution of peptide (final concentration ,100 μM) in 100 mM phosphate buffer (pH 7.4) were added compound (from a 100 mM stock solution in DMSO; final concentration, 1 mM) and hemin (from 1 mM freshly dissolved in DMSO; final concentration, 10 μM) in a 0.6 mL Eppendorf tube. A solution of 100 mM H2O2 in ultrapure water was prepared with dilution from 30% H2O2(aq) just before use. The solution was briefly vortexed, and H 2 O 2 was added (final concentration, 1 mM). For the modification with PTAD (1a), compound 1a (from 100 mM freshly dissolved in acetonitrile; final concentration, 1 mM) was added. After briefly vortexing the reaction mixture, it was allowed to stand at RT for 1 h. The reaction was quenched with DTT (from 1 M solution in water; final concentration, 10 mM) and mixed with internal standard, angiotensin I (0.5−2.0

Figure 6. Immunofluorescent imaging of microtubule formation by an anti-tubulin antibody modified with 18 and DBCO-fluor 488 in HeLa cells. E

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μM CuSO4, 8 mM sodium ascrobate, 80 μM THPTA, 16 μM alkyne reagent) was added, and the mixture was incubated at 4 °C overnight. Excess modifiers were removed with a Bio-Spin 6 column (Bio-Rad). 5× SDS-PAGE sample buffer was added, and the samples were heated at 95 °C for 5 min. Proteins were separated by SDS-PAGE using 10 or 7.5% acrylamide gels. Fluorescence of modified proteins was detected with a Molecular Imager ChemiDoc XRS system (Bio-Rad). After obtaining of fluorescent image, the same gel was visualized with Coomassie brilliant blue (CBB) stain, and the image was obtained with a Molecular Imager ChemiDoc XRS system. For the detection of biotin-modified BSA, SDS-PAGE separated protein was transferred to a PVDF membrane (GE Healthcare). The membrane was blocked with Immuno Block (DS Pharma) and treated with horseradish peroxidase (HRP)-conjugated streptavidin (SAv-HRP, Sigma-Aldrich), and the blot was treated with ECL kit (GE Healthcare). The chemiluminescence images were obtained with a Molecular Imager ChemiDoc XRS system (Bio-Rad). Measurement of CA Activity. After modifying CA with 18 as described above, 4-nitrophenyl acetate (1.1 mM in Tris buffer 50 mM, pH 8.0, 90 μL/well in 96-well plate) was added to the reaction mixture (10 μL/well in 96-well plate). The time-dependent increase in absorbance at 360 nm (4-nitrophenol) was detected using a plate reader (TECAN, Infinite F200). Dual Modification of BSA. To a solution of BSA (final concentration, 10 μM) in 100 mM phosphate buffer (pH 7.4) was added SUNBRIGHT ME-050CS (α-succinimidyloxysuccinyl-ω-methoxy polyoxyethylene, Nichiyu) (from 5 mM freshly dissolved in water; final concentration, 100 μM), and the mixture was incubated at RT for 2 h. The resulting PEG-modified BSA was modified with compound 18 or 19, and the modifications were detected according to the above-described method. Antibody Modification. To a solution of anti-tubulin antibody (Santa Cruz) (final concentration, 10 μg/mL) in 100 mM phosphate buffer (pH 7.4) were added compound 18 (from 1 mM stock solution in DMSO; final concentration, 10 μM) and hemin (from 0.01−1 mM freshly dissolved in DMSO; final concentration, 0.1−10 μM) in a 0.6 mL Eppendorf tube. The solution was mixed with pipetting and added H2O2 (final concentration, 1 mM), incubated at RT for 2 h. Then, DBCO-biotin (Aldrich) or DBCO-fluor 488 (Click Chemistry Tools) (from 10 mM stock solution in DMF, final concentration 200 μM) was added, and the mixture was incubated at 37 °C for 1 h. For the modification using NHS ester, NHS-biotin (Life Technologies, from 1 or 10 mM stock solution in DMSO; final concentration, 10 or 100 μM) was added to the solution of anti-tubulin antibody and incubated at RT for 2 h. After the modification was complete, modified antibodies were prepared by removing excess amounts of small molecules with a Bio-Spin 6 column (Bio-Rad). For the detection of tubulin in HeLa cells, modified antibodies (final concentration, 100 ng/mL) in PBS were incubated at RT for 1 h and detected using SAvHRP (Aldrich) according to the above-described method. Immunofluorescent Imaging. HeLa cells on cover glass (1 × 104 cells/mL) were washed with PBS, fixed with 4% paraformaldehyde in PBS for 15 min at RT, and treated with 0.4% Triton-X for 5 min at RT. Cells were then incubated with anti-tubulin antibody or antibody modified with 18 and DBCO-fluor 488 (final concentration 100 ng/ mL) in 1% BSA Tween-TBS (40 mM Tris, 300 mM NaCl, Tween-20 0.1%, pH 7.5) at 4 °C overnight. For the unmodified antibody, FITCconjugated anti-mouse secondary antibody was treated at RT for 2 h. Nuclei were stained with DAPI (100 nM) at RT for 5 min. Immunostained slides were mounted with Prolong Gold reagent (invigtogen) and examined by fluorescent microscopy (Olympus). Trypsin Modification. To a solution of bovine trypsin (SigmaAldrich) (final concentration, 10 μM) in 100 mM phosphate buffer (pH 7.4) were added compound 18 (from 10 mM stock solution in DMSO; final concentration, 100 μM) and hemin (from 1 mM freshly dissolved in DMSO; final concentration, 10 μM) in a 0.6 mL Eppendorf tube. The solution was mixed with pipetting, H2O2 was added (final concentration, 1 mM), and the mixture was incubated at RT for 1 h. Then, DBCO-biotin (Aldrich) (from 10 mM stock solution in DMF; final concentration, 200 μM) was added to the

equiv., depending on the ionization property of angiotensin II variants). From the reaction mixture 10× diluted with 0.1% TFA, 0.5 μL was mixed with CHCA solution (0.5 mg mL−1 solution in acetonitrile/0.1% TFA = 1:1), and 1 μL was placed on MALDI-TOF plate and dried at RT. The modified protein peaks were detected with MALDI-TOF analysis (Shimadzu AXIMA-CFR), and modification yields were analyzed as shown in Figure S1. LC-ESI Analysis of Modified Peptides. The reaction mixture of angiotensin II and compound 9 was analyzed by LC-ESI performed with a Bruker ESI-TOF-MS (micOTOF II) and VIOLAMO200 C18 HPLC column (2.1 mm i.d. × 250 mm). The micropump gradient method was used, as follows. Mobile phase A was 0.1% FA, and mobile phase B was 100% acetonitrile: 0−2.5 min, 5% B; 2.5−45 min, 5−60% B; 45−50 min, 60−100% B; 50−55 min, 100% B; 55.1−65 min, 5% B. Chemiluminescence Detection. A 10 μL solution of luminol (6a), 9, or 18 (1 mM in 100 mM phosphate buffer (pH7.4)) was mixed with 1 μL of hemin solution (10 mM freshly dissolved in DMSO) in a 96-half-well white plate (Cornig). After the addition of 10 μL of H2O2 solution (100 mM in 100 mM phosphate buffer (pH 7.4)), chemiluminescence was measured immediately with a plate reader (TECAN). Preparation of PTAD Derivatives. PTAD (1a) was purchased from Sigma-Aldrich and used without further purification. Compound 19 was prepared according to procedures reported by Barbas et al.25 and Jessica et al.27 from synthesized compound 19′, a precursor of 19 (see Suporting Information). Solutions of 19′ (50 mM), pyridine (50 mM), and NBS (50 mM) in DMF were mixed in equal amounts. The reaction mixture was vortexed, incubated at RT for 5 min, and kept on ice until it was used. The resulting red solution (19: 16.7 mM in DMF) was used without isolation. Protein Modification. To a solution of BSA, OVA, SAv, or CA (Sigma-Aldrich) (final concentration 10 μM) in 100 mM phosphate buffer (pH 7.4) were added compound 9 or 18 (from 100 mM stock solution in DMSO; final concentration, 1 mM) and hemin (from 1 mM freshly dissolved in DMSO, final concentration 10 μM) in a 0.6 mL Eppendorf tube. The solution was mixed with pipetting and H2O2 was added (final concentration, 1 mM). For the modifcaiton with PTAD (1a) or compound 19, to a solution of BSA (final concentration 10 μM) in 100 mM phosphate buffer (pH 7.4) was added compound 1 (from 100 mM freshly dissolved in acetonitrile; final concentration, 1 mM) or 19 (from 16.7 mM in DMF; final concentration, 1 mM). After mixing the reaction mixture, it was allowed to stand at RT for 1 h. The modification was quenched by removing excess amounts of small molecules with a Bio-Spin 6 column (Bio-Rad). MS Analysis of Modified BSA. The solutions of BSA and modified BSA were desalted using Millipore Zip Tip C4 pipette tip. The resulting solutions were mixed with sinapinic acid (TCI) solution (0.5 mg mL−1 solution in acetonitrile/0.1% TFA = 1:1), and 1 μL was placed on a MALDI-TOF plate and dried at RT. The peaks of BSA and modified BSA were detected by MALDI-TOF analysis (Bruker, UltrafleXtreme). Digestion of Modified BSA. The samples of BSA and modified BSA were treated with 5 mM DTT at 30 °C for 1 h (condition A); 5 mM DTT at 30 °C for 1 h and alkylated with 10 mM iodoacetamide at RT in the dark for 1 h (condition B); or 10 mM DTT at 60 °C for 45 min and alkylated with 20 mM iodoacetamide at RT in the dark for 30 min (condition C). For conditions A and B, resulting solutions were digested with Mono Tip Trypsin (GL Science) by incubating at 37 °C for 20 min. For condition C, resulting solutions were digested with 2.5 μg of Glu-C endoproteinase (Thermo) overnight at 37 °C. The digested peptide mixtures were purified and concentrated on Millipore Zip Tip C18 pipette tip. The peaks were assigned by peptide mass fingerprinting analyzed using Swiss-Prot database. Functionalization of Azide-Modified Proteins. A solution of CuSO4, sodium ascrobate, and Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) in water was mixed with Alexa Fluor 488 alkyne (Invitrogen) or Biotin-PEG4-Alkyne (Click Chemistry Tools) and incubated on ice for 5 min. A freshly prepared solution of BSA modified with compound 18 or 19 (10 μM) (final concentration, 160 F

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ACS Chemical Biology reaction mixture, and it was incubated at 37 °C for 1 h. Immobilization of biotinylated trypsin was performed using FG Streptavidin-coated beads (Tamagawa Seki). The beads were incubated with modified trypsin for 30 min and washed with PBS three times. Protease activities of trypsin and modified trypsin were measured using Z-PheArg-AMC (Aldrich) and a plate reader (TECAN; Ex/Em = 360/430 nm).

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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/acschembio.5b00440. Experimental procedures for compound synthesis, compound characterization, and additional data (Figures S1−S41 and Tables S1 and S2) (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by Grants-in-Aid for Scientific Research on Innovative Areas, “Chemical Biology of Natural Products” (26102721 to H. Nakamura), “Young Scientist (B)” (25810104 to S. Sato), and “Homeostatic Regulation by Various Types of Cell Death” (15H01372 to S. Sato), from MEXT, Japan. We thank Y. Shoji for X-ray crystallography of compound 11a (Chemical Resources Laboratory, Tokyo Institute of Technology).

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DOI: 10.1021/acschembio.5b00440 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acschembio.5b00440 ACS Chem. Biol. XXXX, XXX, XXX−XXX