Articles pubs.acs.org/acschemicalbiology
Second Generation TQ-Ligation for Cell Organelle Imaging Xiaoyun Zhang,†,‡,∥ Ting Dong,†,‡,∥ Qiang Li,†,‡ Xiaohui Liu,‡ Lin Li,‡ She Chen,‡ and Xiaoguang Lei*,†,‡,§ †
Graduate School of Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China National Institute of Biological Sciences (NIBS), Beijing 102206, China § Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China ‡
S Supporting Information *
ABSTRACT: Bioorthogonal ligations play a crucial role in labeling diverse types of biomolecules in living systems. Herein, we describe a novel class of ortho-quinolinone quinone methide (oQQM) precursors that show a faster kinetic rate in the “click cycloaddition” with thio-vinyl ether (TV) than the first generation TQ-ligation in both chemical and biological settings. We further demonstrate that the second generation TQ-ligation is also orthogonal to the widely used strainpromoted azide−alkyne cycloaddition (SPAAC) both in vitro and in vivo, revealing that these two types of bioorthogonal ligations could be used as an ideal reaction pair for the simultaneous tracking of multiple elements within a single system. Remarkably, the second generation TQ-ligation and SPAAC are effective for selective and simultaneous imaging of two different cell organelles in live cells.
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selectivity and good kinetics, the SPAAC reaction is considered to be one of the most widely used bioorthogonal reactions in dual labeling research, and other reactions are chosen for pairing with SPAAC.12,14,29,33,36 A recent example of using SPAAC paired with tetrazine-transcyclooctene for the dual labeling was reported by Hilderbrand and co-workers in which the chosen pairs were used in a simultaneous imaging experiment of two targets in different cell types within a single system.36 More recently, Wittmann and co-workers have demonstrated that SPAAC in combination with the Diels− Alder reaction with inverse electron demand of 1,2,4,5-tetrazine can be used in the simultaneous dual labeling of two different sugars on the cell membrane.33 Although there are several excellent illustrations of using SPAAC paired with other ligations for simultaneous labeling and imaging experiments, they have not been shown to proceed concurrently for tracking multiple elements with different subcellular localization inside cells. We show here that a novel dual labeling strategy employing the SPAAC and TQ-ligation enabled by the click hetero-Diels−Alder (HDA) cycloaddition between thio-vinyl ether (TV) and ortho-quinolinone quinone methide (oQQM; Scheme 1b) for the selective and simultaneous fluorescence labeling of two different cell organelles within a single system. TQ-ligation has been proven to be effective for site-specific labeling experiments in live cells under physiological conditions
ince the introduction of bioorthogonal chemistry a decade ago, the field has matured to a stage in which bioorthogonal ligation is routinely employed in the study of biological processes in complex systems.1−8 However, due to numerous constraints on developing effective bioorthogonal reactions, only a handful of ligations have been discovered to possess excellent biocompatibility and selectivity in living systems. The most widely used reactions include the Staudinger ligation,9,10 tetrazine ligation,11−16 photoinduced tetrazolealkene cycloaddition,17−19 and the strain-promoted copperfree azide−alkyne [3 + 2] cycloaddition.20−23 Each of these reactions is unique and possesses specific key attributes that are well suitable for studying living systems. For example, in comparison to the copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC),24−27 Bertozzi and co-workers utilized ring strain promoted azide−alkyne cycloaddition (SPAAC), which circumvent the cytotoxicity associated with copper-based catalysts.28 The second generation SPAAC, which contains a difluorinated cyclooctyne, possesses good kinetics in living systems.22,29−31 However, a single ligation method cannot meet the requirements of simultaneous multitarget labeling in highly complex biological processes. The demands for developing mutually exclusive bioorthogonal click chemistry reactions and their applications in the multitarget labeling of biomolecules or small molecule drugs within a single system are amplified.12,14,32−39 In particular, the choice of two rapid, selective, and mutually orthogonal reactions for the dual labeling process has gained the most attention. Benefiting from its high © XXXX American Chemical Society
Received: January 26, 2015 Accepted: April 22, 2015
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DOI: 10.1021/acschembio.5b00193 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology Scheme 1. TQ-ligations between oQQM Precursors and TVa
Scheme 2. Synthesis of the Second Generation oQQM Precursor 4a
a
a
(a) First generation TQ-ligation. (b) Second generation TQ-ligation with a more reactive oQQM precursor 4.
Reagents and conditions: (a) MOMCl, NaH, DMF, 1.5 h, 82%; (b) SeO2, 1,4-dioxane, 80 °C, overnight, 86%; (c) NH2OH·HCl, TEA, CH3CN, reflux, 3 h, followed by TsCl, DBU, reflux, 12 h, 94%; (d) AgNO3, (NH4)2S2O8, conc. H2SO4, MeOH, reflux, 6 h, 59%; (e) 0.03 M KOH, Ac2O, 0 °C, 1 h, then r.t., 30 min, 68%; (f) (1) DMP, DCM, 1.5 h; (2) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH/H2O, overnight; (3) TMSCHN2, toluene/MeOH, 2.5 h; (4) NH4OAc, MeOH/H2O, 5 h; 72% yield over four steps; (g) 37% HCHO (aq.), 0.5 M NaOH, 1.5 h, 58%.
and has been demonstrated to be compatible with the widely used SPAAC.40,41 Considering the potential applications of mutually orthogonal ligation methods in targeting specific organelles selectively and simultaneously could provide us tremendous opportunities to dissect crucial biological processes at the subcellular level, we decided to examine whether SPAAC and TQ-ligation could be used as a ligation pair to image two different cell organelles. However, the potential application of the first generation TQ-ligation is hindered by the relatively low kinetic rate as estimated by the second-order rate constant of (1.5 ± 0.1) × 10−3 M−1 s−1 (Scheme 1a).41 Thus, optimizing the kinetic parameter for better pairing is the primary and most urgent task. We therefore sought to develop the second generation TQ-ligation. Here, we describe the design and synthesis of the second generation oQQM precursor 4, followed by its applications in dual labeling with SPAAC in vitro and in vivo. The power of the second generation TQ-ligation has been ultimately demonstrated by pairing with SPAAC for selective and simultaneous imaging of two different cell organelles in live cells.
oxidations and was further methyl esterified with trimethysilydiazomethane to form a methyl ester in which the acetyl group was subsequently deprotected using NH4OAc in aqueous MeOH to afford 12 in 72% yield over four steps. Under basic conditions, hydroxymethylation of 12 with formaldehyde smoothly generated the second generation oQQM precursor 4. Kinetic Evaluation of the TQ-ligation between 4 and Thio-Vinyl Ether 2. With 4 in hand, we first examined whether 4 was able to accelerate the click HDA cycloaddition. We performed the second generation TQ-ligation of 4 and thio-vinyl ether 2 in a 5:1 mixture of H2O and CH3CN to measure the kinetics using 1H NMR. Fortunately, we obtained the second-order rate constant of (2.8 ± 0.1) × 10−2 M−1 s−1 (Figure S1), which showed an approximate 18-fold rate enhancement compared to the first generation TQ-ligation [k2 = (1.5 ± 0.1) × 10−3 M−1 s−1].41 Notably, the enhanced rate constant was even comparable to the widely used second generation SPAAC with a difluorinated cyclooctyne (k2 = 7.6 × 10−2 M−1 s−1 in CH3CN).22 Comparison of the Labeling Efficiency between the First and Second Generation TQ-ligation in Biological Settings. To compare the relative reaction rate of the second generation ligation with the first generation TQ-ligation in a biologically relevant setting, we first synthesized the biotinylated and fluoresceinated derivatives 18 and 20 as the second generation oQQM precursors (Scheme 3). Hydroxymethylation of 10 followed by a diol protection afforded 13, which was subsequently transformed to the activated pentafluorophenyl ester 14 through sequential oxidations and esterification, which could be further coupled with different tags. Treatment of ester 14 with the readily available N-biotinyl-3,6-dioxaoctane-1,8diamine (15) afforded amide 17, which was further deprotected with HCl to generate the desired biotinylated oQQM precursor 18. In addition, coupling of ester 14 with fluorescein piperazine (16) followed by deprotection smoothly afforded the fluoresceinated oQQM precursor 20. With both 18 and 20 in hand, we first evaluated the protein labeling efficiency for the second generation TQ-ligation. We modified the free lysine residues of BSA with TV as reporters (Figure 1a). A standard NHS-ester-coupling reaction was used
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RESULTS AND DISCUSSION Design and Synthesis of the Second Generation oQQM Precursor 4. Given the inverse electron demand characteristics of the hetero-Diels−Alder cycloaddition-based TQ-ligation, we sought to enhance the reaction rate by using the strategy of activating the oQQM precursor. Ultimately, this was achieved with a central effort by introducing electronwithdrawing groups to the quinoline core by installing a cyano group at C2 and an ester group at C4. The synthesis of compound 4 (Scheme 2) commenced with protection of the phenolic hydroxyl group of the previously reported 2-methylquinolin-7-ol (6)42 with chloromethyl methyl ether (MOMCl) under basic conditions, which smoothly produced 7. The methyl group was oxidized with selenium dioxide to yield aldehyde 8. A cyano group was introduced efficiently by treating 8 with hydroxylamine hydrochloride and triethylamine, followed by further elimination with paratoluensulfonyl chloride and DBU in a one pot reaction to form 9 in 95% yield. The dihydroxy compound 10 was then produced by hydroxymethylation of 9 through direct and regioselective C−H functionalization. The phenolic hydroxyl group of 10 was then selectively protected with acetic anhydride in potassium hydroxide solution to provide the benzyl alcohol 11. The primary alcohol of 11 was transformed to carboxylic acid via sequential Dess-Martin and Pinnick B
DOI: 10.1021/acschembio.5b00193 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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ACS Chemical Biology Scheme 3. Syntheses of the Biotinylated and Fluoresceinated oQQM Precursors 18 and 20a
Reagents and conditions: (a) (1) 37% HCHO (aq.), 1 M NaOH, 1.5 h; (2) 2,2-dimethoxypropane, PPTS (cat.), DMF, 80 °C, 5 h; 48% yield over two steps; (b) (1) DMP, DCM, 1 h; (2) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH/H2O, overnight; (3) PFP.TFA, TEA, THF, 3 h; 71% yield over three steps; (c) N-biotinyl-3,6-dioxaoctane-1,8-diamine (15), TEA, DMF, overnight, 91%; (d) fluorescein piperazine (16), DIPEA, DMF, overnight, 72%; (e) 0.5 M HCl/THF, 48 h, 48%; (f) 0.8 M HCl/THF, 16 h, 64%. a
Figure 1. Labeling of TV-modified BSA with 18, 20, and 22. (a) Modification of free lysine residues of BSA (2 mg mL−1) with 21 followed by pulldown assay of the biotinylated, or fluoresceinated oQQM precursor-bound proteins. (b,c) BSA conjugates were prepared by the addition of BSA (2 mg mL−1) with sufficient excess TV NHS-ester 21 and were analyzed via mass spectrometry; then the prepared TV-BSA was incubated with the cognate tag 18, 22, or 20 at the indicated conditions. (b) Coomassie blue staining of the biotin-labeled BSA from the streptavidin pull down experiment. (c) In-gel fluorescence measurements of the fluorescence-labeled BSA. For b and c, protein loading was assessed with Coomassie blue staining.
staining with Coomassie brilliant blue dyes (Figure 1b). Both reagents tested showed time-dependent protein labeling that was consistent with their kinetics in model reactions (Scheme 1). Furthermore, the first generation TQ-ligation enables us to label tubulin in live HeLa cells within 12 h;41 however, the
to attach TV to BSA. The effective TV modification was analyzed by mass spectrometry (Table S1 and Figure S2). Subsequently the prepared TV-labeled BSA was incubated with the cognate tag 18 or 22 (100 μM) for 3, 6, or 12 h, then was pulled down by the streptavidin affinity beads and quantified by C
DOI: 10.1021/acschembio.5b00193 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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clooctyne. Collectively, these results confirm that the second generation TQ-ligation is fully orthogonal to SPAAC in vitro. Dual Labeling of Cellular Membrane Protein EGFR Using TQ-Ligation and SPAAC. In addition to biomolecule modification in vitro, good chemical reporters must be able to traverse metabolic pathways and function in vivo. We next determined whether the second generation TQ-ligation and SPAAC reactions could be utilized simultaneously in vivo. Cetuximab, a well-known antibody drug that specifically targets EGFR in cancer cells, was selected for this study.43,44 Human KB carcinoma cells were chosen due to their high expression level of EGFR receptors.45 We first estimated the relative bioactivity of the modified Cetuximab, and the results confirmed that the chemical modification of Cetuximab did not affect its original activity (Figure 3a). Next, we tested the potential cross-reactions in vitro employing a similar strategy to that shown in Figure 2. The results indicated that TQ-ligation and SPAAC paired well (Figure 3b). We then treated KB cells with both TV-labeled Cetuximab (TV-Cetuximab) and azidelabeled Cetuximab (AZ-Cetuximab) for 30 min and washed the cells before adding the modified reaction partners 20 and 24 for 6 h. Confocal fluorescence microscopy clearly showed good colocalization between the specific TV-Cetuximab associated fluorescein and AZ-Cetuximab associated Sulforhodamine B. In contrast, the control cells were cultured in the absence of TVCetuximab or AZ-Cetuximab but otherwise treated in the same way and showed a negligible background staining. This indicates that the specific EGFR labeling originated from the specific bioorthogonal reaction (Figure 3c). Overall, this result indicated the mutual orthogonality of the TQ-ligation and SPAAC paired effectively in colabeling EGFR. Dual Labeling of Two Cell Organelles Using TQLigation and SPAAC. To further explore the application of TQ-ligation in tracking of multiple elements with different subcellular localization inside cells, a new labeling strategy using two organelle-targeted peptides in a simultaneous double-click experiment was performed. Inspired by the elegant work reported by Kelley and co-workers, which demonstrated the feasibility of using the organelle-specific cell-penetrating peptides RrRK and FrFK to deliver small molecules to the nucleus and mitochondria respectively,46−48 we performed the dual labeling of nucleus and mitochondria using both TQligation and SPAAC. We first synthesized a cell-permeable probe, naphthofluorescein probe 25 (Figure 4c, Figure S6); meanwhile, compound 20 was demonstrated to possess good cellular permeability and minimal cytotoxicity at concentrations up to 160 μM (Figure S7). Furthermore, extensive washing steps significantly reduced the fluorescence signal, which indicated that the widespread fluorescence was not attributed to the nonspecific covalent binding (Figure S8). We next synthesized TV-FrFK (Figure 4a) and AZ-RrRK (Figure 4b) and demonstrated that these two short peptides could successfully recruit their carried chemical reporters to mitochondria and nucleus, respectively (Figure S9). Encouraged by this result, we concentrated our efforts on both mitochondrial and nuclear simultaneous localization experiments. HeLa cells were cotreated with TV-FrFK and AZ-RrRK for 1.5 h, then were treated with 20 and 25 concurrently at 37 °C for 6 h. The images in the green channel showed the localization of TV-FrFK within the mitochondria of HeLa cells, and the fluorescence was originated from the click reaction enabled by TQ-ligation between 20 and TV-FrFK (Figure 4f). On the other hand, the images in the red channel demonstrated
second generation TQ-ligation could achieve a similar labeling effect within 6 h (Figure S3). Collectively, all those results indicated that the second generation TQ-ligation afforded higher labeling sensitivity than the first generation TQ-ligation in vitro and in vivo. Next, the concentration dependence of the reactions was estimated. The reactions were performed with 6.25−200 μM of the fluoresceinated oQQM precursor 20 for a period of 6 h (Figure 1c), and the formation of the ligation product was confirmed by mass spectrometry analysis (Figure S4). The ligation exhibited labeling proportional to reagent concentration. As depicted in Figure 1b,c, the ligations were both timeand dose-dependent, and no reaction was observed in the absence of either TV-BSA or 20. TQ-Ligation Is Orthogonal to SPAAC for Protein Modifications. We next investigated the orthogonality between the second generation TQ-ligation and SPAAC in vitro. The TV and azide reporters on the free lysine residues of BSA and RNase A were introduced with 21 and azide NHSester (23), respectively (Figure 2a). TV-BSA and azide-labeled
Figure 2. Second generation TQ-ligation is orthogonal to SPAAC. (a) Modification of free lysine residues on BSA and RNase A with 21 and 23, respectively, was followed by in-gel fluorescence measurements of the fluoresceinated proteins bound to oQQM precursors. (b) Free lysine residues on BSA or RNase A were modified with TV or azide at 4 °C for 3 h and purified from the unconjugated fluorophore using Amicon centrifugal filters. Then, each sample (2 mg mL−1) was treated with 20 (100 μM), 24 (100 μM), or both 20 and 24 for 6 h at 37 °C before analysis using in-gel fluorescence measurements.
RNase A (AZ-RNase A) conjugates were then combined 1:1 to generate a mixed sample without interfering with each other (Figure 2b). Both functional groups could be selectively targeted with covalent probes (either 20 or a DBCO-PEG4Sulforhodamine B (24)), suggesting that TV and azide can coexist to a certain extent (lane 9, Figure 2b). However, when TV-BSA and AZ-RNase A were treated inversely with 24 and 20, respectively, no signal above the background was observed under the labeling conditions employed (lanes 3 and 7, Figure 2b). In addition, we also examined the cross-reactivities between oQQM precursors and cycloalkyne reagents; as shown in Figure S5, there was no cross-reactivity between these two reagents with oQQM precursor 4 and dibenzocyD
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Figure 3. Dual labeling of cellular membrane protein EGFR using TQ-ligation and SPAAC. (a) KB cells were treated with the indicated concentration of Cetuximab or its variants functionalized with TV or AZ for 24 h. After treatment, the cell lysate was resolved by SDS-PAGE followed by immunoblotting analysis to detect the protein levels of phosphorylated EGFR (pEGFR) and EGFR. (b) Free lysine residues on Cetuximab (100 μg mL−1) were modified with 21 or 23 at 4 °C for 3 h prior to the addition of 20 (100 μM), 24 (100 μM), or both 20 and 24 in the incubator. Finally, the sample was analyzed by in-gel fluorescence measurements on a GE Typhoon TRIO+ Variable Mode Imager. (c) KB cells were treated with or without both TV-Cetuximab and AZ-Cetuximab for 30 min and were washed before the addition of the modified reaction partners (20 and 24) for 6 h. Cells were subsequently costained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) and imaged on a confocal fluorescence microscope. Fluorescent images are shown from (a) TV-Cetuximab-Fluorescein, (b) AZ-Cetuximab-DBCO-PEG4-Sulforhodamine B, (c) the merged channels of a and b, (d) DAPI, (e) DIC, and (f) the merged channels of d and e.
SPAAC could be utilized simultaneously for fluorescent labeling of multiple cell organelles in live cells. In conclusion, we have developed a second generation electron-deficient oQQM precursor, which was efficiently generated by the introduction of a cyano group and an ester group to the quinoline scaffold. Optimization of the kinetic rate
the localization of AZ-RrRK within the nuclei of HeLa cells. In this case, the fluorescence was originated from the click reaction enabled by SPPAC between 25 and AZ-RrRK (Figure 4g). Negligible fluorescence signal was observed in the control group treated with the fluorescent tag alone (Figure 4j and k). Collectively, these results clearly indicated that TQ-ligation and E
DOI: 10.1021/acschembio.5b00193 ACS Chem. Biol. XXXX, XXX, XXX−XXX
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Figure 4. Simultaneous labeling of two organelles using TV-FrFK and AZ-RrRK in live HeLa cells. (a) Structure of TV-FrFK. (b) Structure of AZRrRK. (c) Structure of naphthofluorescein dibenzocyclooctyne conjugate (25). Live HeLa cells were cotreated with 10 μM TV-FrFK and AZ-RrRK for 1.5 h, then were treated with 25 (20 μM) and 20 (20 μM) concurrently at 37 °C for 6 h. The cells were fixed and stained with DAPI before being imaged on a confocal microscope. Shown: fluorescence from (d) the merged three channels of e, f, and g. (e) DAPI. (f) TQ-ligation between 20 and TV-FrFK. (g) SPAAC ligation between 25 and AZ-RrRK. (h) The merged three channels of i, j, and k. (i) DAPI. (j) Addition of 20. (k) Addition of 25. 10% acetic acid) and analyzed by in-gel fluorescence measurements on a GE Typhoon TRIO+ Variable Mode Imager. Fluorescence was measured with a 532 nm excitation wavelength and 580 nm emissions. Total protein loading was confirmed by subsequent staining with Coomassie Brilliant Blue. Cell Culture. HeLa or 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillinstreptomycin in a humidified 5% CO2 incubator (Thermo) at 37 °C. Cell Viability Assay. Cell viability was measured by MTT assay with cell counting kit-8 (Dojindo). A total of 100 μL of cell suspensions (1 × 104 cells mL−1) per well were seeded in 96-well plates, incubated at 37 °C and allowed for attachment for 12 h before treatment. Then, 10 μL of medium per well containing the indicated compounds were added to the wells. Wells containing 110 μL of medium without cells were set as a blank control, and experiment control cells were treated with 10 μL of medium containing 0.1% DMSO. After certain periods of incubation, 10 μL of CCK-8 was added per well. Plates were incubated at 37 °C and 5% CO2 for 3 h. Then the optical density (OD) was recorded by the Paradigm detection platform (Beckman Coulter) at 450 nm. Four wells per dose were conducted in three independent experiments.50 Antibody Labeling. Initially, we prepared the TV-Cetuximab or azide-Cetuximab according to the protein labeling method. Then the KB cells were plated in glass chamber slides at a density of 1 × 105 cells mL−1 in IMEM media. After 24 h of incubation, the cells were treated with the mixture of TV-Cetuximab and AZ-Cetuximab at approximately 10 μg mL−1 for 30 min in IMDM growth media containing 10% FBS, 1% L-Glutaimine, and 1% Penstrep. Then, the cells were washed with IMDM medium three times before adding the modified reaction partners (reagents 20 and 24) for 6 h. After additional washing, the KB cells were fixed and treated with nuclear stain DAPI at 37 °C for 30 min before being visualized on a Nikon eclipse 80i fluorescent microscope. Identical image acquisition settings were used for both the control and the antibody-labeled data sets.36
of the second generation TQ-ligation showed an 18-fold rate enhancement compared with the first generation TQ-ligation. The improved reaction rate of the second generation TQligation has enabled it to become a promising tool for dual bioorthogonal labeling. Ultimately, we have demonstrated that the second generation TQ-ligation is mutually orthogonal with the widely used SPAAC both in vitro and in vivo, and these two ligation methods are effective for selective and simultaneous imaging of two different cell organelles in live cells.
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METHODS
Protein Labeling. Bovine serum albumin conjugates and RNase A conjugates were prepared by the treatment of each chosen model proteins with chemical reporters (21, 23) using a standard coupling protocol.49 Then the modified model proteins were purified from the unconjugated fluorophore using Amicon centrifugal filters, and subsequently reacted with its cognate tag at 37 °C for 6 h. Western Blot. Equal amounts of proteins were loaded in sodium dodecyl sulfate−polyacrylamide gels. After electrophoresis, the gel was transferred to nitrocellulose membranes. The membranes were washed with PBS containing 0.1% Tween 20 (PBST) thrice 10 min each. Membranes were blocked with 5% nonfat powdered milk for 1 h. Then membranes were incubated with specific primary antibodies for 4 h, washed with PBST thrice 10 min each, and further incubated with anti-Biotin for 2.5 h at RT. Finally, the blots were visualized using enhanced chemiluminescence (ECL) kit (GE Healthcare). In-Gel Fluorescence Analysis of Protein Conjugates. Thiovinyl ether labeled protein samples were treated with the tag compound or DMSO. After 6 h, the modified protein samples were measured using a DC Protein Assay kit (BioRad). Protein isolates were analyzed by gel electrophoresis using 12% polyacrylamide gels. Gels were rinsed in destaining buffer (50% D.I. H2O, 40% CH3OH, F
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ACS Chemical Biology Labeling of Mitochondria and Nucleus Using TV-FrFK and AZ-RrRK Respectively in HeLa Cells. We first prepared TV-FrFK and AZ-RrRK conjugated peptides according to the mentioned procedure in the Supporting Information. Then HeLa cells that had been grown to 60% confluence in the chamber were incubated with 10 μM of the indicated peptides for 90 min at 37 °C and then washed with DMEM medium three times. The cells were incubated with their cognate florescent tag 20 or 25 for 6 h. The cells were fixed and stained with DAPI before imaged on a Nikon eclipse 80i confocal microscope.
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ASSOCIATED CONTENT
* Supporting Information S
Additional methods, full experimental details, NMR spectra, mass spectra, and supporting figures and tables. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00193.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions ∥
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank C. Wang (Peking University) for helpful discussion, M. Zhao (NIBS) for NMR and HPLC-MS analysis, J. Zhou (Peking University) for HRMS analysis. Financial support from the National High Technology Projects 973 (2015CB856200, 2012CB837400) and NNSFC (21222209, 91313303, 21472010) is gratefully acknowledged.
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