Bioorganic & Medicinal Chemistry 22 (2014) 6288–6296

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Sequential and parallel dual labeling of nanoparticles using click chemistry Hong Zong a,⇑, Sascha N. Goonewardena a,b, Huai-Ning Chang a, James B. Otis a, James R. Baker Jr. a,⇑ a b

Michigan Nanotechnology Institute for Medicine and Biological Sciences, United States Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, United States

a r t i c l e

i n f o

Article history: Received 11 April 2014 Revised 2 July 2014 Accepted 11 July 2014 Available online 18 July 2014 Keywords: Click chemistry PAMAM dendrimer Fluorescent labeling FRET

a b s t r a c t Bioorthogonal ‘click’ reactions have recently emerged as promising tools for chemistry and biological applications. By using a combination of two different ‘click’ reactions, ‘double-click’ strategies have been developed to attach multiple labels onto biomacromolecules. These strategies require multi-step modifications of the biomacromolecules that can lead to heterogeneity in the final conjugates. Herein, we report the synthesis and characterization of a set of three trifunctional linkers. The linkers having alkyne and cyclooctyne moieties that are capable of participating in sequential copper(I)-catalyzed and copperfree cycloaddition reactions with azides. We have also prepared a linker comprised of an alkyne and a 1,2,4,5-terazine moiety that allows for simultaneous cycloaddition reactions with azides and trans-cyclooctenes, respectively. These linkers can be attached to synthetic or biological macromolecules to create a platform capable of sequential or parallel ‘double-click’ labeling in biological systems. We show this potential using a generation 5 (G5) polyamidoamine (PAMAM) dendrimer in combination with the clickable linkers. The dendrimers were successfully modified with these linkers and we demonstrate both sequential and parallel ‘double-click’ labeling with fluorescent reporters. We anticipate that these linkers will have a variety of application including molecular imaging and monitoring of macromolecule interactions in biological systems. Ó 2014 Published by Elsevier Ltd.

1. Introduction Selective chemical labeling of biomacromolecules, such as proteins, nucleic acids, and lipids is an important goal in basic research, biotechnology, and clinical medicine. This goal requires chemical reactions with high specificity, efficient conversion, and mild reaction conditions compatible with biological matrices.1 Bioorthogonal ‘click’ reactions have provided promising tools to tag biomolecules that overcome many of the existing chemical labeling limitations.2–6 The most widely used ‘click’ reaction is the copper(I)-catalyzed azide and alkyne [3+2] cycloaddition (CuAAC).2 The rarity and inertness of azides and alkynes in biological environments allows for selective labeling without interference from elements in the biological environment. Additionally, Bertozzi et al. have developed a strain-promoted alkyne–azide cycloaddition reaction (SPAAC), which allowed the use of this reaction in living systems because no cytotoxic catalytic Cu(I) is required.7–12 Another intriguing bioorthogonal reaction using a similar concept, ⇑ Corresponding authors. E-mail addresses: [email protected] (H. Zong), [email protected] (J.R. Baker Jr.). http://dx.doi.org/10.1016/j.bmc.2014.07.015 0968-0896/Ó 2014 Published by Elsevier Ltd.

but employing tetrazines and strained trans-cyclooctenes (TCO) for inverse electron demand Diels–Alder reaction, has recently emerged. This reaction has gained popularity due to the extremely fast cycloaddition kinetics allowing modifications of biomacromolecules at extremely low concentrations.13–19 With the development of multiple bioorthogonal reactions, one could perform two or more reactions in sequentially or in parallel in biological systems. Labeling biomacromolecules often requires introducing multiple labels onto the same molecules.20–22 For example, modular labeling of DNA sequences with multiple labels is very important for DNA-based molecular diagnostics and for nanotechnology applications.23–26 Current dual labeling procedures are cumbersome involving multiple coupling reactions, and typically require protection and deprotection steps. Additionally, this strategy frequently suffers from low coupling efficiencies, making the downstream purification process more challenging.27,28 These difficulties necessitate more efficient labeling protocols, especially when the incorporation of multiple labels is required. Over the past few years, progress has been made toward this end in ‘double-click’ labeling of biomacromolecules using a combination of two ‘click’ reactions SPAAC and CuAAC in a sequential

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manner.22,29 Two different labels can be sequentially attached onto biomacromolecules, with the first labeling step being catalyst-free and the second step requiring Cu(I) as a catalyst. Another ‘doubleclick’ labeling strategy using a similar concept, but employing different ligation pairs (tetrazine–TCO and azide–cyclooctyne) has also been developed for simultaneous bioorthogonal labeling.30 These strategies enable observation of multiple targets as well as fluorescence resonance energy transfer (FRET) applications, by using the appropriate pair of fluorescent labels. The ‘double-click’ approach can also be very advantageous in the fabrication of nanomaterials where the controlled immobilization of multiple ligands is needed.31,32 Despite these advances, the ‘double-click’ strategy still has drawbacks that limit its utility, especially when the conjugation of two or more labels in controlled ratios is required on the same biomacromolecule.33 The ‘double-click’ strategy requires two different ‘click’ ligands to be incorporated into the target biomacromolecules first, followed by ‘click’ reactions with corresponding ligation partners. Conjugating multiple ligands to macromolecules complicates the synthesis because of the additional synthetic steps and purifications, which in and of itself can be challenging. Furthermore, the traditional multi-step conjugation strategies can lead to heterogenous populations of macromolecules that differ in the number and ratios of ligands leading to inconsistent results.34,35 Thus, a strategy to introduce multiple labels on to macromolecules with defined ratios preserving the specificity and efficiency of typical click reactions is an important goal.35–38 To address these issues, we have used a trivalent triazine molecule as a linker scaffold.35 Our approach uses a triazine molecule as a trifunctional linker on which two sites are attached with different ‘click’ ligand pairs (alkyne–cyclooctyne or alkyne–tetrazine) in a 1:1 ratio while a hydroxyl or carboxyl ligand is incorporated to its third functional site. These tri-substituted triazines serve as a small-molecule scaffold modified with ‘click’ ligands in defined ratios and controlled spatial topology which can then be attached to biomacromolecules through a one-step coupling reaction using the hydroxyl/carboxyl groups creating a platform for sequential or parallel ‘double-click’ labeling of biomacromolecules. Herein, we report an efficient synthetic strategy to prepare sequential and parallel ‘double-click’ trifunctional linkers. Attaching these linkers onto the biomacromolecules will allow dual labeling with fluorescence reporters that can be then used to monitor biomacromolecules trafficking or interactions with other biomacromolecules in biological systems. We used polyamidoamine (PAMAM) dendrimers as model biomacromolecules because of their well-defined structure, similarity to endogenous biomacromolecules, and the possession of a large number of reactive surface functional groups.39–43 As a proof of concept, we attached the ‘double-click’ linkers onto a folate-targeted G5 PAMAM dendrimer and demonstrated the feasibility of sequential and parallel dual labeling on the dendrimer using fluorescent reporters. We anticipate that these linkers and this approach will have a variety of application including molecular imaging and monitoring of macromolecule interactions in biological systems.

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MS/MS mass spectrometer. Fluorescence studies were performed on a Jobin Yvon FluoroMax-2 fluorimeter. Analytical ultra-performance liquid chromatography (UPLC) was performed on a Waters Acquity Peptide Mapping System equipped with a photodiode array detector and an Acquity BEH C4 column (100  2.1 mm, 1.7 lm) at a flow rate of 0.21 mL/min. Preparative HPLC was performed on a Waters Delta 600 system equipped with a 2996 photodiode array detector, an auto sampler, and an Atlantis Prep T3 column (250  10 mm) at a flow rate of 4.0 mL/min. 2.2. Materials All solvents and chemicals were of reagent grade quality, purchased from Sigma–Aldrich (St. Louis, MO), and used without further purification unless otherwise noted. Cyclooctyne NHS ester was purchased from Berry & Associates (Dexter, MI). trans-Cyclooctene amine (TCO–NH2) was purchased from KeraFAST (Boston, MA). Thin-layer chromatography (TLC) and column chromatography were performed with 25 DC-Plastikfolien Kieselgel 60 F254 (Merck), and Baxter silica gel 60 Å (230– 400 mesh), respectively. 2.3. Synthesis tert-Butyl ((6-methyl-1,2,4,5-tetrazin-3-yl)methyl)carbamate 9,44 G5-NHAc-FA 12,45 3-azido-7-hydroxycoumarin 15,46 and fluorescein-azide 174 were synthesized according to the literature. 2.3.1. Synthesis of N-(but-3-yn-1-yl)-4,6-dichloro-1,3,5-triazin2-amine (Triazine-Alkyne) (1) To a solution of cyanuric chloride (5.34 g, 0.029 mol) in acetone (30 mL) in an ice-water bath was added diisopropylethylamine (DIPEA) (3.75 g, 0.029 mol). 1-amino-butyne (1.00 g, 0.014 mol) in acetone (40 mL) was added slowly over 2 h. The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH2Cl2) to give 1 as a white solid (1.40 g, 45%): 1 H NMR (500 MHz, CDCl3) d 2.04 (t, J = 7.5 Hz, 1H), 2.53 (td, J1 = 6.5 Hz, J2 = 2.5 Hz, 2H), 3.36 (q, J = 6.5 Hz, 2H), 6.77 (m, 1H); 13 C NMR (500 MHz, CDCl3) d 19.0, 40.0, 71.0, 80.3, 165.9, 169.9, 171.1; ESI-MS m/z 217.1 (M+H+) calcd for C7H6Cl2N4 217.0.

2.1. General information

2.3.2. Synthesis of 2-((4-(but-3-yn-1-ylamino)-6-chloro-1,3,5triazin-2-yl)amino)ethanol (Triazine-Alkyne-OH) (2) Ethanolamine (1.28 g, 20.1 mmol) in acetone (10 mL) was added to a solution of 1 (1.04 g, 4.8 mmol) in acetone (10 mL). The reaction was stirred at room temperature for 48 h. Acetone was removed by rotary evaporation and the residue was suspended in 30 mL H2O/CH2Cl2 (50:50). The mixture was filtered off. The solid collected was washed successively with CH2Cl2 and H2O, and dried under reduced pressure to obtain 2 as a white solid (0.96 g, 83%). The product was sufficiently pure for further reactions: 1H NMR (500 MHz, d6-DMSO) 2.37 (m, 2H), 2.83 (m, 1H), 3.25–3.37 (m, 4H), 3.46 (m, 2H), 4.68 (m, 1H), 7.67–7.94 (m, 2H); 13 C NMR (500 MHz, CDCl3) d 13.9, 18.4, 43.0, 59.4, 72.3, 82.1, 165.1, 165.4, 167.6; ESI-MS m/z 242.1 (M+H+) calcd for C9H13ClN5O 242.1.

1 H NMR spectra were obtained using a Varian Inova 500 MHz spectrometer. Matrix-assisted laser desorption ionization timeof-flight mass spectra (MALDI-TOF-MS) were recorded on a PE Biosystems Voyager System 6050, using 2,5-dihydroxybenzoic acid (DHB) as the matrix. Electrospray ionization mass spectra (ESIMS) was recorded using a Micromass Quattro II Electronic HPLC/

2.3.3. Synthesis of tert-butyl (2-(2-(2-((4-(but-3-yn-1-ylamino)6-((2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate (Triazine-Alkyne-OH-NHBoc) (3) DIPEA (161 mg, 1.24 mmol), tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (308 mg, 1.24 mmol), and TriazineAlkyne-OH 2 (150 mg, 0.62 mmol) were dissolved in THF (5 mL).

2. Materials and methods

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The mixture was stirred at 70 °C under N2 overnight. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/CH2Cl2 = 5:95) to give 3 as a white solid (124 mg, 47%): 1H NMR (500 MHz, CDCl3) d 1.46 (s, 9H), 2.20 (m, 1H), 2.47 (m, 2H), 3.33 (m, 3H), 3.50–3.68 (m, 14H), 3.77 (m, 2H), 6.36 (m, 1H), 6.50 (m, 1H), 7.70 (m, 1H), 8.11 (m, 1H); ESIMS m/z 454.2 (M+H+) calcd for C20H36N7O5 454.3. 2.3.4. Synthesis of 6-((4-(but-3-yn-1-ylamino)-6-((1-(1fluorocyclooct-2-yn-1-yl)-1,8-dioxo-12,15-dioxa-2,9diazaheptadecan-17-yl)amino)-1,3,5-triazin-2-yl)amino)hexanoic acid (Triazine-Alkyne-OH-Cyclooctyne) (4) Triazine-Alkyne-OH-NHBoc 3 (30 mg, 0.066 mmol) was dissolved in CH2Cl2 (2 mL). TFA (2 mL) was added and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure. The residue was dissolved in H2O (10 mL) and filtered off. NaOH solution (1 N, aq) was added dropwise to the filtrate until the solution became basic. CH2Cl2 (10 mL) was added and the organic layer was washed with brine and H2O, dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was dissolved in dimethylformamide (DMF) (1.0 mL). Cyclooctyne-NHS (51 mg, 0.13 mmol), and DIPEA (26 mg, 0.11 mmol) were added. The mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/CH2Cl2 = 5:95) to give 7 as a white solid (9.8 mg, 48%): 1H NMR (500 MHz, d6-acetone) d 1.26 (m, 2H), 1.32 (m, 2H), 1.52 (m, 2H), 1.63 (m, 2H), 2.02 (m, 1H), 2.16 (t, J = 7.5 Hz, 2H), 2.32 (m, 6H), 2.47 (m, 4H), 3.27 (m, 2H), 3.48(t, J = 5.5 Hz, 2H), 3.49 (s, 1H), 3.77 (m, 2H), 3.51–3.66 (m, 14H), 6.53 (m, 2H), 8.02 (m, 1H), 8.21 (m, 1H), 8.39 (m, 1H); ESI-MS m/z 619.3 (M+H+) calcd for C30H48FN8O5 619.4. 2.3.5. Synthesis of 6-((4-(but-3-yn-1-ylamino)-6-chloro-1,3,5triazin-2-yl)amino)hexanoic acid (Triazine-Alkyne-COOH) (5) 6-Aminohexanoic acid (1.78 g, 13.6 mmol) in H2O (30 mL) was added to a solution of 1 (0.98 g, 4.5 mmol) and Et3N (1.37 g, 13.5 mmol) in acetone (30 mL). The reaction was stirred at room temperature for 24 h. Acetone was removed by rotary evaporation. HCl (1 N, aq) was added into the residue dropwise to adjust the pH to 5. The mixture was filtered off. The solid collected was washed successively with CH2Cl2 and H2O, and dried under reduced pressure to obtain 5 as a white solid (1.25 g, 89%). The product was sufficiently pure for further reactions: 1H NMR (500 MHz, CDCl3) d 1.17 (m, 4H), 1.41 (m, 2H), 1.63 (m, 4H), 2.35 (td, J1 = 7.5 Hz, J2 = 3.0 Hz, 2H), 3.35 (m, 1H), 3.43 (m, 2H), 7.43 (m, 1H), 7.63 (t, J = 5.5 Hz, 1H), 12.11 (br s, 1H); 13C NMR (500 MHz, CDCl3) d 24.5, 26.4, 28.8, 29.0, 30.5, 34.1, 41.2, 55.6, 67.6, 166.6, 170.2, 171.4, 179.0; ESI-MS m/z 310.2 (MH+) calcd for C13H17ClN5O2 310.2. 2.3.6. Synthesis of 6-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-6-(but-3-yn-1-ylamino)-1,3,5-triazin-2-yl)amino)hexanoic acid (Triazine-Alkyne-COOH-NH2) (6) Triazine-Alkyne-COOH 5 (100 mg, 0.32 mmol), tert-butyl 2-(2(2-aminoethoxy)ethoxy)ethylcarbamate (160 mg, 0.64 mmol), and DIPEA (125 mg, 0.97 mmol) were suspended in THF (10 mL). The mixture was stirred at 70 °C under N2 overnight. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2 (3 mL). TFA (3 mL) was added and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure. The residue was dissolved in H2O (20 mL) and

filtered off. The sample was purified by HPLC using a two-step linear gradient beginning with 90:10 (v/v) water/ACN and ending with 75:25 (v/v) water/ACN over 20 min. The gradient was gradually changed to 25:75 (v/v) water/ACN over the next 10 min. TFA at 0.10% concentration in water as well as in ACN was used as a counter ion. The desired product 6 was collected as TFA salt (white solid, 55 mg, 32%): 1H NMR (500 MHz, CDCl3) d 1.39 (m, 4H), 1.60 (m, 4H), 2.29 (m, 2H), 3.27 (m, 2H), 3.52 (m, 1H), 3.65 (m, 8H), 3.81 (m, 6H), 7.04–7.56 (m, 3H), 8.56 (br s, 3H); ESI-MS m/z 424.1 (M+H+) calcd for C19H34N7O4 424.3. 2.3.7. Synthesis of 6-((4-(but-3-yn-1-ylamino)-6-((1-(1fluorocyclooct-2-yn-1-yl)-1,8-dioxo-12,15-dioxa-2,9-diazaheptadecan-17-yl)amino)-1,3,5-triazin-2-yl)amino)hexanoic acid (Triazine-Alkyne-COOH-Cyclooctyne) (7) Triazine-Alkyne-OH-NH2 6 (16 mg, 0.038 mmol), CyclooctyneNHS (28.7 mg, 0.075 mmol), and DIPEA (14.6 mg, 0.11 mmol) were dissolved in DMF (500 lL). The mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/ CH2Cl2 = 7:93) to give 7 as a white solid (9.8 mg, 48%): 1H NMR (500 MHz, CDCl3) d 1.34 (m, 2H), 1.46 (m, 2H), 1.55 (m, 2H), 1.67 (m, 4H), 1.98 (m, 2H), 2.08 (m, 2H), 2.19–2.26 (m, 12H), 2.50 (m, 2H), 3.28 (m, 2H), 3.36 (m, 1H), 3.48 (m, 4H), 3.58–3.68 (m, 12H), 6.52 (m, 2H), 6.97 (m, 1H), 7.54 (m, 1H), 8.22 (m, 1H); ESIMS m/z 689.5 (M+H+) calcd for C34H54FN8O6 689.4. 2.3.8. Synthesis of 6,60 -((6-(but-3-yn-1-ylamino)-1,3,5-triazine2,4-diyl)bis(azanediyl))dihexanoic acid (Triazine-Alkyne-COOHCOOH) (8) The solvent was removed under reduced pressure. 6-Aminohexanoic acid (302 mg, 2.3 mmol) in H2O (3 mL) was added to a solution of DIPEA (295 mg, 2.28 mmol) and 1 (100 mg, 0.46 mmol) in THF (3 mL). The mixture was stirred at 60 °C under N2 overnight. THF was removed by rotary evaporation. HCl (1 N, aq) was added into the residue dropwise to adjust the pH to 5. The mixture was filtered off. The solid collected was washed successively with CH2Cl2 and H2O, and dried under reduced pressure to obtain 8 as a white solid (111 mg, 59%). The product was sufficiently pure for further reactions: 1H NMR (500 MHz, d6-DMSO) d 1.26 (m, 4H), 1.49 (m, 600H), 2.19 (t, J = 7.5 Hz, 4H), 2.38 (m, 4H), 2.50 (m, 1H), 3.17 (m, 2H), 3.34 (m, 4H), 6.60 (m, 2H), 7.84 (m, 1H), 11.98 (br s, 2H); 13C NMR (500 MHz, d6-DMSO) d 24.2, 24.3, 25.9, 26.1, 28.4, 29.1, 33.6, 72.2, 158.5, 165.3, 167.6, 174.5; ESI-MS m/z 407.1 (M+H+) calcd for C19H31N6O4 407.2. 2.3.9. Synthesis of 2,5-dioxopyrrolidin-1-yl 6-((4-(but-3-yn-1ylamino)-6-((6-(((6-methyl-1,2,4,5-tetrazin-3-yl)methyl)amino)-6-oxohexyl)amino)-1,3,5-triazin-2-yl)amino)hexanoate (Triazine-Alkyne-NHS-Tetrazine) (11) tert-Butyl ((6-methyl-1,2,4,5-tetrazin-3-yl)methyl)carbamate 9 (25 mg, 0.066 mmol) was dissolved in CH2Cl2 (1 mL). TFA (1 mL) was added and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure. The deprotected product (6-methyl-1,2,4,5-tetrazin-3-yl)methanamine 10 will be used in the following reaction without further purification. Triazine-Alkyne-COOH-COOH 8 (18 mg, 0.044 mmol), N,N0 -Dicyclohexylcarbodiimide (DCC) (9.1 mg, 0.088 mmol), and NHydroxysuccinimide (NHS) (5.1 mg, 0.088 mmol) were dissolved in DMF (500 mL) and stirred at room temperature under N2 for 1.5 h. (6-Methyl-1,2,4,5-tetrazin-3-yl)methanamine 10 in DMF (500 ll) was added. The mixture was stirred at room temperature for another 3 h. The solvent was removed under reduced pressure.

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The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/ CH2Cl2 = 5:95) to give 11 as a purple solid (8.3 mg, 31%): 1H NMR (500 MHz, d6-acetone) d 0.89 (m, 6H), 1.11 (t, J = 7.5 Hz, 4H), 2.35 (m, 3H), 2. 52 (m, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.90 (s, 2H), 3.06 (m, 1H), 3.07 (m, 2H), 3.25 (q, 2H), 3.36 (m, 2H), 3.57 (m, 3H), 3.68 (m, 2H), 5.08 (m, 2H), 5.14 (s, 1H), 5.29 (m, 1H), 7.19 (m, 1H), 7.47 (m, 1H), 8.17 (m, 1H); ESI-MS m/z 611.0 (M+H+) calcd for C27H39N12O5 611.3. 2.3.10. SPAAC and CuAAC sequential labeling of ‘double-click’ linker Triazine-Alkyne-OH-Cyclooctyne 4 Triazine-Alkyne-OH-Cyclooctyne 4 (1.0 mg) was dissolved in 200 ll DMF. 3-Azido-7-hydroxycoumarin 14 (5 equiv) in 300 ll DMF was added and the solution was allowed to stir at room temperature for 6 h. The solvent was removed under reduced pressure. The resulting residue was purified by TLC on silica gel (eluent CH3OH/CH2Cl2 = 5:95) to give Triazine-Alkyne-OH-Coumarin 16 as a brown solid: ESI-MS m/z 822.3 (M+H+) calcd for C39H53FN11O8 822.4. Triazine-Alkyne-OH-Coumarin 16 (0.5 mg) and fluoresceinazide 17 (5 equiv) were dissolved in 200 ll DMF. Cu(II) sulfate (0.5 equiv, 1.0 mg/mL H2O) and sodium ascorbate (3 equiv, 2.0 mg/mL H2O) were added. The mixed solution was allowed to stir at RT for 4 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by TLC on silica gel (eluent CH3OH/CH2Cl2 = 5:95) to give Triazine-Fluorescein-OH-Coumarin 18 as a yellow solid: ESI-MS m/z 1280.0 (M+H+) calcd for C63H71FN15O14 1280.5. 2.3.11. Synthesis of Fluorescein-TCO (19) TCO–NH2 HCl salt (10 mg, 0.038 mmol) was suspended in CH2Cl2 (500 ll). Et3N (15.4 mg, 0.15 mmol) was added and the mixture turned to clear. Fluorescein isothiocyanate (FITC) (29.6 mg, 0.076 mmol) in acetone (500 ll) was then added and the solution was allowed to stir at RT for 4 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2, washed with water, dried over Na2SO4, and rotary evaporated. The resulting residue was purified by column chromatography on silica gel (eluent CH3OH/CH2Cl2 = 10:90) to give 19 as a yellow solid (10.6 mg, 45%): 1H NMR (500 MHz, d4-methanol) d 1.50 (m, 4H), 1.62–1.79 (m, 5H), 1.97 (m, 1H), 2.08 (m, 2H), 2.26 (m, 1H), 3.07 (m, 2H), 3.58 (m, 2H), 5.51 (quint, J = 8.0 Hz, 1H), 5.56 (quint, J = 6.5 Hz, 1H), 6.46 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 2H), 6.59 (d, J = 2.5 Hz, 2H), 6.62 (d, J = 9.0 Hz), 7.08 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.98 (s, 1H); ESI-MS m/z 616.1 (M+H+) calcd for C33H34N3O7S 616.2. 2.4. General procedure for the syntheses of dendrimer conjugates 2.4.1. Characterization All the conjugates were analyzed by MALDI-TOF and NMR, the methods of which have been previously described.34,35,45 The number of ligands that attached to the dendrimer was obtained from the integration of the methyl protons of the terminal acetyl groups to the aromatic protons on the conjugated ligands. The number of acetyl groups per dendrimer was determined by first computing the total number of end groups from the number average molecular weight from gel permeation chromatography (GPC) and potentiometric titration data for G5-NH2 (100%) as previously described.45 The total number of end groups was applied to the ratio of primary amines to acetyl groups, obtained from the 1H

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NMR of the partially acetylated dendrimer, to compute the average number of acetyl groups per dendrimer. 2.4.2. Synthesis G5-NHAc-FA 12 (yellow solid): MALDI-TOF mass 32774. The 1H NMR integration determined the mean number of acetyl groups per dendrimer is 80.1. The mean number of FA per dendrimer is 3.6. 2.4.3. Synthesis of G5-FA-Alkyne-Cyclooctyne (13) Triazine-Alkyne-COOH-Cyclooctyne 7 (1.1 mg, 1.6 lmol), N-(3Dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) (0.63 mg, 3.3 lmol), and NHS (0.38 mg, 3.3 lmol) in DMSO (500 ll) was added to a solution of G5-NHAc-FA 12 (10 mg, 0.32 lmol) in H2O (300 ll). The reaction mixture was stirred at room temperature under N2 overnight. Excess glycidol (10 ll) was added to cap the remaining free amino group on the dendrimer surface. The reaction mixture was stirred at room temperature for an additional 6 h. Samples were purified using 10,000 MWCO centrifugal filtration devices. Purification consisted of 10 cycles (20 min at 4800 rpm) using PBS (5 cycles) and DI water (5 cycles). The purified dendrimer samples were lyophilized to yield 13 as yellow solids (6.5 mg, 59%): MALDI-TOF mass 32374; the 1H NMR integration determined the mean number of linker is 3.2. 2.4.4. Synthesis of G5-FA-Alkyne-Tetrazine (14) Triazine-Alkyne-NHS-Tetrazine 11 (1.0 mg, 1.6 lmol) in DMSO (300 ll) was added to a solution of G5-NHAc-FA 12 (10 mg, 0.32 lmol) in H2O (300 ll). The reaction mixture was stirred at room temperature under N2 overnight. Excess glycidol (10 ll) was added to cap the remaining free amino group on the dendrimer surface. The reaction mixture was stirred at room temperature for an additional 6 h. Samples were purified using 10,000 MWCO centrifugal filtration devices. Purification consisted of 10 cycles (20 min at 4800 rpm) using PBS (5 cycles) and DI water (5 cycles). The purified dendrimer samples were lyophilized to yield 14 as yellow solids (6.0 mg, 55%): MALDI-TOF mass 33160; the 1H NMR integration determined the mean number of linker is 3.5. 2.4.5. Sequential dual labeling of dendrimer nanoparticles G5-FA-Alkyne-Cyclooctyne 13 5.0 mg was dissolved in H2O (5.0 mg/mL H2O). 3-Azido-7-hydroxycoumarin 15 (10 azide mole ratio to G5 dendrimer, 5.0 mg/mL DMSO solution) was added. The reaction mixture was stirred at room temperature for 6 h. Samples were purified using 10,000 MWCO centrifugal filtration devices. Purification consisted of 10 cycles (20 min at 4800 rpm) using PBS (5 cycles) and DI water (5 cycles). The purified dendrimer samples were lyophilized to yield coumarin labeled dendrimer nanoparticle as yellow solids (3.8 mg). Coumarin labeled dendrimer nanoparticle 2.0 mg was dissolved in Cu(II) sulfate (20 mol % per alkyne ligand, 1.0 mg/mL H2O) and sodium ascorbate (120 mol % per alkyne ligand, 1.0 mg/mL H2O) solution. Fluorescein-azide 17 (10 azide mole ratio to G5 dendrimer, 5.0 mg/mL DMSO solution) was added. The reaction mixture was stirred at room temperature for 6 h. Samples were purified using 10,000 MWCO centrifugal filtration devices. Purification consisted of 10 cycles (20 min at 4800 rpm) using PBS (5 cycles) and DI water (5 cycles). The purified dendrimer samples were lyophilized to yield 20 as yellow solids (1.8 mg): MALDI-TOF mass 33470. 2.4.6. Parallel dual labeling of dendrimer nanoparticles G5-FA-Alkyne-Tetrazine 14 3.0 mg was dissolved in Cu(II) sulfate (20 mol % per alkyne ligand, 1.0 mg/mL H2O) and sodium ascorbate (120 mol % per alkyne ligand, 1.0 mg/mL H2O) solution. Both 3-azido-7-hydroxycoumarin 15 and Fluorescein-TCO 19 (10 mole ratio to G5 dendrimer, 5.0 mg/mL DMSO solution) were

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added at the same time. The reaction mixture was stirred at room temperature for 6 h. Samples were purified using 10,000 MWCO centrifugal filtration devices. Purification consisted of 10 cycles (20 min at 4800 rpm) using PBS (5 cycles) and DI water (5 cycles). The purified dendrimer samples were lyophilized to yield 21 as yellow solids (2.5 mg): MALDI-TOF mass 33897.

The tetrazine derivative was attached through direct coupling of (6-methyl-1,2,4,5-tetrazin-3-yl)methanamine 10 to TriazineAlkyne-COOH-COOH 8 using DCC/NHS. Partial acetylation of the G5 PAMAM dendrimer was used to neutralize a fraction of the surface primary amino groups, reducing the cytotoxicity of the cationic dendrimers, while also enhancing the solubility of the dendrimer during the conjugation reaction of folic acid (FA).45 The remaining non-acetylated primary amino groups were used to conjugate the ‘double-click’ linkers. The linker-modified dendrimer conjugates G5-FA-Alkyne-Cyclooctyne 13 and G5-FA-Alkyne-Tetrazine 14 were synthesized by direct coupling of G5-FA 12 with the corresponding triazine linkers 7 and 11, respectively (Schemes 4 and 5).

3. Results and discussion 3.1. Synthesis The trifunctional triazine linkers 4, 7, and 11 were synthesized from 2,4,6-trichloro-1,3,5-triazine by consecutive aromatic nucleophilic substitution reactions in a stepwise manner, and these reactions were controlled by the temperature employed (Schemes 1–3). The ease of displacement of chlorine atoms in 2,4,6-trichloro1,3,5-triazine by various nucleophiles in a controlled manner makes this reagent useful for making tri-substituted triazines.47–50 The monosubstituted triazine, Triazine-Alkyne 1, was synthesized by adding but-3-yn-1-amine to a solution of 2,4,6-trichloro-1,3,5-triazine in acetone at 0 °C. A large excess of 2,4,6trichloro-1,3,5-triazine was used to minimize the disubstituted byproduct. The second substitution was carried out in the same solvent at room temperature. For the sequential ‘double-click’ linkers, we have prepared two disubstituted triazines, TriazineAlkyne-OH 2 and Triazine-Alkyne-COOH 5, to show the flexibility of our synthetic procedure (Schemes 1 and 2). The hydroxylmodified linker can be attached to macromolecules through an ester bond, while the carboxyl-modified linker can be attached through amide bond. The disubstituted Triazine-Alkyne-OH 2 and Triazine-Alkyne-COOH 5 were treated with tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate in the presence of DIPEA in THF at 60 °C to form Triazine-Alkyne-OH-NHBoc 3 and Triazine-Alkyne-OH-NHBoc 6, which contained a protected primary amine that is used for conjugation with cyclooctyne. The Boc-protected di-amine compound was necessary because efforts to make trifunctional triazine using unprotected 2,20 -(ethane-1,2diylbis(oxy))diethanamine were unsuccessful due to the formation of triazine dimers. After removal of the BOC protecting group, the triazine derivatives were reacted with cyclooctyne NHS ester to yield the sequential ‘double-click’ linkers 4 and 7. The synthetic strategy was modified for the synthesis of the parallel ‘double-click’ linker 11. The mono-substituted TriazineAlkyne 1 was treated with 6-aminohexanoic acid in the presence of DIPEA in THF at 60 °C to form Triazine-Alkyne-COOH-COOH 8.

N

Cl N

Cl +

N Cl

To test the feasibility of dual labeling on the ‘double-click’ linkers, we first performed the SPAAC and CuAAC reactions directly on the sequential ‘double-click’ linker 4. We used fluorescent azide 3azido-7-hydroxycoumarin 14 for the copper-free labeling and fluorescein-azide 17 for the copper-mediated labeling (Scheme 6). Fluorescent azide 3-azido-7-hydroxycoumarin 14 has unique fluorogenic properties in that only the click product is fluorescent, thus eliminating the background fluorescence of the unreacted starting material.46 Fluorescent azide 14 readily reacted with the sequential ‘double-click’ linker 4, in the absence of Cu(I) catalyst, to give the fluorescent triazole linked adduct 16 (Fig. 1). The ESI-MS spectrum obtained for the crude product was in full agreement with the expected mass of the product (m+H+/z 822.3) and no ‘double-click’ product was found (Supporting information, Fig. S1). The second click reaction was performed by mixing the coumarin ‘clicked’ product 16 (DMSO solution) with a solution of Cu2SO4, sodium ascorbate, and fluorescein-azide 17 (Scheme 6). This reaction was also successful and the expected compound 18 was identified by mass spectrometry (Supporting information, Fig. S2), showing that two fluorescent labels can be introduced sequentially onto the ‘double-click’ linker. The successful labeling was also confirmed by fluorescence studies. The fluorescence signal (kex at 360 nm for coumarin, kex at 490 nm for fluorescein) generated by the labeled ‘double-click’ linker was monitored by fluorimeter (Jobin Yvon FluoroMax-2). As shown in Figure 1, the coumarin labeled product 16 has strong blue fluorescence at 410 nm that reflects the successful SPAAC reaction. For the dual labeled product 18, if there is FRET between the two attached fluorescent labels, excitation at the absorbance

N

Cl

0°C DIPEA

H2N

3.2. Fluorescent labeling of the ‘double-click’ linkers

N

Cl + H2N

N

OH

HN 1

r.t.

H N

HO

N

Cl

N N HN

+ H2N

O

O

60 °C DIPEA

NHBoc

2 O HO

H N

H N

N N

O

O

N

NHBoc

TFA

HO

H N

N N

HN

N

O

O

NH2

HN

3

HO

H N

O H N

H N

N N

N

O

O

O N H

H N O

F NH

+ O

NO O

F

HN 4

Scheme 1. Synthesis of the sequential ‘double-click’ linker Triazine-Alkyne-OH-Cyclooctyne 4.

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Cl N

Cl

Cl

N

Cl

0°C DIPEA

+ HN 2

N

N

Cl

OH

+ H2N

N

O

HN 1

O r.t.

H N

HO

N

Cl

H2N

+

N N HN

Et3N

O

O

NHBoc

5

O O

60 °C DIPEA

TFA

H N

HO

H N

N N

N

NH

O

O

NH2

+ O

HN 6 O

H N

HO

H N

N N

O

N

O

O

O

F

H N

N H

O

NO O

F

HN 7

Scheme 2. Synthesis of the sequential ‘double-click’ linker Triazine-Alkyne-COOH-Cyclooctyne 7.

Cl

O

Cl

N N

N

OH

+ H 2N

O

HN

H N

HO

60 °C DIPEA

N

OH

N

HN 8

1 BocHN

O

H N

N

H2N

N N

TFA

N N

N N

N N 10

9

O

DCC/NHS

8 + 10

H N

O

DMF

N

O

N

O

O

H N

N

N H

N

N N

HN

N

N

11

Scheme 3. Synthesis of the parallel ‘double-click’ linker Triazine-Alkyne-NHS-Cyclooctyne 11.

Folic acid

O

O HN

+

NH2

H N

HO

H N

N N N HN

12

O

O

O

H N

N H

O

F

7 Folic acid

1) EDC/NHS

O

2) Glycidol

O

HN HO

N H OH

N

OH

OH

H N

H N

N N

N

O

O

O N H

H N

OF

HN

13

Scheme 4. Synthesis of the sequential ‘double-click’ linker 7 modified dendrimer conjugate G5-FA-Alkyne-Cyclooctyne 13.

Folic acid

O

O HN

NH2

H N

O

+ O

N

H N

N

O N H

N N HN

O

12

NN

NN

11 Folic acid

1) DMSO

O

2) Glycidol

HN HO

O N H OH

N

OH OH

H N

H N

N N

N

HN

O N H

N N

N

N

14

Scheme 5. Synthesis of the parallel ‘double-click’ linker 11 modified dendrimer conjugate G5-FA-Alkyne-Tetrazine 14.

wavelength of donor fluorescent label coumarin (360 nm) will cause emission by acceptor fluorescent label fluorescein (510 nm) due to FRET. As expected, excitation at 360 nm led to green fluorescence (510 nm) on the dual labeled product 18. Importantly, no noticeable blue fluorescence (360 nm) was detected indicating the presence of FRET. The FRET efficiency depends on the distance between the donor and the acceptor. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET exquisitely sensitive to small variations in distances. Typical effective distances between the donor and acceptor molecules are in the 1–10 nm range. Our linkers can synthetically fix two ‘click’ labels in a very short distance, which is expected to provide a well-defined system for FRET studies in biomacromolecules.

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O

HO

H N N

O

H N

N

O

O

N

N H

Copper-free Click

HF N

HO

O

O

O

HN

O

H N

N N

O

O

N

N H

O

O

HN

N3

15

4

H N

HO

N N N HF N

16

OH

O HO

O

OH O

16 +

O

O

CuSO 4 aq.

HO

Copper Click

H N N

O

H N

N

O

O

N

N H

N N N HF N

O

O

HN

NH

N N N

17 N3

18 HN

O

HO O

O

O

OH

Scheme 6. SPAAC and CuAAC sequential labeling of ‘double-click’ linker 4.

Figure 1. Emission spectra of ‘double-click’ linker 4 after sequential ‘click’ reactions: (a) after SPAAC with 3-azido-7-hydroxycoumarin 15 (kex at 360 nm, blue); (b) after CuAAC with fluorescein-azide 17 (kex at 360 nm, green; kex at 490 nm, red). For the dual labeled linker 18, excitation at the absorbance wavelength of 3-azido-7hydroxycoumarin 15 (360 nm) led to only green fluorescence (510 nm), indicating the presence of FRET. This signal matches the fluorescence signal observed with excitation at the absorbance wavelength of fluorescein-azide 17 (490 nm). Samples were dissolved in DMSO.

3.3. Dual fluorescent labeling of PAMAM dendrimer nanoparticles After confirming the functionality of the ‘double-click’ linkers, we then sought to evaluate the efficiency of dual labeling on a G5 PAMAM dendrimer as a model biomacromolecule. Leveraging the FRET capabilities of the small-molecule triazine scaffold,

we used sequential and parallel linker-modified dendrimers to construct FRET systems using the appropriate combinations of fluorescent labels. To demonstrate sequential labeling, G5-FAAlkyne-Cyclooctyne 13 was first reacted with the fluorescent azide 3-azido-7-hydroxycoumarin 14 (Fig. 2). The modified dendrimer was then separated from unreacted reagents by centrifugation. The mono-labeled dendrimer was then treated with the second

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a)

HO

Folic acid

O O

1)

O HN

Folic acid

15

N3

O HO

Copper-free Click

O O

2)

H N

13 N3

FRET

HN

OH 17

20

O O

Copper-mediated Click

b) Folic acid

HO

O HN

Folic acid

O O

15

N3

N N N N

Copper-mediated Click HO

14

O

OH O

O N O H

Coumarin-azide

S N N H H

O

O FRET

HN

21

19

Fluorescein-azide

Fluorescein-TCO

Figure 2. Schematic representation of sequential and parallel dual labeling of targeted dendrimer nanoparticles: (a) dendrimer G5-FA-Alkyne-Cyclooctyne 13 was treated with 3-azido-7-hydroxycoumarin 15 and fluorescein-azide 17 sequentially; (b) G5-FA-Alkyne-Tetrazine 14 was treated with 3-azido-7-hydroxycoumarin 15 and fluoresceinTCO 19 concurrently.

label fluorescein-azide 17 in the presence of CuSO4 and sodium ascorbate at room temperature for 6 h (Fig. 2). The crude sample was purified and analyzed by fluorimeter. Fluorescence analyses indicated that both ‘click’ reactions proceeded successfully. In Figure 3, the mono-labeled dendrimer emits strong blue fluorescence when excited at 360 nm. We noticed that the blue fluorescence from mono-labeled dendrimer has red-shifted compared with that of the small molecule 16 (kem 473 nm vs 405 nm). This shift may introduced by interaction between clicked coumarins since there are multiple coumarins on each dendrimer nanoparticle. As expected, the blue fluorescence signal was quenched and green fluorescence (510 nm) was detected after the second click reaction (Fig. 3), which indicated the presence of FRET. The fluorescence results of the dendrimer were in agreement with that of the small molecule linker (Fig. 1). A labeling experiment using G5-FA-Alkyne-Tetrazine 14 in a simultaneous ‘double-click’ experiment was also undertaken (Fig. 2). Dual labeling of the dendrimer 14 was carried out in

DMSO/water. The aq solution containing the dendrimer and Cu(I) catalyst was treated with an equimolar quantity of 3-azido-7hydroxycoumarin 15 and Fluorescein-TCO 19 in DMSO. The solutions were stirred at room temperature for 6 h, and then the crude products were purified using centrifugal filtration devices. Although it is known that azide could react with TCO, the cycloaddition kinetics of azide–TCO reaction (second-order rate constant 0.0064 ± 0.002 L mol1 s1) is much slower than that of tetrazine–TCO (second-order rate constant 210–30 000 L mol1 s1).30 Also the undesired crosslinking small molecule byproduct azide– TCO will be cleared during the macromolecule purification procedure. Fluorescence results of the simultaneously labeled dendrimer were similar to those of the sequential dual-labeled G5-FAAlkyne-Cyclooctyne 13. Due to FRET, green fluorescence (510 nm) was observed in the modified dendrimer when excited at coumarin absorption wavelength (kex at 360 nm) (Fig. 4). Also, no noticeable blue fluorescence was detected, indicating that the absence of any

5

5x10

b 5

6

4x10

1.5x10

5

3x10

6

1.0x10

a 5

2x10

5

5.0x10

5

1x10

0 400

450

500

550

600

650

Wavelength (nm)

0.0 400

450

500

550

600

650

Wavelength (nm) Figure 3. Emission spectra of the dendrimer nanoparticle 13 after sequential ‘click’ reactions. G5-FA-Alkyne-Cyclooctyne 13 was treated with 3-azido-7-hydroxycoumarin 15, and followed by fluorescein-azide 17, kex at 360 nm: (a) after 3azido-7-hydroxycoumarin 15 ‘clicked’ (blue); (b) after fluorescein-azide 17 ‘clicked’ (green). Samples were dissolved in H2O.

Figure 4. Emission spectra of the dendrimer nanoparticle 14 after simultaneous ‘click’ reactions. G5-FA-Alkyne-Tetrazine 14 was treated with 3-azido-7-hydroxycoumarin 15 and fluorescein-TCO 19 concurrently, kex at 360 nm. Samples were dissolved in H2O.

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mono-labeled (only coumarin labeled) dendrimer conjugates (Fig. 4). These results suggest that (1) simultaneous dual labeling has been performed successfully on biomacromolecules, (2) tetrazine–TCO ‘click’ reaction is highly efficient. All coumarin labeled dendrimers have been labeled with the second fluorescent reporter fluorescein-TCO 19, (3) azide modified coumarin won’t react with tetrazine groups on the dendrimer, which further confirms the mutual orthogonality of the tetrazine–TCO and azide–alkyne reaction pairs on a biomacromolecule. The dual fluorescent labeling studies on linker modified PAMAM dendrimer confirms that the functionality of the ‘double-click’ linkers was preserved after attaching on to a model biomacromolecule. These linkers enable the biomacromolecules the ability to perform sequential or simultaneous ‘click’ reactions with a high degree of specificity. Further studies are ongoing to test the functionality of these conjugates in biological systems to monitor macromolecular interactions and intracellular trafficking. 4. Conclusion In summary, we have developed sequential and parallel ‘double-click’ linkers that can be easily attached on to biomacromolecules. The suitability of these linkers for labeling of biomacromolecules was demonstrated using a G5 PAMAM dendrimer as a model biomacromolecule. Preliminary studies with the linker-modified dendrimers using fluorescent labels showed excellent specificity and FRET effect. We have shown that these ‘doubleclick’ linkers are viable tools to modify biomacromolecules, on which multiple labels can be attached in series or in parallel without the need for complicated synthetic strategies. We anticipate that these linkers will have a variety of application including molecular imaging and monitoring of macromolecule interactions in biological systems. Acknowledgment S.N.G. was supported by the Cardiovascular Center Inaugural Grant from the University of Michigan. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.07.015. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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