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Photo-triggering of the metal-free azide to acetylene cycloaddition reaction was achieved by masking the triple bond of dibenzocyclooctynes as cyclopropenone. Such masked cyclooctynes do not react with azides in the dark. Irradiation of cyclopropenones results in the efficient (Φ355 = 0.33) and clean regeneration of the corresponding dibenzocyclooctynes, which then undergo facile catalyst-free cycloadditions with azides to give corresponding triazoles under ambient conditions. In-situ light activation of a cyclopropenone linked to biotin made it possible to label living cells expressing glycoproteins containing N-azidoacetyl-sialic acid. The cyclopropenone-based photo-triggered click chemistry offers exciting opportunities to label living organisms in a temporally and spatially controlled manner and may facilitate the preparation of microarrays.
The bioorthogonal chemical reporter strategy is emerging as a versatile method for labeling of biomolecules such as nucleic acids, lipids, proteins, and carbohydrates.1,2 In this approach, a unique chemical functionality is incorporated into a targeted biomolecule, preferably by the biosynthetic machinery of the cell, followed by a specific chemical reaction of the functional group with an appropriate probe. In particular, the azide is an attractive chemical reporter because of its small size, diverse mode of reactivity, and bio-orthogonality. Azides can be incorporated into biomolecules using a variety of strategies such as post synthetic modification,3 in-vitro enzymatic transfer,4 the use of covalent inhibitors,5 and metabolic labeling by feeding cells a biosynthetic precursor modified with an azido function.1
The most commonly employed bioorthogonal reactions with azides include the Staudinger ligation with phosphines,6 copper(I)-catalyzed cycloaddition with terminal alkynes,7 and strain-promoted cycloaddition with cyclooctynes.8,9 The latter type of reaction, which was coined copper-free click chemistry, does not require a cytotoxic metal catalyst thereby offering a unique opportunity for labeling living cells. The attraction of this type of technology was elegantly demonstrated by a study of the Bertozzi laboratory in which glycans of the developing zebrafish were imaged using a difluorinated cyclooctyne derivative.10 We have recently demonstrated that derivatives of 4-dibenzocyclooctynol (1a,b; DIBO, Scheme 1) react exceptionally fast in the absence of a CuI catalyst with azido-containing saccharides and amino acids, and can be employed for visualizing glycoconjugates of living cells that are metabolically labeled with azido-containing monosaccharides.9
The utility of azide-based bioorthogonal reporter strategy can be further extended by the development of a photochemically-triggered click reaction as this approach provide opportunities for the spatial and temporal control of the labeling of the target substrates. In fact, photochemical release or generation of an active molecule is a widely employed strategy to deliver bioactive compounds to addressable target sites in a time-controlled manner.11 To achieve this goal, we have explored photochemical generation of reactive dibenzocyclooctynes. It is known that single12,13 or two-photon14 excitation of cyclopropenones results in the formation of corresponding acetylenes. Photochemical decarbonylation of thermally stable diaryl-substituted cyclopropenones is especially efficient (Φ = 0.2 – 1.0) and produces alkynes in a quantitative yield.13 This reaction is also extremely fast and is complete within few hundred picoseconds after excitation.15 We have already employed cyclopropenone moiety in the development of photoswitchable enediynes.16 Here we report a novel photo-triggered click strategy for metal-free ligation of azides (Scheme 1). Cyclopropenones, such as 2, do not react with azides under ambient conditions in the dark but efficiently produce reactive dibenzocyclooctynes 3 upon irradiation. The latter type of compound could be employed for labeling of living cells modified with azido-containing cell surface saccharides.
Friedel-Crafts alkylation of appropriate substrates with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropene offers a convenient synthesis of aromatic cyclopropenones.13 Thus, the target cyclopropenone 2a was obtained by treatment of 3,3′-bisbutoxybibenzyl (5) with tetrachlorocyclopropene in the presence of aluminum chloride followed by in situ hydrolysis of the intermediate dichlorocyclopropene (Scheme 2). In addition to 2a, a small amount of a bis-butoxy analog (2c) was isolated.
Biotinylated cyclopropenone 2b was prepared to explore the utility of the photo-triggered click chemistry for the light controlled labeling of living cells (Scheme 2). Thus, cyclopropenone 2a was coupled with diethylene glycol acetate under Mitsunobu conditions to give 6 in 92% yield. The carbonyl moiety of cyclopropenone 6 was protected as a neopentyl glycol acetal by treatment with neopentyl glycol in the presence of BF4O(C2H5)3 and the acetyl ester of the resulting compound 7 was saponified with sodium methoxide in methanol to produce 8. Treatment of 8 with 4-nitrophenyl chloroformate gave activated intermediate 9, which was immediately reacted with N-biotinyl-3,6-dioxaoctane-1,8-diamine to provide carbamate 10. Finally, the acetal-protecting group of 10 was removed to give the required cyclopropenone-biotin conjugate 2b by the treatment with Amberlyst 15 in acetone. The performance of the photo-triggered click reagent 2b was compared to the known labeling reagent 1b9 and to biotinylated dibenzocyclooctyne 3b (Scheme 3) prepared by an independent route. For this purpose, cyclopropenone 6 was converted into dibenzocyclooctyne 11 by preparative photolysis, which was modified with a biotin moiety to give compound 3b by a similar procedure employed for the conversion of acetal 7 into compound 10.
The UV spectra of methanol solutions of cyclopropenones 2a–c showed two close-lying intense bands (λmax = 331 nm and 347 nm, logε ~ 4.5, Fig. 1). Irradiation of 2a–c with 350 nm light resulted in efficient (Φ355 = 0.33) decarbonylation of the starting material, which could be observed by bleaching of the 331–347 nm bands, and the quantitative formation of acetylenes 3a–c. The thermal stability of the cyclopropenone 2c in aqueous solution was tested by incubating 1 mM solutions of 2c at 60°C. After 12 h at this temperature, negligible loss of starting material was observed in aqueous solution (ca. 3%) and methanol (ca. 4%). It should be noted that thermal decomposition of cyclopropenones in nucleophilic solvents results in ring opening and the formation of acrylic acid derivatives rather than decarbonylation and thus will not produce an alkyne.13 Incubation of methanolic solutions of cyclopropenone 2a–c and benzyl- or phenyl azide in the dark for several days did not result in the detectable changes in UV absorbance and HPLC analysis of the mixture showed only the presence of starting materials. Upon irradiation of the solutions, however, the azides rapidly reacted with photo-generated cycloalkyne 3a–c to produce the corresponding triazoles 4a–c in quantitative yields. It is important to note that photoproducts 3a–c and 4a–c have virtually no absorbance above 340 nm (Fig. 1), thus allowing for selective irradiation of cyclopropenones 2a–c in their presence and for the convenient monitoring of the reaction progress.
The rate measurements of cycloaddition of acetylenes 3c and 1a were conducted by UV spectroscopy at 25±0.1°C. A calculated amount of 0.25 M solutions of an azide required to achieve desired azide concentration (6 ×10−4 - 1.5 ×10−2 M) was added to a thermally equilibrated 6 ×10−5 M solution of acetylene in MeOH. Reactions were monitored by following the decay of the characteristic absorbance of acetylenes ca. 317 nm (Fig. 1). Consumption of starting material followed a first order equation and the pseudo-first order rate constants were obtained by least-square fitting of the data to a single exponential equation. The rate dependence as a function of the concentration of azide was linear. Least-squares fitting of the data to a linear equation produced bimolecular rate constants summarized in Table 1. It was found that this method gives more accurate rate constants compared to the use of NMR.8,9 In this respect, the UV spectroscopic method can be performed under pseudo first order conditions over a wide range of reagent concentrations making the analysis of second-order kinetic curves more reliable. Interestingly, the rate constants for cycloaddition of acetylene 3c with benzyl azide were very similar to that of dibenzocyclooctynol (1a),9 and thus, the aromatic alkoxy-substitutents of 3a–c do not appear to influence the rate constants.
Having established that light activation of cyclopropenones results in the clean formation of the corresponding dibenzocyclooctynes, which can undergo metal-free cycloadditions with azides to give corresponding triazoles, attention was focused on labeling living cells modified with azido moieties. Thus, Jurkat cells were cultured in the presence of 25 μM of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) for 3 days to metabolically introduce N-azidoacetyl-sialic acid (SiaNAz) moieties into glycoproteins and glycolipids.17 As a negative control, Jurkat cells were employed that were grown in the presence of peracetylated N-acetylmannosamine (Ac4ManNAc). The cells were exposed to 30 μM of compound 1b, 2b, and 3b for 1 h at room temperature. In addition, cells and cyclopropenone 2b were exposed to light (350 nm) for 1 min to form in-situ cyclooctyne 3b and then incubated for 1 h at room temperature. Next, the cells were washed and stained with avidin-fluorescein isothiocyanate (FITC) for 15 min at 4°C. The efficiency of the two-step cell surface labeling was determined by measuring the fluorescence intensity of the cell lysates. Cyclooctynes 1b and 3b exhibited strong labeling of the cells (Fig. 2a). Furthermore, in-situ activation of 2b to give 3b resulted in equally efficient cell labeling. As expected, low fluorescence intensities were measured when cells were exposed to cyclopropenone 2b in the dark demonstrating that this compound can be selectively activated by a short irradiation with 350 nm light. Similar staining patterns were obtained when the living cells were analyzed by flow cytometry (Fig. S1).18
Some background labeling was observed when the control cells (labeled with Ac4ManNAc) were exposed to 2b or 3b and then treated with avidin-FITC (Fig. 2a). To exclude the possibility that the background labeling is due to unwanted side reactions of the compounds with protein, the cell lysates were analyzed by Western blotting using an anti-biotin antibody conjugated to HRP (Fig. 2b). Gratifyingly, the control cells gave negligible staining, demonstrating that background staining is not due to chemical reactions of the compounds with protein and probably arises from non-covalent interactions with the cell membrane. As expected, similar patterns of staining were observed for cells labeled with Ac4ManNAz and then exposed to 3b or in-situ activated 2b.
The concentration-dependency of the cell surface labeling was examined by incubating cells with various concentrations of 1b, in-situ activated 2b, and 3b, followed by staining with avidin-FTIC (Fig. 2c). The cells displaying azido moieties showed a dose-dependent increase in fluorescence intensity. Reliable fluorescent labeling was achieved at a concentration of 3 μM, however, optimal results were obtained at concentrations ranging from 10 to 100 μM. Interestingly, at low concentration, 3b gave a somewhat higher fluorescent reading than 1b.
A time course experiment demonstrated that labeling with 1b and 3b (30 μm) at 25°C reaches an apparent plateau after an incubation time of approximately 45 min, which gradually increased after prolonged exposure (Fig. 2d). A similar experiment at a lower temperature (4°C) also showed an initial fast- followed by a slow and gradual increase in fluorescent intensity; however, the responses were somewhat lower compared to the reaction at 25°C (Fig. S2).18
Light activation of cyclopropenone 2b provides and attractive opportunity for labeling cells in a temporal controlled manner. To establish a proof of principle for such labeling, a time course experiment was performed whereby cells were first incubated in the presence of 2b for 30 min in the dark, and then exposed to UV light to form in-situ alkyne 3b, which was allowed to react with cell surface azide moieties for different periods of time. Importantly, an identical pattern of labeling was observed compared to cells immediately exposed to UV light, however, with a 30 min delay (Fig. 2d).
The heat-sensitivity of the cyclopropenone extrusion reaction was examined by exposing cells labeled with Ac4ManNAz to 2b at 37°C in the dark, and no significant increase in fluorescence was observed compared to exposure at room temperature (Fig. S4).18 To ensure that in situ activation of 2b had no effect on cell viability and morphology, cells were assessed for the ability to exclude trypan blue and fortunately no changes were observed compared to cells that were not exposed to 2b both with and without UV light activation (Fig. S5).18 Cell viability was also examined after incubation with 2b with and without light activation followed by reincubation for 5 and 24 h (Fig. S6).18 In each case, there was no significant difference in the ability of the cells to reduce MTT to its insoluble formazan salt as compared to control cells.
Finally, attention was focused on visualizing azido-containing glycoconjugates of cells by confocal microscopy. Thus, adherent Chinese hamster ovary (CHO) cells were cultured in the presence of Ac4ManNAz (100 μM) for three days. The resulting cell surface azido moieties were reacted with in situ generated 3b (30 μM) and then visualized with avidin-Alexa fluor 488. As expected, staining was only observed at the cell surface (Fig. 3) and showed similar cell surface labeling as obtained by staining with 1b.9 Cells cultured in the presence of Ac4ManNAc (100 μM) exhibited very low fluorescence staining. As expected, cells metabolically labeled with Ac4ManNAz and exposed to 2b in the dark showed also negligible staining (data not shown).
It has been shown that light activation of cyclopropenones 2a–c results in the clean formation of the corresponding dibenzocyclooctynes 3a–c, which can undergo fast and catalyst-free cycloadditions with azides to give corresponding triazoles. In-situ light activation of 2b made it possible to efficiently label living cells expressing glycoproteins containing N-azidoacetyl-sialic acid. It is to be expected that the properties of compounds such as 2b will make it possible to label living organisms in a temporal and/or spatial controlled manner.
It has already been demonstrated that glycoconjugates of model organisms, such as zebrafish, can be metabolically labeled with azido-containing sugars and such an approach has been employed to demonstrate tissue specific expression of glycoconjugates.10 It is to be expected that the use of compounds such as 2b will make it possible to visualize azido-labeled biomolecules in model organisms or tissues in a more controlled and reliable manner. In this respect, differences in staining intensities and patterns may arise when classical metal-free click reagents19 such as 1b are employed due to possible concentration gradients. On the other hand, the use of a photo-triggered click reaction will make it possible to achieve a homogeneous concentration of reagent before initiating the click reaction. The photo-triggered click reagent reported here has a much higher quantum yield than the previously described photo-activated Diels-Alder reaction and hence will exhibit much less light induced toxicity.
It is to be expected that compounds such as 2b can be activated in a spatial controlled manner, however, the resulting alkyne (3b) is a stable derivative, which may diffuse to surrounding space thereby reducing the resolution of labeling. Although future studies will need to establish the spatial resolution of the photo-triggered click reaction, it is to be expected that it can selectively label organs or tissues of model organisms. Such an approach provides a unique opportunity for biotinylation of glycoconjugates of specific organs or tissues, which can then be isolated for glycomics or glycoproteomics studies. Wong and coworkers have already reported a combined use of metabolic labeling, Cu-mediated click reactions and glycoconjugate isolation for glycomics.20 However, such an approach cannot be employed for living organisms.
It is to be expected that other fields of science such as the fabrication of microarrays and the preparation of multifunctional materials, may benefit from photo-triggered click chemistry. In this respect, Cu-mediated click reactions have been used for the fabrication of saccharide microarrays by offering a convening approach to immobilize azide-modified saccharides to an alkyne-modified surface.21 It is to be expected that surface modification with compounds 2a will offer exciting opportunities for spatially controlled ligand immobilization using light activation followed by copper-free ligation. Furthermore, metal-free click reactions have been applied in material chemistry,22 and the obvious advantage of such a synthetic approach is that it offers a reliable approach for macromolecule modification without the need of using toxic reagents. Therefore, it is to be expected that the combined use of traditional- and photo-activated metal-free click reactions will offer an attractive approach for multi-functionalization of polymers and macromolecules.23
All NMR spectra were recorded in CDCl3 and referenced to TMS unless otherwise noted. Melting points are uncorrected. Purification of products by column chromatography was performed using 40–63 μm silica gel. Tetrahydrofuran was distilled from sodium/benzophenone ketyl; ether and hexanes were distilled from sodium. Other reagents were obtained from Aldrich or VWR and used as received unless otherwise noted. 11,12-didehydro-5,6-dihydro-dibenzo[a,e]cycloocten-5-ol (1a) and 11,12-didehydro-5,6-dihydrodibenzo [a,e]cycloocten-5-yl ester of 19-[(3aS,4S,6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-15-oxo-5,8,11-trioxa-2,14-diazanonadecanoic acid (1b) were prepared as reported previously.9
BBr3 (11.3 g, 45 mmol) was added to a solution of 1,2-bis(3-methoxyphenyl)ethane24 (11.56 g, 47.8 mmol) in CH2Cl2 at −78°C. The reaction mixture was slowly warmed to r.t., and stirred overnight. The reaction mixture was quenched with water, diluted with CH2Cl2, and the reaction mixture extracted with 2 M solution of NaOH (3 × 100 mL). The aqueous layer was slowly acidified at 0°C with concentrated HCl to c.a. pH =1, the grey precipitate was filtered, washed with water, dried in the air at r.t., and then under vacuum at 85°C over 5 h to provide 10.3 g of crude 1,2-bis(3-hydroxyphenyl)ethane as grey solid. A suspension of crude 1,2-bis(3-hydroxyphenyl)ethane (10.3 g), BuBr (6.50 g, 143.4 mmol), and K2CO3 (20.08 g, 143.4 mmol) in DMF (70 mL) was stirred overnight at 75°C, cooled to r.t., diluted with hexanes (~150 mL) and water (~250 mL). The organic layer was separated, washed with water, brine, dried over anhydrous MgSO4, and concentrated. The residue was separated by chromatography (Hex:EtOAc 40:1) to provide of 1,2-bis(3-butoxyphenyl)ethane (11.22 g, 72%,) as slightly yellow oil that slowly crystallizes on standing. 1H NMR: δ 7.18 (dt, J = 8.8, 1.2 Hz, 2 H), 6.77 (d, J = 8.0 Hz, 2 H), 6.75-6.70 (m, 4 H), 3.93 (t, J = 6.4 Hz, 4 H), 2.88 (s, 4 H), 1.75 (5, J = 6.4 Hz, 4 H), 1.48 (six, J = 7.2 Hz, 4 H), 0.98 (t, J = 6.8 Hz, 6 H), 1.60-1.55 (m, 4 H), 0.87 (s, 9 H), 0.03 (s, 6 H); 13C NMR: 159.4, 143.6, 129.5, 120.9, 115.0, 112.1, 67.8, 38.1, 31.6, 19.5, 14.1; MS calc for C22H30O2 (M+) 326.2246, EI-HRMS found 326.2280.
Tetrachlorocyclopropene was added to a suspension of AlCl3 (2.45 g, 13.76 mmol) in CH2Cl2 (200 mL), the reaction mixture was stirred for 10 min at r.t., and then cooled to −78°C. A solution of 5 (4.48 g, 13.76 mmol) in CH2Cl2 (~10 mL) was added dropwise, and the reaction mixture was stirred for ~2 h. at −78°C, slowly warmed to r.t., and stirred for an extra hour at r.t. The reaction was quenched by 5% aqueous HCl solution, the organic layer was separated, washed with water, dried over anhydrous MgSO4, and concentrated. The residue was separated by chromatography (CH2Cl2: MeOH 20: 1) to provide 2a (0.997 g, 23%) as a yellow powder and 2c (0.628 g, 12%) as a white powder.
1H NMR (DMSO): δ 10.41 (s, 1 H), 7.73 (d, J = 8.4 Hz, 1 H), 7.66 (d, J = 8.4 Hz, 1 H), 7.05 (d, J = 2.4Hz, 1 H), 6.97 (dd, J = 8.8, 2.4 Hz, 1 H), 6.86 (d, J = 2.4 Hz, 1 H), 6.80 (dd, J = 8.4, 2.4 Hz, 1 H), 4.05 (t, J = 6.4 Hz, 2 H), 3.42-3.35 (m, 1 H) 2.45-2.35 (m, 3 H), 1.69 (p, J = 7.2 Hz, 2 H), 1.41 (sxt, J = 7.6 Hz, 2 H), 0.91 (t, J = 7.2 Hz, 3 H), 1.60-1.55 (m, 4 H), 0.87 (s, 9 H), 0.03 (s, 6 H); 13C NMR: 158.9, 155.42, 155.19, 155.07, 127.1, 126.9, 117.5, 116.96, 116.72, 116.1, 113.3, 112.1, 110.79, 110.34, 68.1, 36.8, 36.7, 31.5, 19.5, 14.1. MS calc for C21H21O3 (MH+) 321.1491, APCI-HRMS found 321.1482.
H1 NMR: δ 7.73 (d, J = 9.6 Hz, 2 H), 6.69 (m, 4 H), 4.04 (t, J = 6.0 Hz, 4 H), 3.33 (d, J = 10.4 Hz, 2 H), 2.63 (d, J = 10.4 Hz, 2 H), 1.80 (p, J = 6.0 Hz, 4 H), 1.52 (s, J = 7.6 Hz, 4 H), 1.00 (t, J = 7.6 Hz, 6 H); 13C NMR: 162.3, 154.0, 148.0, 142.3, 136.0, 116.5, 112.5, 68.2, 37.4, 31.4, 19.42, 14.03.
A solution of DEAD (0.635 g, 3.75 mmol) in THF (5 mL) was added to a suspension of 2a (0.75 g, 2.34 mmol), PPh3 (0.983 g, 3.75 mmol), and 2-(2-hydroxyethoxy)ethyl acetate (0.44 g, 3.0 mmol) in THF (100 mL), and the reaction mixture was stirred for 30 min. Solvents were removed in vacuo, and the residue purified by silica gel chromatography (Hex:EtOAc 2:1 → Hex:EtOAc:CH2Cl2 4:3:1 → Hex:EtOAc:(CH2Cl2+5% of MeOH) 5:5:4) to give 6 (0.971 g, 92%) as slightly yellow oil that crystallizes on standing. 1H NMR: δ 7.93 (d, J = 8.4 Hz, 2 H), 6.94-6.86 (m, 4 H), 4.27 (t, J = 4.4 Hz, 2 H), 4.22 (t, J = 4.4 Hz, 2 H), 4.04 (t, J = 6.0 Hz, 2 H), 3.90 (t, J = 4.4 Hz, 2 H), 3.72 (t, J = 4.4 Hz, 2 H), 3.33 (d, J = 10.4 Hz, 2 H), 2.62 (d, J = 11.2 Hz, 2 H), 2.09 (s, 3 H), 1.80 (p, J = 7.2 Hz, 2 H), 1.52 (sxt, J = 7.6 Hz, 2 H), 1.00 (t, J = 7.2 Hz, 3 H); 13C NMR: 171.3, 162.1, 161.5, 153.5, 147.81, 147.78, 142.5, 135.8, 135.7, 116.7, 116.4, 116.36, 116.13, 112.32, 112.30, 69.43, 69.39, 68.0, 67.6, 63.5, 37.2, 31.1, 21.0, 19.2, 13.8.
BF4O(C2H5)3 (0.45 g, 2.38 mmol) was added to a solution of cyclopropenone 6 (0.97 g, 2.16 mmol) in CH2Cl2 (5 mL), and the resulting solution was stirred for 20 min at r.t. A solution of neopentyl glycol (0.27 g, 2.59 mmol) and Et3N (0.33 g, 3.24 mmol) in CH2Cl2 (1.5 mL) was added, the reaction mixture was stirred for 20 min, and the solvents were then removed under reduced pressure. The residue was purified by silica gel column chromatography (Hex:EtOAc 5:1+1.5% of Et3N → Hex:EtOAc 1:1+1.5% of Et3N → Hex:EtOAc:CH2Cl2 5:5:4+5% of MeOH and1.5% of Et3N) to provide 7 (0.593 g, 96% calculated on consumed substrate) as an oil, and unreacted cyclopropenone 6 (0.431 g, 0.96 mmol). 1H NMR: δ 7.65 (dd, J = 8.4, 2.4 Hz, 2 H), 6.92-6.82 (m, 4 H), 4.26 (t, J = 4.4 Hz, 2 H), 4.18 (t, J = 4.4 Hz, 2 H), 4.00 (t, J = 6.4 Hz, 2 H), 3.9a (m, 4 H), 3.88 (t, J = 4.4 Hz, 2 H), 3.78 (t, J = 4.4 Hz, 2 H), 3.24 (d, J = 10.4 Hz, 2 H), 2.41 (d, J = 11.2 Hz, 2 H), 2.08 (s, 3 H), 1.79 (p, J = 7.2 Hz, 2 H), 1.51 (sxt, J = 7.6 Hz, 2 H), 1.21 (s, 3 H), 1.19 (s, 3 H), 0.99 (t, J = 7.2 Hz, 3 H); 13C NMR: 171.1, 159.6, 159.0, 147.1, 131.5, 131.4, 124.2, 123.4, 119.5, 118.9, 116.05, 115.94, 111.97, 111.92, 83.9, 79.2, 69.6, 69.4, 63.5, 36.9, 31.3, 30.6, 22.62, 22.59, 21.0, 19.2, 13.9.
A solution of NaOH (1.2 mL, 1.2 mmol, 1 M aqueous solution) was added to 7 (0.593 g, 1.11 mmol) in a mixture of MeOH and THF (13 ml, 10/3, v/v) at r.t., and the reaction mixture was stirred for 30 min. The reaction mixture was partially concentrated under reduced pressure, diluted with EtOAc (~25 mL) and washed with water (~10 mL). The organic layer was separated, washed with brine, and dried (MgSO4), filtered and the filtrate concentrated under reduced pressure. The residue was purified by silica gel column chromatography (Hex:EtOAc:CH2Cl2 3:2:1+1.5% of Et3N) to provide 8 (0.493 g, 81%) as an oil that crystallized on standing. 1H NMR: δ 7.65 (dd, J = 8.4, 2.4 Hz, 2 H), 6.92-6.82 (m, 4 H), 4.18 (t, J = 4.4 Hz, 2 H), 4.04 (t, J = 6.4 Hz, 2 H), 3.92 (m, 4 H), 3.88 (t, J = 4.4 Hz, 2 H), 3.77 (t, J = 4.4 Hz, 2 H), 3.68 (t, J = 4.4 Hz, 2 H), 3.24 (d, J = 10.8 Hz, 2 H), 2.41 (d, J = 10.8 Hz, 2 H), 1.76 (p, J = 7.2 Hz, 2 H), 1.50 (sxt, J = 7.6 Hz, 2 H), 1.21 (s, 3 H), 1.19 (s, 3 H), 0.99 (t, J = 7.2 Hz, 3 H); 13C NMR: 159.8, 159.2, 147.4, 131.84, 131.75, 131.67, 131.57, 124.4, 123.6, 119.8, 119.1, 116.3, 116.2, 112.2, 84.1, 79.4, 72.8, 69.8, 68.0, 76.7, 62.0, 37.1, 31.5, 30.8, 22.9, 19.4, 14.2.
A solution of alcohol 8 (0.439 g, 0.89 mmol) and pyridine (0.25 g, 3.21 mmol) in CH2Cl2 (5 mL) was added to a solution of 4-nitrophenyl chloroformate (0.30 g, 1.49 mmol) in CH2Cl2 (25 mL) at r.t., and the reaction mixture was stirred for 20 min. Solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (Hex:EtOAc 4:1+1.5% of Et3N) to provide 9 (0.317 g, 80%) and starting material 8 (0.113 g, 0.23 mmol). 1H NMR: δ 8.25 (d, J = 8.8 Hz, 2 H) 7.65 (dd, J = 8.4, 2.0 Hz, 2 H), 7.35, (d, J =9.2, 2 H), 6.92-6.82 (m, 4 H), 4.43 (t, J = 4.4 Hz, 2 H), 4.19 (t, J = 6.4 Hz, 2 H), 3.98 (t, J = 4.4 Hz, 2 H), 3.92 (m, 7 H), 3.22 (d, J = 10.8 Hz, 2 H), 2.43 (d, J = 10.8 Hz, 2 H), 1.75 (p, J = 7.2 Hz, 2 H), 1.51 (sxt, J = 7.6 Hz, 2 H), 1.21 (s, 3 H), 1.19 (s, 3 H), 0.98 (t, J = 7.2 Hz, 3 H); 13C NMR: 159.9, 159.2, 155.7, 152.7, 150.0 147.4, 145.6, 131.77, 131.63, 125.5, 124.6, 123.4, 122.0, 119.8, 119.1, 116.25, 116.19, 112.2, 112, 15, 84.1, 79.4, 70.0, 69.1, 68.4, 70.0, 67.8, 37.1, 31.5, 30.8, 22.87, 22.79, 19.5, 14.1.
A solution of cyclopropenone acetal 9 (0.21 g, 0.312 mmol) in DMF (2 mL) was added to a solution of Et3N (0.18 g, 1.75 mmol) and N-biotinyl-3,6-dioxaoctane-1,8-diamine9 (0.13 g, 0.35 mmol) in DMF (35 mL) at r.t. The reaction mixture was stirred for 18 h and then most of the solvent was evaporated under reduced pressure. The residue was passed through a short column of silica gel (CH2Cl2:MeOH 25:1+1.5% of Et3N) to provide crude 10 (0.275 g) that was used in the next step without further purification. A suspension of crude cyclopropenone acetal 10 (0.199 g) and Amberlyst 15 (0.10 g) in Me2CO (10 mL) was stirred for 60 min at r.t. Solids were removed by filtration, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (CH2Cl2:MeOH, 10:1) to provide cyclopropenone 2b (17 mg) as an amorphous solid. 1H NMR: δ 7.65 (dd, J = 8.4, 3.0 Hz, 2 H), 6.93-6.87 (m, 4 H), 6.66 (s, b, 1 H), 6.25, (s, b, 1 H) 5.61 (m, b, 1 H) 5.39 (s, b, 1 H) 4.48 (m, b, 1 H), 4.30-4.24 (m, 4 H), 4.21 (t, J = 5.0 Hz, 2 H), 4.05 (t, J = 7.5 Hz, 2 H), 3.88 (t, J = 5.5 Hz, 2 H), 3.78 (m, 2 H), 3.60 (s, 4 H), 3.44 (q, J = 6.5 Hz, 2 H), 3.40-3.30 (m, 4 H), 3.18-3.1 (m, 3 H), 2.27 (dd, J = 16.0, 6.0 Hz, 1 H), 2.73 (d, J = 16.0 Hz, 1 H), 2.62 (d, J = 14.0 Hz, 2 H), 2.20 (t, J = 9.0 Hz, 2 H), 2.19-2.02 (m, 4 H), 1.81 (p, J = 8.5 Hz, 2 H), 1.74-1.60 (m, 4 H), 1.51 (six, J = 9.0 Hz, 2 H), 1.46-1.4 (m, 2 H), 1.36 (t, J = 9 Hz, 2 H), 1.00 (t, J = 9.5 Hz, 3 H); 13C NMR: 173.4, 163.8, 162.2, 161.5, 156.5, 153.8, 147.86, 147.83, 142.5, 141.9, 135.85, 135.76, 116.71, 116.4, 116.28, 116.14, 112.39, 112.34, 70.13, 70.07, 69.99, 69.88, 69.4, 68.0, 67.7, 63.9, 62.8, 60.2, 55.5, 45.8, 40.8, 40.5, 39.1, 37.20, 37.15, 35.8, 31.1, 28.13, 28.07, 25.5, 19.2, 13.8, 8.6; MS calc for C41H56N4O9S (M+-CO+Na) 803.3666, ESI-HRMS found 803.3677.
A solution of cyclopropenone 6 (0.54 g, 1.35 mmol) in MeOH:THF (1:1, v:v, 60 mL) was irradiated with 350 nm lamps for 20 min. The solution was concentrated under reduced pressure to 10 mL, and 1 M aqueous NaOH solution (1.68 mL, 1.68 mmol) was added to the mixture and stirring was continued for 30 min. Ethyl acetate was added, and the organic layer was separated, washed with water, brine, dried (MgSO4), filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc:Hex 1:1.5) to provide 12 (0.375 g, 73%) as an amorphous white solid. 1H NMR: δ 7.20 (dd, J = 8.4, 0.8 Hz, 2 H), 6.87 (dd, J = 11.2 Hz, 2.0, 2 H), 6.75 (td, J = 8.0, 2.4 Hz, 2 H), 4.15 (t, J = 4.4 Hz, 2 H), 3.97 (t, J = 6.0 Hz, 2 H), 3.87 (t, J = 4.4 Hz, 2 H), 3.76 (s, b, 2 H), 3.68 (d, J = 4.4 Hz, 2 H), 3.17 (d, J = 11.2 Hz, 2 H), 2.43 (d, J = 10.4 Hz, 2 H), 1.77 (p, J = 7.2 Hz, 2 H), 1.50 (six, J = 7.2 Hz, 4 H), 0.98 (t, J = 7.2 Hz, 6 H); 13C NMR: 158.9, 158.3, 155.1, 126.99, 126.84, 117.05, 116.93, 116.10, 112.08, 112.05, 110.91, 110.39, 72.8, 69.8, 68.0, 67.7, 62.0, 36.94, 36.77, 31.5, 19.5, 14.1, 14.01. MS calc for C24H28O4 (M+) 380.1988, EI-HRMS found 380.1982.
A solution of pyridine (0.20 g, 2.60 mmol) in CH2Cl2 (~1 mL) was added to 12 (0.24 g, 0.63 mmol) and 4-nitrophenyl chloroformate (0.20 g, 1.00 mmol) in CH2Cl2 (5 mL), and the reaction mixture was stirred for 3 h. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (Hex:EtOAc 4:1) to provide 13 (0.34 g, 99%) as an oil. 1H NMR: δ 8.25 (d, J = 8.8 Hz, 2 H), 7.36 (d, J = 9.2 Hz, 2 H), 7.19 (d, J = 8.8 Hz, 2 H), 6.89 (dd, J = 14.0, 2.4 Hz, 2 H), 6.79-6.75 (m, 2 H), 4.47 (t, J = 4.4 Hz, 2 H), 4.18 (t, J = 4.4 Hz, 2 H), 3.97 (t, J = 6.6 Hz, 2 H), 3.92-3.88 (m, 4 H), 3.17 (d, J = 10.8 Hz, 2 H), 2.42 (d, J = 10.8 Hz, 2 H), 1.77 (p, J = 7.2 Hz, 2 H), 1.49 (six, J = 7.2 Hz, 4 H), 0.98 (t, J = 7.2 Hz, 6 H); 13C NMR: 158.9, 158.3, 155.7, 155.13, 155.08, 152.7, 145.6, 127.0, 126.9, 112.1, 121.9, 117.0, 116.97, 116.94, 112.15, 112.11, 112.00, 111.0, 110.3, 70.1, 69.1, 68.5, 68.0, 67.8, 36.9, 36.7, 31.5, 19.5, 14.2, 14.0.
A solution of carbonate 13 (0.15 g, 0.28 mmol) in DMF (2 mL) was added to a solution of Et3N (0.5 g, 4.95 mmol) and N-biotinyl-3,6-dioxaoctane-1,8-diamine9 (0.01g, 0.28 mmol) in DMF (10 mL) The reaction mixture was stirred for 18 h at ambient temperature and then the solvents were evaporated under reduced pressure, and the residue purified by silica gel chromatography (CH2Cl2:MeOH 30:1) to provide 3b (0.164 g, 75%). 1H NMR: 7.19 (d, J = 8.4 Hz, 2H), 6.88 (dd, J = 9.5, 2.5 Hz, 2H), 6.76 (td, J = 8.2, 2.5, 2H), 6.74 – 6.65 (m, 1H), 6.54 (s, b, 1H), 5.74 (s, b, 1H), 5.60 (s, b 1 H), 4.49 – 4.43 (m, 1H), 4.29-4.22 (m, 3H), 4.16 – 4.10 (m, 2H), 3.97 (t, J = 6.5, 2H), 3.87 – 3.81 (m, 2H), 3.76 (m, 2H), 3.59-3.48 (m, 10H), 3.42 (m, 2H), 3.37 – 3.12 (m, 2H), 3.21 – 3.09 (m, 4H), 2.86 (dd, J = 12.6, 4.7 Hz, 1H), 2.72 (d, J = 12.7, 1H), 2.42 (d, J = 10.9, 2H), 2.21 (t, J = 7.4, 4H), 1.81-1.56 (m, 6H), 1.48 (six, J = 7.4 Hz, 2H), 1.44-1.36 (m, 2H), 1.32 (t, J = 7.4 Hz, 1H), 0.98 (t, J = 7.4, 3H); 13C NMR: 173.4, 164.1, 158.7, 158.1, 156.5, 154.8, 126.66, 126.63, 116.80, 116.72, 116.59, 115.8, 111.91, 111.83, 110.67, 110.14, 70.09, 70.04, 69.95, 69.90, 69.80, 69.54, 67.78, 67.52, 63.88, 61.80, 60.2, 55.6, 45.6, 40.8, 40.5, 39.1, 36.63, 36.61, 35.9, 31.3, 28.22, 28.08, 25.6, 19.2, 13.8, 8.5. MS calc for C41H56N4O9S (M+ +Na) 803.3666, ESI-HRMS found 803.3672.
A solution of cyclopropenone 2c (0.20 g, 0.532 mmol) in MeOH (20 mL, 2.72×10−2M) was irradiated (4 × 350 nm) for 20 min at r.t. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (Hex:EtOAc 1:20) to provide 3c (0.160 g, 86%) as slightly yellow oil. 1H NMR: 7.19 (d, J = 8.4 Hz, 2 H), 6.87 (d, J = 2.4 Hz, 2 H), 6.75 (dd, J = 8.4, 2.4 Hz, 2 H), 3.97 (t, J = 6.4 Hz, 4 H), 3.18 (d, J = 11.2 Hz, 2 H), 2.44 (d, J = 11.2 Hz, 2 H), 1.77 (p, J = 7.2 Hz, 4 H), 1.52 (six, J = 7.2 Hz, 4 H), 0.98 (t, J = 7.2 Hz, 6 H); 13C NMR: 158.9, 155.1, 126.9, 116.9, 116.2, 112.0, 110.6, 68.0, 36.9, 31.5, 19.5, 14.1.
A solution of 3c (0.5 mmol) and appropriate organic azide (0.75 mmol) in MeOH was stirred for 18 h at r.t. The solvent was evaporated under reduced pressure, and the excess of azide was removed by silica gel column chromatography.
1H NMR: δ 7.53 (d, J = 8.8 Hz, 1 H), 7.39 (s, 5 H), 6.85 (d, J = 2.4 Hz, 1 H), 6.79 (dd, J = 8.4, 2.4 Hz, 1 H), 6.74 (d, J = 2.4 Hz, 1 H), 6.62 (d, J = 8.8 Hz, 1 H), 6.51 (dd, J = 8.4, 2.8 Hz, 1 H), 3.94 (t, J = 6.4 Hz, 2 H), 3.89 (t, J = 6.4 Hz, 2 H), 3.50-3.30 (m, 2 H), 3.17-2.92 (m, 2 H), 1.78-1.68 (m, 4 H), 1.46 (sep, J = 7.2 Hz, 4 H), 0.96 (t, J = 7.2 Hz, 3 H), 0.95 (t, J = 7.2 Hz, 3 H); 13C NMR: 159.9, 159.2, 147.0, 142.5, 139.7, 137.0, 133.6, 133.0, 131.8, 129.5, 128.8, 124.8, 122.5, 118.8, 116.5115.8, 112.8, 112.6, 67.81, 67.77, 36.2, 34.2, 31.5, 19.47, 19.45, 14.10, 14.07.
1H NMR: δ 7.43 (d, J = 8.4 Hz, 1 H), 7.06 (d, J = 8.4 Hz, 1 H), 6.87 (d, J = 2.4 Hz, 1 H), 6.78 (dd, J = 8.4, 2.4 Hz, 1 H), 6.75 (dd, J = 8.4, 2.4 Hz, 1 H) 6.67 (d, J = 2.4 Hz, 1 H), 4.42-4.24 (m, 2 H), 3.96 (t, J = 6.4 Hz, 2 H), 3.93 (t, J = 6.8 Hz, 2 H), 3.40-3.32 (m, 1 H), 3.14-2.98 (m, 2 H), 2.88-2.78 (m, 1 H), 1.86-1.68 (m, 6 H), 1.54-1.41 (m, 4 H), 1.34-1.18 (m, 2 H), 0.98 (t, J = 7.6 Hz, 3 H), 0.95 (t, J = 7.2 Hz, 3 H), 0.85 (t, J = 7.2 Hz, 3 H); 13C NMR: 160.1, 158.9, 146.6, 143.3, 139.2, 133.6, 133.2, 130.2, 122.8, 118.9, 116.6, 115.9, 112.9, 112.567.9, 67.7, 48.2, 36.9, 33.4, 32.3, 31.55, 31.50, 19.8, 19.5, 14.1, 13.7.
Synthetic compounds 1b, 2b, and 3b were reconstituted in DMF and stored at −80°C. Final concentrations of DMF never exceeded 0.56% to avoid toxic effects. The in situ photo-activation of biotinylated cyclopropenone 2b was performed using a mini-Rayonet® photoreactor equipped with two 350 nm florescent tubes (4W). The irradiated cell suspensions were kept in plastic vials, which served as an additional short band-path filter. The vial wall absorbs ca. 60% of light at 350 nm, 70% at 300 nm, and is virtually not transparent below 275 nm.
Human Jurkat cells (Clone E6-1; ATCC) were cultured in RPMI 1640 medium (ATCC) with L-glutamine (2 mM), adjusted to contain sodium bicarbonate (1.5 g/L), glucose (4.5 g/L), HEPES (10 mM), and sodium pyruvate (1 mM) and supplemented with penicillin (100 u/ml)/streptomycin (100 μg/mL; Mediatech) and fetal bovine serum (FBS, 10%; Hyclone). Chinese hamster ovary (CHO) cells (Clone K1; ATCC) were cultured in Kaighn’s modification of Ham’s F-12 medium (F-12K) with L-glutamine (2 mM), adjusted to contain sodium bicarbonate (1.5 g L−1) and supplemented with penicillin (100 u mL−1)/streptomycin (100 μg mL−1) and FBS (10%). Cells were maintained in a humid 5% CO2 atmosphere at 37°C.
Jurkat cells were seeded at a density of 75,000 cells mL−1 in a total volume of 40 mL culture medium in the presence of peracetylated N-azidoacetylmannosamine (Ac4ManNaz; 25 μM final concentration) and grown for 3 days, leading to the metabolic incorporation of the corresponding N-azidoacetyl sialic acid (SiaNAz) into their cell surface glycoproteins. Control cells were grown in the presence of peracetylated N-acetylmannosamine (Ac4ManNac; 25 μM final concentration) for 3 days. Similarly, CHO cells were grown for 3 days in the presence of Ac4ManNaz (100 μM final concentration) or Ac4ManNac (100 μM final concentration).
Jurkat cells bearing azides and control cells were washed with labeling buffer (DPBS, pH 7.4 containing 1% FBS and 1% BSA), transferred to round bottom tubes (1 ×106 cells/sample) and incubated with the biotinylated compounds 1b, 2b, or 3b (0–100 μM) in labeling buffer for 0–90 min at r.t. To activate 2b in situ, the cell suspension was subjected to UV light (350 nm) for 1 min immediately after adding the compound to the cells, unless stated otherwise. The cells were washed three times with cold labeling buffer and then incubated with avidin conjugated with fluorescein (0.5 μg/ml; Molecular Probes) for 15 min at 4°C. Following three washes, cells were either lysed in passive lysis buffer (Promega) and cell lysates were analysed for fluorescence intensity (485 ex/520 em) using a microplate reader (BMG Labtech) or live cells were assessed by flow cytometry using the FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems) and data analysis was performed with FlowJo software (Tree Star, Inc.). Data points were collected in triplicate and are representative of three separate experiments. Fluorescence of Jurkat cell lysates was expressed as fluorescence (arbitrary units; AU) per 800,000 cells.
Cell viability and cell morphology were assessed by exclusion of trypan blue and microscopic evaluation immediately after photoactivation or after reincubation of the labeled cells in cell culture medium for 5 or 24 h. After the reincubation, viability was measured by quantifying the cellular ability to reduce the water-soluble tetrazolium dye 3–4,5-dimethylthiazole-2,5-diphenyl tetrazolium bromide (MTT) to its insoluble formazan salt.25 Data points were collected in triplicate and expressed as normalized values for control cells (100%).
Jurkat cells were harvested by centrifugation (5 min at 500 × g) and resuspended as 5 ×106 cells/mL. The cell suspensions (200 μL per sample) were incubated with biotin-conjugated alkynes 1b, 2b, and 3b (30 μM) or without compound as control for 1 h. To activate 2b in situ, immediately after adding the compound to the cells, the cell suspension was subjected to UV light (350 nm) for 1 min. The cells were washed (4 × 10 min) with cold DPBS, pH 7.4 containing FBS (1%) and lysed in passive lysis buffer. The cell lysates were clarified by centrifugation at 22,000 × g for 15 min and the total protein content of the clear supernatants was assessed using the bicinchonic acid assay (BCA; Pierce Biotechnology). Cell lysate samples (20 μg protein) in SDS-PAGE sample buffer containing 2-mercaptoethanol were boiled for 5 min, resolved on a 4–20% Tris-HCl gel (Bio-Rad) and transferred to nitrocellulose membrane. Next the membrane was blocked in blocking buffer (non-fat dry milk (5%; Bio-Rad) in PBST (PBS containing 0.1% Tween-20 and 0.1% Triton X-100)) for 2 h at r.t. The blocked membrane was incubated for 1 h at r.t. with an anti-biotin antibody conjugated to horseradish peroxidase (HRP) (1:100,000; Jackson ImmunoResearch Lab, Inc.) in blocking buffer and washed with PBST (4 × 10 min). Final detection of HRP activity was performed using ECL Plus chemiluminescent substrate (Amersham™), exposure to film (Kodak) and development using a digital X-ray imaging machine (Kodak). Next the blot was stripped and reprobed for loading control (β-actin) as described above. Coomassie staining was used to confirm total protein loading.
CHO cells bearing azides and untreated control cells were transferred to glass coverslips and cultured for 36 h in their original medium. Live CHO cells were treated with the biotinylated compound 2b (30 μM) in labeling buffer (DPBS, supplemented with FBS (1%)) for 1 h at r.t. To activate 2b in situ, immediately after adding the compound to the cells, the cells were subjected to UV light (350 nm) for 1 min. Next, the cells were incubated with avidin conjugated with Alexa Fluor 488 (Molecular Probes) for 15 min at 4°C. Cells were washes 3 times with labeling buffer and fixed with formaldehyde (3.7% in PBS). The nucleus was labeled with the far red-fluorescent TO-PRO-3 dye (Molecular Probes). The cells were mounted with PermaFluor (Thermo Electron Corporation) before imaging. Initial analysis was performed on a Zeiss Axioplan2 fluorescent microscope. Confocal images were acquired using a 60X (NA1.42) oil objective. Stacks of optical sections were collected in the z dimensions. The step size, based on the calculated optimum for each objective, was between 0.25 and 0.5 μm. Subsequently, each stack was collapsed into a single image (z-projection). Analysis was performed offline using ImageJ 1.39f software (National Institutes of Health, USA) and Adobe Photoshop CS3 Extended Version 10.0 (Adobe Systems Incorporated), whereby all images were treated equally.
Statistical significance between groups was determined by two-tailed, unpaired Student’s t test. Differences were considered significant when P<0.05.
This research was supported by National Science Foundation (Grant No. CHE-0449478, V.V.P.), Georgia Cancer Coalition (V.V.P.), the National Cancer Institute of the US National Institutes of Health (Grant No. RO1 CA88986, G.-J.B.), and the National Institute of General Medical Sciences of the US National Institutes of Health (Grant No. R01 GM61761, G.-J.B.).