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Stabilized Wittig olefination for bioconjugation† Cite this: Chem. Commun., 2013, 49, 11188
Kenneth M. Lum, Vanessa J. Xavier, Michelle J.-H. Ong, Charles W. Johannes and Kok-Ping Chan*
Received 5th August 2013, Accepted 11th October 2013 DOI: 10.1039/c3cc45961f www.rsc.org/chemcomm
Stabilized Wittig olefination holds great potential as a bioconjugation reaction. We demonstrate that the reaction of stabilized phosphorus ylides (or phosphonium salts) with aryl aldehydes is sufficiently robust to be used for live cell affinity isolation and fluorescence tagging of a protein, FKBP12.
Bioconjugation reactions are indispensable for studying biology.1 Current gold standards in the field have been developed from decades-old name reactions: [3+2] azide–alkyne ‘‘Click’’ reaction (Huisgen condensation),1b,2 tetrazine–cycloocetene [4+2] addition (Diels–Alder cycloaddition),3 and azide–ester Staudinger ligation (Staudinger reaction, mediated by phosphine).4 Advances in these strategies such as the strain-promoted azide–alkyne condensation5 have obviated harsh conditions (e.g. high temperatures, cytotoxic metal catalysts) and enabled tremendous progress in understanding native cellular processes. Unsurprisingly, chemists are actively investigating a variety of other reagent pairs,6 including aldehydes,7 as chemical reporters in biological systems. These efforts underscore the increasing demand for new strategies to do chemistry in vivo. Despite such efforts, the Wittig reaction8 has not been considered as a bioconjugation strategy. However, recent evidence has shown that the reaction of stabilized phosphorus ylides with aldehydes not only tolerates water but also benefits from a rate enhancement in aqueous media.9 Therefore, we envisaged that we might exploit stabilized Wittig olefination to modify aldehydes in biological matrices. Partway into our investigation, we were gratified to learn that others too had begun to consider stabilized Wittig olefination, utilising it to modify proteins in aqueous–organic mixtures;10 this report provided strong support for our efforts in developing this methodology for bioconjugation. We describe herein a strategy for tagging proteins that is based on Wittig olefination of a functionalized phosphoranylidene or a phosphonium salt probe with an aryl aldehyde bait. The only Division of Organic Chemistry, Institute of Chemical and Engineering Sciences, Agency for Science Technology and Research Singapore, 11 Biopolis Way, Helios Block #03-08, Singapore. E-mail: [email protected]; Fax: +65 6874 5870; Tel: +65 6799 8599 † Electronic supplementary information (ESI) available: Full experimental details and compound characterization. See DOI: 10.1039/c3cc45961f
11188
Chem. Commun., 2013, 49, 11188--11190
byproduct of the reaction is triphenylphosphine oxide, which is also produced in the well-explored Staudinger ligation.4 Although these coupling partners are not bioorthogonal in the strictest sense,11 we show that they are sufficiently robust for protein affinity isolation and fluorescence labeling in live cells. From the outset, we recognized the inherent challenge of using an electrophile–nucleophile pair to do chemistry in biological matrices. Aldehydes are increasingly being recognized as mild ‘‘bioorthogonal’’ electrophiles,7a although they are well-known to form reversible adducts with cellular nucleophiles.11,12 Likewise, stabilized phosphorus ylides are moderate nucleophiles, but little is known about their reactivity with biologically-relevant electrophiles. Collectively, these challenges have perhaps undermined the potential of Wittig olefination as a bioconjugation strategy. Notwithstanding, we hoped to leverage on (1) the kinetic preference of stabilized phosphoranylidenes for aldehydes, (2) the irreversibility of the Wittig reaction due to the formation of strong PQO and CQC bonds, and (3) the use of an aryl aldehyde coupling partner such that extensive p-conjugation in the final cinnamate adduct would provide an additional driving force for preferential conjugation with the phosphoranylidene (Scheme 1). Indeed, our preliminary studies (see ESI†) revealed that stabilized phosphorus ylides readily undergo Wittig olefination with aldehydes in common culture media and cell lysis buffers. The reaction of methyl 2-triphenylphosphoranylideneacetate with benzaldehyde in various aqueous buffers afforded the corresponding (E)-cinnamate adduct stereoselectively (approximate E/Z = 9 : 1), with excellent
Scheme 1 Mechanistic rationale behind the development of stabilized Wittig olefination for bioconjugation.
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The Royal Society of Chemistry 2013
View Article Online
Communication
ChemComm Table 1
Wittig-mediated bioconjugation of aldehydes in Tris buffer
Published on 14 October 2013. Downloaded by UNIVERSIDAD DE GIRONA on 28/10/2014 15:35:52.
Aldehyde
7 Scheme 2 Synthesis of Wittig probes. (a) BrCH2COBr, Et3N, CH2Cl2. (b) PPh3, PhMe. (c) 2 M NaOH–CH2Cl2 (1 : 1). (d) (D)-biotin, K2CO3, DMF, 60 1C. (e) 1 M HCl. (f) N-dansyl6-aminohexanoic acid, K2CO3, DMF, 60 1C. (g) (D)-biotin, K2CO3, DMSO, 50 1C, then 1 M HCl. (h) N-dansyl-3-aminopropanoic acid, K2CO3, DMF, 60 1C, then 1 M HCl.
chemoselectivity towards aldehydes and no observable reaction with simple ketones. In rate studies of stabilized Wittig olefination in Tris buffer, we found that the second order rate constant at 4 1C was comparable to that of the mechanistically-related Staudinger ligation (see ESI†). Encouraged by these results, we developed a rapid synthesis to access probes bearing a stabilized phosphoranylidene joined to a functional tag by a nonyl linker (Scheme 2A). These phosphoranylidene probes underwent Wittig olefination smoothly with aldehydes of varying structural complexity, in Tris buffer at 4 1C (Table 1). Leveraging on this synthetic approach, we developed a second generation of probes linked by tetraethylene glycol (Scheme 2B), which we hoped would improve probe uptake for cellular bioconjugation.13 A similar synthetic route starting from mono-tosylated tetraethylene glycol allowed quick access to biotin probe 13 and dansyl probe 14, analogues of 3a and 4 respectively (Scheme 3). Having established the effectiveness of our probes in buffered media, we turned our attention to demonstrating proof of concept in biological matrices. We conducted streptavidin-mediated affinity isolation on cell lysates treated with bait molecule 7 followed by phosphoranylidene probe 3. Western blot analysis revealed bands at 12 kDa indicating successful isolation of FKBP12 through Wittig conjugation of 3 and 7 in vitro. The intensity of the FKBP12 band increased in a dose dependent manner with probe concentration at fixed bait concentration (Fig. 1A). Likewise, band intensity also increased with bait concentration when probe concentration was fixed, appearing to saturate at bait concentrations exceeding 0.5 mM (Fig. 1B). Phosphonium probe 3a was likewise effective (Fig. 1C, lane 2). Neither 3 nor 3a would effect a significant level of pulldown with unmodified FK506 that lacked aldehyde functionality (Fig. 1C, This journal is
c
The Royal Society of Chemistry 2013
Phosphoranylidene probe
Product
Yield (%)
3
78
3
94
3
73
4
76
FK506 is a small-molecule macrolide with high affinity for FKBP12. The aryl aldehyde analog 7 is designed to demonstrate Wittig olefination in biological systems (see ESI for respective structures).
Scheme 3 Synthesis of dansyl probe 17 bearing an ethylenediamine linker. (a) BrCH2COBr, Et3N, CH2Cl2. (b) PPh3, PhMe.
lanes 3 and 4), confirming that the probes react preferentially with aldehydes over ketones. With positive results from the affinity isolation of FKBP12 in cell lysates, we investigated if the reaction would tolerate cellular matrices. HeLa cells were incubated with bait 7 for 8 hours, followed by probe 3 or 13 in fresh media for 15 hours. We were delighted to find that both probes 3 and 13 were able to isolate FKBP12, indicating successful Wittig olefination within the cells (Fig. 1D). The relative amount of FKBP12 isolated with 13 (C log P = 4.7 of ylide) was higher than that of 3 (C log P = 8.2),‡ consistent with the notion that the linkers alter solubility and membrane permeability. Interestingly, the relative amount of FKBP12 pulled down with either probe was less than that obtained with pre-conjugated adduct 8, suggesting that further optimization is required to improve the bioconjugation yields in live cells (see ESI†). Nevertheless, successful pulldown of FKBP12 from the complex environment of a living cell indicates that stabilized Wittig olefination is indeed a viable bioconjugation strategy. Concurrently, we were interested to show that stabilized Wittig olefination might be used for fluorescently labeling proteins in live cells. To this end, we conducted imaging experiments with bait 7 and dansyl probes 4 and 14 to explore if we could fluorescently label FKBP12 in live cells. HeLa cells were dosed with either bait 7 or DMSO as a control, followed by Chem. Commun., 2013, 49, 11188--11190
11189
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Fig. 1 Affinity isolation of FKBP12. Titration experiments with (A) increasing probe concentration and (B) increasing bait concentration. The intensity of the FKBP12 band increased in a dose-dependent manner in these experiments. (C) Comparison of phosphoranylidene probe 3 and phosphonium probe 3a. Both probes react with bait 7, but not unmodified FK506. (D) Pulldown of FKBP12 from live HeLa cells using probes 3 and 13.
the respective dansyl probe in a similar manner to the affinity isolation assays. We hypothesized that cells treated with both bait 7 and a dansyl probe would be more fluorescent than cells treated with a probe alone: the irreversible reaction of 7 with the probe traps the fluorophore in the cell as the dansylatedFK506–FKBP12 complex, consequently shifting the equilibrium to favor the diffusion of more probe into the cell. Unfortunately, dansyl probes 4 and 14 were unsuitable for this purpose, since cells treated with bait 7 and these probes displayed only weak fluorescence (see Fig. 2A and B). These findings prompted us to further synthesize dansyl probe 17 (Scheme 3), which bears an ethylenediamine linker, in the hope that (1) a shorter linker would decrease non-radiative modes of relaxation and lead to a stronger fluorescence signal, and (2) the solubility properties associated with this linker would further improve membrane permeability of the probe. Satisfyingly, cells treated with both 7 and 17 (Fig. 2D) showed a marked increase in fluorescence intensity compared to cells treated with 17 alone (Fig. 2C). Taken together with the pulldown results, this observation provides further evidence of stabilized Wittig olefination in live cells, and alludes to its capacity for other imaging applications.
Fig. 2 Differential Interference Contrast (DIC) and fluorescence images of live HeLa cells treated with (A) bait 7 and probe 4 only (B) bait 7 and probe 14 (C) probe 17 only, and (D) bait 7 and probe 17. The scale bar represents 25 mm. The concentrations of bait and probe were 1 and 5 mM respectively.
11190
Chem. Commun., 2013, 49, 11188--11190
Communication Given the various genetic7a,14 and chemical methods for incorporating aldehyde reporters into cells, we anticipate that stabilized Wittig olefination will find increased use in chemical biology, both on its own and possibly in tandem with other bioorthogonal reactions. Towards this goal, we have demonstrated that stabilized Wittig olefination possesses desirable attributes for bioconjugation: (1) the reacting functionalities are sufficiently stable and chemoselective in complex environments, and (2) when installed on appropriately tuned scaffolds, the resulting reagents can be used to biotinylate or fluorescently-label target proteins in live cells. This research was supported by A*STAR ICES (Project code ICES/11-240B08). We thank Michelle Su (Singapore BioImaging Corsortium) for her assistance in the cell biology experiments and Graham Wright (Institute of Medical Biology, Microscopy Unit) for his invaluable microscopy advice.
Notes and references ‡ C log P values were calculated using ChemBioDraw Ultra. 1 (a) N. Stephanopoulos and M. B. Francis, Nat. Chem. Biol., 2011, 7, 876; (b) K. Nwe and M. W. Brechbiel, Cancer Biother.Radiopharm., 2009, 24, 289; (c) J. A. Prescher and C. R. Bertozzi, Nat. Chem. Biol., 2005, 1, 13. 2 (a) E. Lallana, R. Riguera and E. Fernandez-Megia, Angew. Chem., Int. Ed., 2011, 50, 8794; (b) C. Besanceney-Webler, H. Jiang, T. Zheng, L. Feng, D. Soriano del Amo, W. Wang, L. M. Klivansky, F. L. Marlow, Y. Liu and P. Wu, Angew. Chem., Int. Ed., 2011, 50, 8051; (c) V. Hong, S. I. Presolski, C. Ma and M. G. Finn, Angew. Chem., Int. Ed., 2009, 48, 9879; (d) C. D. Hein, X. M. Liu and D. Wang, Pharm. Res., 2008, 25, 2216. 3 (a) T. Reiner, E. J. Keliher, S. Earley, B. Marinelli and R. Weissleder, Angew. Chem., Int. Ed., 2011, 50, 1922; (b) N. K. Devaraj and R. Weissleder, Acc. Chem. Res., 2011, 44, 816; (c) M. F. Debets, S. S. van Berkel, J. Dommerholt, A. J. Dirks, F. P. J. T. Rutjes and F. L. van Delft, Acc. Chem. Res., 2011, 44, 805. 4 (a) S. S. van Berkel, M. B. van Eldijk and J. van Hest, Angew. Chem., Int. Ed., 2011, 50, 8806; (b) F. L. Lin, H. M. Hoyt, H. van Halbeek, R. G. Bergman and C. R. Bertozzi, J. Am. Chem. Soc., 2005, 127, 2686; ¨hn and R. Breinbauer, Angew. Chem., Int. Ed., 2004, 43, 3106. (c) M. Ko 5 (a) J. van Hest and F. L. van Delft, ChemBioChem, 2011, 12, 1309; (b) E. M. Sletten and C. R. Bertozzi, Acc. Chem. Res., 2011, 44, 666; (c) D. C. Kennedy, C. S. McKay, M. C. B. Legault, D. C. Danielson, J. A. Blake, A. F. Pegoraro, A. Stolow, Z. Mester and J. P. Pezacki, J. Am. Chem. Soc., 2011, 133, 17993; (d) P. V. Chang, J. A. Prescher, E. M. Sletten, J. M. Baskin, I. A. Miller, N. J. Agard, A. Lo and C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 1821; (e) S. S. van Berkel, A. T. J. Dirks, M. F. Debets, F. L. van Delft, J. J. L. M. Cornelissen, R. J. M. Nolte and F. P. J. T. Rutjes, ChemBioChem, 2007, 8, 1504. 6 (a) D. M. Patterson, L. A. Nazarova, B. Xie, D. N. Kamber and J. A. Prescher, J. Am. Chem. Soc., 2012, 134, 18638; (b) R. L. Simmons, R. T. Yu and A. G. Myers, J. Am. Chem. Soc., 2011, 133, 15870. 7 (a) P. Agarwal, J. van der Weijden, E. M. Sletten, D. Rabuka and C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 46; (b) D. Rabuka, J. S. Rush, G. W deHart, P. Wu and C. R. Bertozzi, Nat. Protocols, 2012, 7, 1052. 8 (a) G. Wittig and W. Haag, Chem. Ber., 1955, 88, 1654; (b) G. Wittig ¨llkopf, Chem. Ber., 1954, 87, 1318. and U. Scho 9 A. El-Batta, C. Jiang, W. Zhao, R. Anness, A. L. Cooksy and M. Bergdahl, J. Org. Chem., 2007, 72, 5244. 10 M.-J. Han, D.-C. Xiong and X.-S. Ye, Chem. Commun., 2012, 48, 11079. 11 E. M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed., 2009, 48, 6974. 12 W. P. Jencks, in Progress in Physical Organic Chemistry, ed. S. G. Cohen, A. Streitwieser, Jr. and R. W. Taft, John Wiley & Sons, Inc., 1964, pp. 63–128. 13 (a) N. V. Katre, Adv. Drug Delivery Rev., 1993, 10, 91; (b) F. M. Veronese and G. Pasut, Drug Discovery Today, 2005, 10, 1451. 14 J. Xie and P. G. Schultz, Curr. Opin. Chem. Biol., 2005, 9, 548.
This journal is
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Published on 14 October 2013. Downloaded by UNIVERSIDAD DE GIRONA on 28/10/2014 15:35:52.
COMMUNICATION
View Journal | View Issue
Stabilized Wittig olefination for bioconjugation† Cite this: Chem. Commun., 2013, 49, 11188
Kenneth M. Lum, Vanessa J. Xavier, Michelle J.-H. Ong, Charles W. Johannes and Kok-Ping Chan*
Received 5th August 2013, Accepted 11th October 2013 DOI: 10.1039/c3cc45961f www.rsc.org/chemcomm
Stabilized Wittig olefination holds great potential as a bioconjugation reaction. We demonstrate that the reaction of stabilized phosphorus ylides (or phosphonium salts) with aryl aldehydes is sufficiently robust to be used for live cell affinity isolation and fluorescence tagging of a protein, FKBP12.
Bioconjugation reactions are indispensable for studying biology.1 Current gold standards in the field have been developed from decades-old name reactions: [3+2] azide–alkyne ‘‘Click’’ reaction (Huisgen condensation),1b,2 tetrazine–cycloocetene [4+2] addition (Diels–Alder cycloaddition),3 and azide–ester Staudinger ligation (Staudinger reaction, mediated by phosphine).4 Advances in these strategies such as the strain-promoted azide–alkyne condensation5 have obviated harsh conditions (e.g. high temperatures, cytotoxic metal catalysts) and enabled tremendous progress in understanding native cellular processes. Unsurprisingly, chemists are actively investigating a variety of other reagent pairs,6 including aldehydes,7 as chemical reporters in biological systems. These efforts underscore the increasing demand for new strategies to do chemistry in vivo. Despite such efforts, the Wittig reaction8 has not been considered as a bioconjugation strategy. However, recent evidence has shown that the reaction of stabilized phosphorus ylides with aldehydes not only tolerates water but also benefits from a rate enhancement in aqueous media.9 Therefore, we envisaged that we might exploit stabilized Wittig olefination to modify aldehydes in biological matrices. Partway into our investigation, we were gratified to learn that others too had begun to consider stabilized Wittig olefination, utilising it to modify proteins in aqueous–organic mixtures;10 this report provided strong support for our efforts in developing this methodology for bioconjugation. We describe herein a strategy for tagging proteins that is based on Wittig olefination of a functionalized phosphoranylidene or a phosphonium salt probe with an aryl aldehyde bait. The only Division of Organic Chemistry, Institute of Chemical and Engineering Sciences, Agency for Science Technology and Research Singapore, 11 Biopolis Way, Helios Block #03-08, Singapore. E-mail: [email protected]; Fax: +65 6874 5870; Tel: +65 6799 8599 † Electronic supplementary information (ESI) available: Full experimental details and compound characterization. See DOI: 10.1039/c3cc45961f
11188
Chem. Commun., 2013, 49, 11188--11190
byproduct of the reaction is triphenylphosphine oxide, which is also produced in the well-explored Staudinger ligation.4 Although these coupling partners are not bioorthogonal in the strictest sense,11 we show that they are sufficiently robust for protein affinity isolation and fluorescence labeling in live cells. From the outset, we recognized the inherent challenge of using an electrophile–nucleophile pair to do chemistry in biological matrices. Aldehydes are increasingly being recognized as mild ‘‘bioorthogonal’’ electrophiles,7a although they are well-known to form reversible adducts with cellular nucleophiles.11,12 Likewise, stabilized phosphorus ylides are moderate nucleophiles, but little is known about their reactivity with biologically-relevant electrophiles. Collectively, these challenges have perhaps undermined the potential of Wittig olefination as a bioconjugation strategy. Notwithstanding, we hoped to leverage on (1) the kinetic preference of stabilized phosphoranylidenes for aldehydes, (2) the irreversibility of the Wittig reaction due to the formation of strong PQO and CQC bonds, and (3) the use of an aryl aldehyde coupling partner such that extensive p-conjugation in the final cinnamate adduct would provide an additional driving force for preferential conjugation with the phosphoranylidene (Scheme 1). Indeed, our preliminary studies (see ESI†) revealed that stabilized phosphorus ylides readily undergo Wittig olefination with aldehydes in common culture media and cell lysis buffers. The reaction of methyl 2-triphenylphosphoranylideneacetate with benzaldehyde in various aqueous buffers afforded the corresponding (E)-cinnamate adduct stereoselectively (approximate E/Z = 9 : 1), with excellent
Scheme 1 Mechanistic rationale behind the development of stabilized Wittig olefination for bioconjugation.
This journal is
c
The Royal Society of Chemistry 2013
View Article Online
Communication
ChemComm Table 1
Wittig-mediated bioconjugation of aldehydes in Tris buffer
Published on 14 October 2013. Downloaded by UNIVERSIDAD DE GIRONA on 28/10/2014 15:35:52.
Aldehyde
7 Scheme 2 Synthesis of Wittig probes. (a) BrCH2COBr, Et3N, CH2Cl2. (b) PPh3, PhMe. (c) 2 M NaOH–CH2Cl2 (1 : 1). (d) (D)-biotin, K2CO3, DMF, 60 1C. (e) 1 M HCl. (f) N-dansyl6-aminohexanoic acid, K2CO3, DMF, 60 1C. (g) (D)-biotin, K2CO3, DMSO, 50 1C, then 1 M HCl. (h) N-dansyl-3-aminopropanoic acid, K2CO3, DMF, 60 1C, then 1 M HCl.
chemoselectivity towards aldehydes and no observable reaction with simple ketones. In rate studies of stabilized Wittig olefination in Tris buffer, we found that the second order rate constant at 4 1C was comparable to that of the mechanistically-related Staudinger ligation (see ESI†). Encouraged by these results, we developed a rapid synthesis to access probes bearing a stabilized phosphoranylidene joined to a functional tag by a nonyl linker (Scheme 2A). These phosphoranylidene probes underwent Wittig olefination smoothly with aldehydes of varying structural complexity, in Tris buffer at 4 1C (Table 1). Leveraging on this synthetic approach, we developed a second generation of probes linked by tetraethylene glycol (Scheme 2B), which we hoped would improve probe uptake for cellular bioconjugation.13 A similar synthetic route starting from mono-tosylated tetraethylene glycol allowed quick access to biotin probe 13 and dansyl probe 14, analogues of 3a and 4 respectively (Scheme 3). Having established the effectiveness of our probes in buffered media, we turned our attention to demonstrating proof of concept in biological matrices. We conducted streptavidin-mediated affinity isolation on cell lysates treated with bait molecule 7 followed by phosphoranylidene probe 3. Western blot analysis revealed bands at 12 kDa indicating successful isolation of FKBP12 through Wittig conjugation of 3 and 7 in vitro. The intensity of the FKBP12 band increased in a dose dependent manner with probe concentration at fixed bait concentration (Fig. 1A). Likewise, band intensity also increased with bait concentration when probe concentration was fixed, appearing to saturate at bait concentrations exceeding 0.5 mM (Fig. 1B). Phosphonium probe 3a was likewise effective (Fig. 1C, lane 2). Neither 3 nor 3a would effect a significant level of pulldown with unmodified FK506 that lacked aldehyde functionality (Fig. 1C, This journal is
c
The Royal Society of Chemistry 2013
Phosphoranylidene probe
Product
Yield (%)
3
78
3
94
3
73
4
76
FK506 is a small-molecule macrolide with high affinity for FKBP12. The aryl aldehyde analog 7 is designed to demonstrate Wittig olefination in biological systems (see ESI for respective structures).
Scheme 3 Synthesis of dansyl probe 17 bearing an ethylenediamine linker. (a) BrCH2COBr, Et3N, CH2Cl2. (b) PPh3, PhMe.
lanes 3 and 4), confirming that the probes react preferentially with aldehydes over ketones. With positive results from the affinity isolation of FKBP12 in cell lysates, we investigated if the reaction would tolerate cellular matrices. HeLa cells were incubated with bait 7 for 8 hours, followed by probe 3 or 13 in fresh media for 15 hours. We were delighted to find that both probes 3 and 13 were able to isolate FKBP12, indicating successful Wittig olefination within the cells (Fig. 1D). The relative amount of FKBP12 isolated with 13 (C log P = 4.7 of ylide) was higher than that of 3 (C log P = 8.2),‡ consistent with the notion that the linkers alter solubility and membrane permeability. Interestingly, the relative amount of FKBP12 pulled down with either probe was less than that obtained with pre-conjugated adduct 8, suggesting that further optimization is required to improve the bioconjugation yields in live cells (see ESI†). Nevertheless, successful pulldown of FKBP12 from the complex environment of a living cell indicates that stabilized Wittig olefination is indeed a viable bioconjugation strategy. Concurrently, we were interested to show that stabilized Wittig olefination might be used for fluorescently labeling proteins in live cells. To this end, we conducted imaging experiments with bait 7 and dansyl probes 4 and 14 to explore if we could fluorescently label FKBP12 in live cells. HeLa cells were dosed with either bait 7 or DMSO as a control, followed by Chem. Commun., 2013, 49, 11188--11190
11189
View Article Online
Published on 14 October 2013. Downloaded by UNIVERSIDAD DE GIRONA on 28/10/2014 15:35:52.
ChemComm
Fig. 1 Affinity isolation of FKBP12. Titration experiments with (A) increasing probe concentration and (B) increasing bait concentration. The intensity of the FKBP12 band increased in a dose-dependent manner in these experiments. (C) Comparison of phosphoranylidene probe 3 and phosphonium probe 3a. Both probes react with bait 7, but not unmodified FK506. (D) Pulldown of FKBP12 from live HeLa cells using probes 3 and 13.
the respective dansyl probe in a similar manner to the affinity isolation assays. We hypothesized that cells treated with both bait 7 and a dansyl probe would be more fluorescent than cells treated with a probe alone: the irreversible reaction of 7 with the probe traps the fluorophore in the cell as the dansylatedFK506–FKBP12 complex, consequently shifting the equilibrium to favor the diffusion of more probe into the cell. Unfortunately, dansyl probes 4 and 14 were unsuitable for this purpose, since cells treated with bait 7 and these probes displayed only weak fluorescence (see Fig. 2A and B). These findings prompted us to further synthesize dansyl probe 17 (Scheme 3), which bears an ethylenediamine linker, in the hope that (1) a shorter linker would decrease non-radiative modes of relaxation and lead to a stronger fluorescence signal, and (2) the solubility properties associated with this linker would further improve membrane permeability of the probe. Satisfyingly, cells treated with both 7 and 17 (Fig. 2D) showed a marked increase in fluorescence intensity compared to cells treated with 17 alone (Fig. 2C). Taken together with the pulldown results, this observation provides further evidence of stabilized Wittig olefination in live cells, and alludes to its capacity for other imaging applications.
Fig. 2 Differential Interference Contrast (DIC) and fluorescence images of live HeLa cells treated with (A) bait 7 and probe 4 only (B) bait 7 and probe 14 (C) probe 17 only, and (D) bait 7 and probe 17. The scale bar represents 25 mm. The concentrations of bait and probe were 1 and 5 mM respectively.
11190
Chem. Commun., 2013, 49, 11188--11190
Communication Given the various genetic7a,14 and chemical methods for incorporating aldehyde reporters into cells, we anticipate that stabilized Wittig olefination will find increased use in chemical biology, both on its own and possibly in tandem with other bioorthogonal reactions. Towards this goal, we have demonstrated that stabilized Wittig olefination possesses desirable attributes for bioconjugation: (1) the reacting functionalities are sufficiently stable and chemoselective in complex environments, and (2) when installed on appropriately tuned scaffolds, the resulting reagents can be used to biotinylate or fluorescently-label target proteins in live cells. This research was supported by A*STAR ICES (Project code ICES/11-240B08). We thank Michelle Su (Singapore BioImaging Corsortium) for her assistance in the cell biology experiments and Graham Wright (Institute of Medical Biology, Microscopy Unit) for his invaluable microscopy advice.
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