CHEMBIOCHEM COMMUNICATIONS DOI: 10.1002/cbic.201402051
Comparison of the Reactivity of Carbohydrate Photoaffinity Probes with Different Photoreactive Groups Kaori Sakurai,* Shimpei Ozawa, Rika Yamada, Tomoki Yasui, and Sakae Mizuno[a] A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule–protein interactions. A set of carbohydrate-based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand-dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a lowaffinity binding protein in cell lysate.
Photoaffinity labeling (PAL) has re-emerged in recent years as a powerful approach for direct isolation of cellular targets of bioactive small molecules and elucidation of their mechanisms of action.[1] PAL is based on a small molecule with a photoreactive label, which upon photoactivation, can nonselectively form covalent bonds with proximal amino acid residues of the bound proteins. Its ability to covalently crosslink the binding proteins is a considerable advantage over conventional affinity purification methods when targeting low abundance or lowaffinity proteins, membrane proteins, or proteins in live-cell settings.[2] However, due to the low crosslinking efficiency and selectivity of PAL, technical challenges remain significant in many cases where the binding affinity of small molecules are weak.[1, 3] Excess quantities of photoaffinity probes (at a concentration greater than the Kd value of the parent ligand) are often used in order to maximize formation of a labile probe– protein complex. Such reaction conditions would also increase ligand-independent, nonspecific crosslinking. As a result, product analysis for target identification would become complicated. Although the low efficiency of PAL has been addressed by the design of new photoaffinity probes, factors which can be exploited to overcome low selectivity have scarcely been investigated.[4] One way to improve low selectivity of PAL involves a judicious choice of the photoreactive group most suited for crosslinking of low-affinity proteins. The most commonly used photoreactive groups to date are aryl azide, benzophenone, and diazirine, which respectively generate nitrene, ketyl diradical, [a] Prof. K. Sakurai, S. Ozawa, R. Yamada, T. Yasui, S. Mizuno Department of Biotechnology and Life Science Tokyo University of Agriculture and Technology Naka-cho, 2-24-16, Koganei, Tokyo 184-8588 (Japan) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402051.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
and carbene as reactive intermediates (Scheme 1).[1, 5] It is known that the type of photoreactive group can influence the efficiency and selectivity of PAL. For example, arylazide-derived nitrene groups can insert into X H bonds (X = C, N, O, S) of proximal amino acid residues but can also rapidly rearrange to 1,2-didehydroazepine (Scheme 1), a more stable electrophile, which decreases crosslinking efficiency and selectivity.[6] Benzophenone-derived ketyl diradicals can insert into C H bonds, which display higher chemoselectivity compared to other photoreactive groups.[5a] Due to the reversible nature of its activation, benzophenone often requires a long reaction time for optimal crosslinking efficiency. Such reaction conditions also give rise to ligand-independent crosslinking. Diazirine generates highly reactive carbene, which can insert into proximal X H bonds (X = C, N, O, S) of amino acid residues. It can also rapidly rearrange to a relatively inert diazo intermediate, which would result in low crosslinking yield.[5c–e] However, there have been a few comparative studies on different photoreactive groups, which would be helpful for choosing a suitable group to efficiently capture binding proteins of a small molecule of interest.[7] As a consequence, researchers often test several different types of photoreactive groups in order to optimize the chance of crosslinking specific binding proteins.[8] In this study, we report a comparative analysis of the effects of different photoreactive groups on the efficiency and selectivity of crosslinking a low-affinity binding protein. We designed and synthesized a set of trifunctional photoaffinity probes (1–3) bearing a carbohydrate ligand and a reporter group, which differ only in the photoreactive group (Scheme 1). We chose three photoreactive groups—benzophenone, aryl azide, and alkyl diazirine groups—on the bases of different functionality, ready availability, and different molecular sizes. Carbohydrate–protein interactions represent an example of weak binding interactions in nature.[9] With our particular interest in the identification of novel carbohydrate-binding proteins, we aimed to develop selective PAL methods for lowaffinity proteins. d-Lactose (lactose), which is a common subunit found in glycolipids and glycoproteins on cell surfaces, was chosen as a model ligand. Lactose is known to bind peanut agglutinin (PNA, Kd = 770 mm),[10] which belongs to a lectin family of carbohydrate binding proteins with specificity toward terminal d-galactose residues. An alkyne group was employed as a reporter group to readily introduce a fluorescent tag through CuI-catalyzed azide–alkyne cycloaddition (CuAAC)[11] to the probe-crosslinked proteins, subsequent to PAL reaction. Although PAL is ideally based on a pseudo-intramolecular reaction between a probe and a protein within a bound complex (ligand-dependent reactivity), it has been suggested that photo-crosslinking can also occur between a free probe and ChemBioChem 2014, 15, 1399 – 1403
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Scheme 1. Structures of lactose-based photoaffinity probes (1–3) and control probes (4–6) with three different types of photoreactive groups.
a protein by diffusion (ligand-independent reactivity), depending on the type of photoreactive groups.[5, 12] To estimate the ligand-independent reactivity based on intermolecular reactions between a free probe and PNA, control probes were also prepared, which were analogues of 1–3 but without the carbohydrate ligand (4–6, Scheme 1). Typical PAL experiments were performed with a photoaffinity probe and PNA, which were first allowed to bind at 4 8C for 1 h, then were reacted by irradiating with a UV lamp (365 nm, 15 W) at a distance of 5 cm at 0 8C for 1 h (Scheme 2). Unreacted probes were removed by acetone precipitation, and the resulting mixture was separated by SDS-PAGE. After conjugating BODIPY-azide (7) through CuAAC in gel,[13] the probe-cross-
linked PNA was detected by fluorescence imaging analysis. The amount of crosslinked PNA was quantitated by fluorescence intensity of a corresponding band in the SDS-PAGE gel, and the crosslinking yields were calculated based on the molar amounts of PNA used for a given reaction. We initially investigated how different photoreactive groups affect the relationship between probe concentration and the efficiency and selectivity of PAL by using photoaffinity probes 1–3 and PNA. Varied concentrations of a photoaffinity probe were reacted with PNA, the concentration of which was kept below the Kd value so that the relative concentration of the probe–protein complex would be dependent on the probe concentration. For all three probes (1–3), formation of the
Scheme 2. A photoaffinity labeling reaction between the lactose-based photoaffinity probe and a binding protein. a) Binding, 4 8C, 1 h; b) hn (365 nm), 4 8C, 1 h; c) SDS-PAGE; d) CuAAC, BODIPY-N3 (7); e) fluorescence imaging.
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probe-crosslinked PNA was observed only under UV irradiation conditions (UV+) and not under dark conditions (UV ; see the Supporting Information). Titration graphs were obtained to express the relationship between probe concentration and the amounts of probe-crosslinked PNA, as determined by the fluorescence intensity of conjugated BODIPY. Increasing amounts of crosslinked PNA were generated with increasing probe concentration for all three probes, with aryl azide 2 being the highest yielding of all when compared at the same concentration (Figure 1). In an ideal PAL reaction, crosslinking occurs
Figure 2. Time course of PAL reaction between PNA (0.4 mm) and the photoaffinity probes (10 mm). A) 1: *, B) 2: &, C) 3: ~.
Figure 1. Concentration dependence of PAL reactions between PNA (0.4 mm) and varied concentrations of photoaffinity probes (1: *, 2: &, 3: ~) in solid lines and control probes (4: *, 5: &, 6: ~) in dashed lines.
only when a photoaffinity probe and a protein specifically form a bound complex. However, control experiments with 4– 6, which display no specific ligands, showed that crosslinking by benzophenone 4 and aryl azide 5 becomes significant at concentrations above 100 mm (Figure 1). The data indicated that formation of crosslinked PNA by benzophenone 1 or aryl azide 2 were also likely increased by the presence of the ligand-independent reaction between the free probe in solution and PNA. Therefore, the apparent crosslinking efficiency of benzophenone 1 and aryl azide 2 might also be influenced by factors other than binding affinity of the parent ligand.[3b] In contrast, diazirine 6 was unreactive toward PNA at concentrations up to 1 mm. By comparing the titration graphs for 1–3 with those for 4–6 in Figure 1, it was concluded that the selectivity of ligand-dependent over ligand-independent reactions with PNA was highest when using diazirine 3. The crosslinking efficiencies of different photoreactive groups were also compared by studying the time course of PAL reactions at 0 8C with probes 1–3 (10 mm) and PNA (0.4 mm). The yields of crosslinking PNA were different among the three probes (Figure 2). The highest yield was achieved by aryl azide 2 and the lowest by benzophenone 1 for a reaction time up to 60 min. On the other hand, the fastest initial rate of reaction and the shortest reaction time (10 min) was observed for diazirine 3. Its initial rate of reaction and the lower crosslinking yields (~ 20 %) could be explained by competition between the crosslinking reaction of a highly reactive carbene intermediate with proximal amino acid residues and the quenching reaction with solvent water. In terms of both initial rate of 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
reaction and crosslinking yield (~ 30 %), aryl azide 2 provided the best results with optimal reaction time of around 30 min. Nitrenes generated from aryl azide can instantaneously rearrange into the more stable 1,2-didehydroazepine intermediates through ring expansion.[6] 1,2-Didehydroazepine is less reactive with water molecules and yet more selectively reactive toward nucleophilic functionalities of proteins. It has been reported that the lifetime of didehydroazepine is much longer (~ ms) than that of nitrene (~ ns).[6] The presence of the long-lived reactive intermediate might contribute to higher crosslinking efficiency compared to carbene. PAL reactions by benzophenone 1 were the slowest, and a longer reaction time (> 60 min) was necessary to achieve comparable crosslinking yields (~ 30 %) to those of 2 or 3. Product formation by 1 did not saturate after up to 100 min. This is consistent with previous reports that increased crosslinking efficiency is expected with prolonged UV irradiation due to the reversible nature of benzophenone activation and the absence of a quenching pathway by solvent water.[5a] To compare the ability of probes 1–3 to photo-crosslink a binding protein in a complex mixture of proteins, we combined the probes at 10 mm concentration with PNA spiked in a protein mixture from cell lysate at a ratio of 1:100 (w/w). For all three probes, reactions were conducted under both UV irradiation and dark conditions in parallel so that BODIPY-labeled proteins formed independently of PAL could be discriminated. Figure 3 shows that diazirine 3 enabled crosslinking of PNA
Figure 3. Products for PAL reactions of probes 1–3 (10 mm) with a mixed solution containing 0.5 mg of PNA and 50 mg of HeLa cell lysate. A reaction condition involving UV irradiation is shown as UV+ and a dark condition as UV . Left: in-gel fluorescence imaging data. Right: Coomassie Brilliant Blue (CBB) staining data.
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CHEMBIOCHEM COMMUNICATIONS with high selectivity. In contrast, benzophenone 1 and aryl azide 2 yielded a multitude of photoadducts, which made it difficult to identify whether PNA was specifically labeled. The results showed that, among the three photoaffinity probes with different photoreactive groups, only diazirine 3 was able to achieve the most selective photoaffinity labeling of PNA, which represented low-affinity binding proteins. The high selectivity observed for the diazirine probe is remarkable, given that the binding affinity of PNA with lactose is very weak (Kd value of 770 mm). Taken together, our results revealed considerably different outcomes of PAL with the three photoreactive groups when crosslinking a low-affinity protein. Diazirine 3 was characterized by the fastest initial rate of reaction, rapid termination of reaction, and low ligand-independent reactivity. In an ideal PAL reaction, the photoactivated probe should rapidly crosslink with any proximal amino acid residues or should be quenched by solvent before diffusing to crosslink with nonspecific proteins. The reactivity features observed for the diazirine probe in this study are consistent with the ideal mechanism of PAL and provide a basis for selective photoaffinity labeling of PNA in cell lysate. Although the drawback of using the diazirine probe is its low crosslinking efficiency, it should be possible to address the problem by using a higher concentration for probe 3, given its highly selective reactivity. Aryl azide 2 provided the best results, based on the fast initial rate of reaction and high crosslinking yields in PAL experiments with a single protein. However, high levels of ligand-independent reactivity were also observed with an increasing concentration of aryl azide control probe 5, which explained the apparently low crosslinking selectivity in PAL experiments in cell lysate. Optimization of the probe concentration and reaction time are thus required for successful use of the aryl azide probe for capturing lowaffinity proteins. The crosslinking yields of benzophenone 1 were lower than those for aryl azide 2 or diazirine 3, due to its slow reaction rate. It appears difficult to use the benzophenone probe to target low-affinity proteins in cellular context. Employing higher probe concentrations or longer reaction times would result in low selectivity while improving crosslinking yields. The alkyl diazirine group has not been the first choice for a photoreactive group in many PAL studies because of the generally low crosslinking yields and the need for synthetic preparation.[1d, 5f] Our findings bring new light to the utility of the diazirine group in PAL studies, particularly in the exploration of low-affinity binding proteins, which remains a challenging task with conventional affinity purification methods. In summary, we designed and synthesized a set of carbohydrate-based photoaffinity probes to compare the effects of different photoreactive groups on the efficiency and selectivity of photoaffinity labeling a low-affinity binding protein. We showed that, although the diazirine probe gave low crosslinking yields, it displayed highly ligand-dependent reactivity consistent with the principle of PAL. Moreover, we demonstrated the strikingly different outcome of using different photoreactive groups for PAL experiments in cell lysate: only the diazirine probe achieved highly selective crosslinking of a low-affinity binding protein, which could not be unambiguously detect 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org ed by aryl azide or benzophenone probes. Our findings should be relevant to the future design of photoaffinity probes toward rapid identification of small-molecule binding proteins.
Experimental Section Experimental details for the synthesis of chemical probes, photocrosslinking reactions, and their analyses are provided in the Supporting Information.
Acknowledgements This work was funded in part by a young investigator grant from the Human Frontier Science Program Foundation. S.O. is supported by the Ajinomoto scholarship, and R.Y. is supported by the Teijin-Kumura scholarship. Keywords: binding proteins · carbohydrates · chemical probes · photoaffinity labeling · photoreactive groups [1] a) V. Chowdhry, F. H. Westheimer, Annu. Rev. Biochem. 1979, 48, 293 – 325; b) R. J. Guillory, Pharmacol. Ther. 1989, 41, 1 – 25; c) J. Brunner, Annu. Rev. Biochem. 1993, 62, 483 – 514; d) F. Kotzyba-Hibert, I. Kapfer, M. Goeldner, Angew. Chem. Int. Ed. Engl. 1995, 34, 1296 – 1312; Angew. Chem. 1995, 107, 1391 – 1408; e) Y. Tanaka, M. R. Bond, J. J. Kohler, Mol. BioSyst. 2008, 4, 473 – 480; f) P. P. Geurink, L. M. Prely, G. A. van der Marel, R. Bischoff, H. S. Overkleeft, Top. Curr. Chem. 2012, 324, 85 – 113. [2] a) S. Sato, A. Murata, T. Shirakawa, M. Uesugi, Chem. Biol. 2010, 17, 616 – 623; b) S. Ziegler, V. Pries, C. Hedberg, H. Waldmann, Angew. Chem. Int. Ed. 2013, 52, 2744 – 2792; Angew. Chem. 2013, 125, 2808 – 2859. [3] a) Y. Nakamura, R. Miyatake, M. Ueda, Angew. Chem. Int. Ed. 2008, 47, 7289 – 7292; Angew. Chem. 2008, 120, 7399 – 7402; b) A. Kawamura, S. Hindi, D. M. Mihai, L. James, O. Aminova, Bioorg. Med. Chem. 2008, 16, 8824 – 8829; c) K. Sakurai, R. Yamada, A. Okada, M. Tawa, S. Ozawa, M. Inoue, ChemBioChem 2013, 14, 421 – 425. [4] Recently, the use of multivalent photoaffinity probes has been reported as a promising approach to overcome the low affinity of the probe. See: a) L. Ballell, M. van Scherpenzeel, K. Buchalova, R. M. J. Liskamp, R. J. Pieters, Org. Biomol. Chem. 2006, 4, 4387 – 4394; b) T.-C. Chang, C.H. Lai, C.-W. Chien, C.-F. Liang, A. K. Adak, Y.-J. Chuang, Y.-J. Chen, C.-C. Lin, Bioconjugate Chem. 2013, 24, 1895 – 1906. [5] a) G. Dormn, G. D. Prestwich, Biochemistry 1994, 33, 5661 – 5673; b) S. A. Fleming, Tetrahedron 1995, 51, 12479 – 12520; c) A. Blencowe, W. Hayes, Soft Matter 2005, 1, 178 – 205; d) M. Hashimoto, Y. Hatanaka, Eur. J. Org. Chem. 2008, 2513 – 2523; e) J. Das, Chem. Rev. 2011, 111, 4405 – 4417; f) L. Dubinsky, B. P. Krom, M. M. Meijler, Bioorg. Med. Chem. 2012, 20, 554 – 570; g) A. Kawamura, D. M. Mihai in Methods in Molecular Biology, Vol. 803 (Eds.: G. Drewes, M. Bantscheff), Humana Press, Totowa, NJ, 2012, pp. 65 – 75. [6] M. S. Rizk, X. Shi, M. S. Platz, Biochemistry 2006, 45, 543 – 551. [7] a) P. J. Weber, A. G. Beck-Sickinger, J. Pept. Res. 1997, 49, 375 – 383; b) J. J. Tate, J. Persinger, B. Bartholomew, Nucleic Acids Res. 1998, 26, 1421 – 1426; c) J. T. Bush, L. J. Walport, J. F. McGouran, I. K. H. Leung, G. Berridge, S. S. van Berkel, A. Basak, B. M. Kessler, C. J. Schofield, Chem. Sci. 2013, 4, 4115 – 4120. [8] a) M. Wiegand, T. K. Lindhorst, Eur. J. Org. Chem. 2006, 4841 – 4851; b) C.-Y. Jiao, I. D. Alves, V. Point, S. Lavielle, S. Sagan, G. Chassaing, Bioconjugate Chem. 2010, 21, 352 – 359; c) P. P. Geurink, B. I. Florea, G. A. Van der Marel, B. M. Kessler, H. S. Overkleeft, Chem. Commun. 2010, 46, 9052 – 9054; d) Y. Nakamura, S. Inomata, M. Ebine, Y. Manabe, I. Iwakura, M. Ueda, Org. Biomol. Chem. 2011, 9, 83 – 85; e) B. Albertoni, J. S. Hannam, D. Ackermann, A. Schmitz, M. Famulok, Chem. Commun. 2012, 48, 1272 – 1274; f) H. Fuwa, Y. Takahashi, Y. Konno, N. Watanabe, H. Miyashita, M. Sasaki, H. Natsugari, T. Kan, T. Fukuyama, T. Tomita, T. Iwatsubo, ACS Chem. Biol. 2007, 2, 408 – 418.
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CHEMBIOCHEM COMMUNICATIONS [9] H. Lis, N. Sharon, Chem. Rev. 1998, 98, 637 – 674. [10] K. J. Neurohr, D. R. Bundle, N. M. Young, H. H. Mantsch, Eur. J. Biochem. 1982, 123, 305 – 310. [11] a) F. Himo, T. Lovell, R. Hilgraf, V. V. Rostovtsev, L. Noodleman, K. B. Sharpless, V. V. Fokin, J. Am. Chem. Soc. 2005, 127, 210 – 216; b) M. Meldal, C. W. Tornøe, Chem. Rev. 2008, 108, 2952 – 3015; c) J. E. Hein, V. V. Fokin, Chem. Soc. Rev. 2010, 39, 1302 – 1315. [12] A. E. Ruoho, H. Kiefer, P. E. Roeder, S. J. Singer, Proc. Natl. Acad. Sci. USA 1973, 70, 2567 – 2571. [13] In this study, we chose to conjugate BODIPY-azide with the probe-labeled PNA in gel rather than prior to gel loading based on our previous
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www.chembiochem.org findings: significant sample loss accompanied the CuAAC reaction subsequent to PAL and prior to gel, loading due to precipitation of proteins under CuAAC reaction conditions. This problem was circumvented by performing CuAAC in the gel after separating the PAL reaction mixture by SDS-PAGE. See ref. [3 c].
Received: February 21, 2014 Published online on May 27, 2014
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Comparison of the Reactivity of Carbohydrate Photoaffinity Probes with Different Photoreactive Groups Kaori Sakurai,* Shimpei Ozawa, Rika Yamada, Tomoki Yasui, and Sakae Mizuno[a] A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule–protein interactions. A set of carbohydrate-based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand-dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a lowaffinity binding protein in cell lysate.
Photoaffinity labeling (PAL) has re-emerged in recent years as a powerful approach for direct isolation of cellular targets of bioactive small molecules and elucidation of their mechanisms of action.[1] PAL is based on a small molecule with a photoreactive label, which upon photoactivation, can nonselectively form covalent bonds with proximal amino acid residues of the bound proteins. Its ability to covalently crosslink the binding proteins is a considerable advantage over conventional affinity purification methods when targeting low abundance or lowaffinity proteins, membrane proteins, or proteins in live-cell settings.[2] However, due to the low crosslinking efficiency and selectivity of PAL, technical challenges remain significant in many cases where the binding affinity of small molecules are weak.[1, 3] Excess quantities of photoaffinity probes (at a concentration greater than the Kd value of the parent ligand) are often used in order to maximize formation of a labile probe– protein complex. Such reaction conditions would also increase ligand-independent, nonspecific crosslinking. As a result, product analysis for target identification would become complicated. Although the low efficiency of PAL has been addressed by the design of new photoaffinity probes, factors which can be exploited to overcome low selectivity have scarcely been investigated.[4] One way to improve low selectivity of PAL involves a judicious choice of the photoreactive group most suited for crosslinking of low-affinity proteins. The most commonly used photoreactive groups to date are aryl azide, benzophenone, and diazirine, which respectively generate nitrene, ketyl diradical, [a] Prof. K. Sakurai, S. Ozawa, R. Yamada, T. Yasui, S. Mizuno Department of Biotechnology and Life Science Tokyo University of Agriculture and Technology Naka-cho, 2-24-16, Koganei, Tokyo 184-8588 (Japan) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402051.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
and carbene as reactive intermediates (Scheme 1).[1, 5] It is known that the type of photoreactive group can influence the efficiency and selectivity of PAL. For example, arylazide-derived nitrene groups can insert into X H bonds (X = C, N, O, S) of proximal amino acid residues but can also rapidly rearrange to 1,2-didehydroazepine (Scheme 1), a more stable electrophile, which decreases crosslinking efficiency and selectivity.[6] Benzophenone-derived ketyl diradicals can insert into C H bonds, which display higher chemoselectivity compared to other photoreactive groups.[5a] Due to the reversible nature of its activation, benzophenone often requires a long reaction time for optimal crosslinking efficiency. Such reaction conditions also give rise to ligand-independent crosslinking. Diazirine generates highly reactive carbene, which can insert into proximal X H bonds (X = C, N, O, S) of amino acid residues. It can also rapidly rearrange to a relatively inert diazo intermediate, which would result in low crosslinking yield.[5c–e] However, there have been a few comparative studies on different photoreactive groups, which would be helpful for choosing a suitable group to efficiently capture binding proteins of a small molecule of interest.[7] As a consequence, researchers often test several different types of photoreactive groups in order to optimize the chance of crosslinking specific binding proteins.[8] In this study, we report a comparative analysis of the effects of different photoreactive groups on the efficiency and selectivity of crosslinking a low-affinity binding protein. We designed and synthesized a set of trifunctional photoaffinity probes (1–3) bearing a carbohydrate ligand and a reporter group, which differ only in the photoreactive group (Scheme 1). We chose three photoreactive groups—benzophenone, aryl azide, and alkyl diazirine groups—on the bases of different functionality, ready availability, and different molecular sizes. Carbohydrate–protein interactions represent an example of weak binding interactions in nature.[9] With our particular interest in the identification of novel carbohydrate-binding proteins, we aimed to develop selective PAL methods for lowaffinity proteins. d-Lactose (lactose), which is a common subunit found in glycolipids and glycoproteins on cell surfaces, was chosen as a model ligand. Lactose is known to bind peanut agglutinin (PNA, Kd = 770 mm),[10] which belongs to a lectin family of carbohydrate binding proteins with specificity toward terminal d-galactose residues. An alkyne group was employed as a reporter group to readily introduce a fluorescent tag through CuI-catalyzed azide–alkyne cycloaddition (CuAAC)[11] to the probe-crosslinked proteins, subsequent to PAL reaction. Although PAL is ideally based on a pseudo-intramolecular reaction between a probe and a protein within a bound complex (ligand-dependent reactivity), it has been suggested that photo-crosslinking can also occur between a free probe and ChemBioChem 2014, 15, 1399 – 1403
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Scheme 1. Structures of lactose-based photoaffinity probes (1–3) and control probes (4–6) with three different types of photoreactive groups.
a protein by diffusion (ligand-independent reactivity), depending on the type of photoreactive groups.[5, 12] To estimate the ligand-independent reactivity based on intermolecular reactions between a free probe and PNA, control probes were also prepared, which were analogues of 1–3 but without the carbohydrate ligand (4–6, Scheme 1). Typical PAL experiments were performed with a photoaffinity probe and PNA, which were first allowed to bind at 4 8C for 1 h, then were reacted by irradiating with a UV lamp (365 nm, 15 W) at a distance of 5 cm at 0 8C for 1 h (Scheme 2). Unreacted probes were removed by acetone precipitation, and the resulting mixture was separated by SDS-PAGE. After conjugating BODIPY-azide (7) through CuAAC in gel,[13] the probe-cross-
linked PNA was detected by fluorescence imaging analysis. The amount of crosslinked PNA was quantitated by fluorescence intensity of a corresponding band in the SDS-PAGE gel, and the crosslinking yields were calculated based on the molar amounts of PNA used for a given reaction. We initially investigated how different photoreactive groups affect the relationship between probe concentration and the efficiency and selectivity of PAL by using photoaffinity probes 1–3 and PNA. Varied concentrations of a photoaffinity probe were reacted with PNA, the concentration of which was kept below the Kd value so that the relative concentration of the probe–protein complex would be dependent on the probe concentration. For all three probes (1–3), formation of the
Scheme 2. A photoaffinity labeling reaction between the lactose-based photoaffinity probe and a binding protein. a) Binding, 4 8C, 1 h; b) hn (365 nm), 4 8C, 1 h; c) SDS-PAGE; d) CuAAC, BODIPY-N3 (7); e) fluorescence imaging.
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probe-crosslinked PNA was observed only under UV irradiation conditions (UV+) and not under dark conditions (UV ; see the Supporting Information). Titration graphs were obtained to express the relationship between probe concentration and the amounts of probe-crosslinked PNA, as determined by the fluorescence intensity of conjugated BODIPY. Increasing amounts of crosslinked PNA were generated with increasing probe concentration for all three probes, with aryl azide 2 being the highest yielding of all when compared at the same concentration (Figure 1). In an ideal PAL reaction, crosslinking occurs
Figure 2. Time course of PAL reaction between PNA (0.4 mm) and the photoaffinity probes (10 mm). A) 1: *, B) 2: &, C) 3: ~.
Figure 1. Concentration dependence of PAL reactions between PNA (0.4 mm) and varied concentrations of photoaffinity probes (1: *, 2: &, 3: ~) in solid lines and control probes (4: *, 5: &, 6: ~) in dashed lines.
only when a photoaffinity probe and a protein specifically form a bound complex. However, control experiments with 4– 6, which display no specific ligands, showed that crosslinking by benzophenone 4 and aryl azide 5 becomes significant at concentrations above 100 mm (Figure 1). The data indicated that formation of crosslinked PNA by benzophenone 1 or aryl azide 2 were also likely increased by the presence of the ligand-independent reaction between the free probe in solution and PNA. Therefore, the apparent crosslinking efficiency of benzophenone 1 and aryl azide 2 might also be influenced by factors other than binding affinity of the parent ligand.[3b] In contrast, diazirine 6 was unreactive toward PNA at concentrations up to 1 mm. By comparing the titration graphs for 1–3 with those for 4–6 in Figure 1, it was concluded that the selectivity of ligand-dependent over ligand-independent reactions with PNA was highest when using diazirine 3. The crosslinking efficiencies of different photoreactive groups were also compared by studying the time course of PAL reactions at 0 8C with probes 1–3 (10 mm) and PNA (0.4 mm). The yields of crosslinking PNA were different among the three probes (Figure 2). The highest yield was achieved by aryl azide 2 and the lowest by benzophenone 1 for a reaction time up to 60 min. On the other hand, the fastest initial rate of reaction and the shortest reaction time (10 min) was observed for diazirine 3. Its initial rate of reaction and the lower crosslinking yields (~ 20 %) could be explained by competition between the crosslinking reaction of a highly reactive carbene intermediate with proximal amino acid residues and the quenching reaction with solvent water. In terms of both initial rate of 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
reaction and crosslinking yield (~ 30 %), aryl azide 2 provided the best results with optimal reaction time of around 30 min. Nitrenes generated from aryl azide can instantaneously rearrange into the more stable 1,2-didehydroazepine intermediates through ring expansion.[6] 1,2-Didehydroazepine is less reactive with water molecules and yet more selectively reactive toward nucleophilic functionalities of proteins. It has been reported that the lifetime of didehydroazepine is much longer (~ ms) than that of nitrene (~ ns).[6] The presence of the long-lived reactive intermediate might contribute to higher crosslinking efficiency compared to carbene. PAL reactions by benzophenone 1 were the slowest, and a longer reaction time (> 60 min) was necessary to achieve comparable crosslinking yields (~ 30 %) to those of 2 or 3. Product formation by 1 did not saturate after up to 100 min. This is consistent with previous reports that increased crosslinking efficiency is expected with prolonged UV irradiation due to the reversible nature of benzophenone activation and the absence of a quenching pathway by solvent water.[5a] To compare the ability of probes 1–3 to photo-crosslink a binding protein in a complex mixture of proteins, we combined the probes at 10 mm concentration with PNA spiked in a protein mixture from cell lysate at a ratio of 1:100 (w/w). For all three probes, reactions were conducted under both UV irradiation and dark conditions in parallel so that BODIPY-labeled proteins formed independently of PAL could be discriminated. Figure 3 shows that diazirine 3 enabled crosslinking of PNA
Figure 3. Products for PAL reactions of probes 1–3 (10 mm) with a mixed solution containing 0.5 mg of PNA and 50 mg of HeLa cell lysate. A reaction condition involving UV irradiation is shown as UV+ and a dark condition as UV . Left: in-gel fluorescence imaging data. Right: Coomassie Brilliant Blue (CBB) staining data.
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CHEMBIOCHEM COMMUNICATIONS with high selectivity. In contrast, benzophenone 1 and aryl azide 2 yielded a multitude of photoadducts, which made it difficult to identify whether PNA was specifically labeled. The results showed that, among the three photoaffinity probes with different photoreactive groups, only diazirine 3 was able to achieve the most selective photoaffinity labeling of PNA, which represented low-affinity binding proteins. The high selectivity observed for the diazirine probe is remarkable, given that the binding affinity of PNA with lactose is very weak (Kd value of 770 mm). Taken together, our results revealed considerably different outcomes of PAL with the three photoreactive groups when crosslinking a low-affinity protein. Diazirine 3 was characterized by the fastest initial rate of reaction, rapid termination of reaction, and low ligand-independent reactivity. In an ideal PAL reaction, the photoactivated probe should rapidly crosslink with any proximal amino acid residues or should be quenched by solvent before diffusing to crosslink with nonspecific proteins. The reactivity features observed for the diazirine probe in this study are consistent with the ideal mechanism of PAL and provide a basis for selective photoaffinity labeling of PNA in cell lysate. Although the drawback of using the diazirine probe is its low crosslinking efficiency, it should be possible to address the problem by using a higher concentration for probe 3, given its highly selective reactivity. Aryl azide 2 provided the best results, based on the fast initial rate of reaction and high crosslinking yields in PAL experiments with a single protein. However, high levels of ligand-independent reactivity were also observed with an increasing concentration of aryl azide control probe 5, which explained the apparently low crosslinking selectivity in PAL experiments in cell lysate. Optimization of the probe concentration and reaction time are thus required for successful use of the aryl azide probe for capturing lowaffinity proteins. The crosslinking yields of benzophenone 1 were lower than those for aryl azide 2 or diazirine 3, due to its slow reaction rate. It appears difficult to use the benzophenone probe to target low-affinity proteins in cellular context. Employing higher probe concentrations or longer reaction times would result in low selectivity while improving crosslinking yields. The alkyl diazirine group has not been the first choice for a photoreactive group in many PAL studies because of the generally low crosslinking yields and the need for synthetic preparation.[1d, 5f] Our findings bring new light to the utility of the diazirine group in PAL studies, particularly in the exploration of low-affinity binding proteins, which remains a challenging task with conventional affinity purification methods. In summary, we designed and synthesized a set of carbohydrate-based photoaffinity probes to compare the effects of different photoreactive groups on the efficiency and selectivity of photoaffinity labeling a low-affinity binding protein. We showed that, although the diazirine probe gave low crosslinking yields, it displayed highly ligand-dependent reactivity consistent with the principle of PAL. Moreover, we demonstrated the strikingly different outcome of using different photoreactive groups for PAL experiments in cell lysate: only the diazirine probe achieved highly selective crosslinking of a low-affinity binding protein, which could not be unambiguously detect 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org ed by aryl azide or benzophenone probes. Our findings should be relevant to the future design of photoaffinity probes toward rapid identification of small-molecule binding proteins.
Experimental Section Experimental details for the synthesis of chemical probes, photocrosslinking reactions, and their analyses are provided in the Supporting Information.
Acknowledgements This work was funded in part by a young investigator grant from the Human Frontier Science Program Foundation. S.O. is supported by the Ajinomoto scholarship, and R.Y. is supported by the Teijin-Kumura scholarship. Keywords: binding proteins · carbohydrates · chemical probes · photoaffinity labeling · photoreactive groups [1] a) V. Chowdhry, F. H. Westheimer, Annu. Rev. Biochem. 1979, 48, 293 – 325; b) R. J. Guillory, Pharmacol. Ther. 1989, 41, 1 – 25; c) J. Brunner, Annu. Rev. Biochem. 1993, 62, 483 – 514; d) F. Kotzyba-Hibert, I. Kapfer, M. Goeldner, Angew. Chem. Int. Ed. Engl. 1995, 34, 1296 – 1312; Angew. Chem. 1995, 107, 1391 – 1408; e) Y. Tanaka, M. R. Bond, J. J. Kohler, Mol. BioSyst. 2008, 4, 473 – 480; f) P. P. Geurink, L. M. Prely, G. A. van der Marel, R. Bischoff, H. S. Overkleeft, Top. Curr. Chem. 2012, 324, 85 – 113. [2] a) S. Sato, A. Murata, T. Shirakawa, M. Uesugi, Chem. Biol. 2010, 17, 616 – 623; b) S. Ziegler, V. Pries, C. Hedberg, H. Waldmann, Angew. Chem. Int. Ed. 2013, 52, 2744 – 2792; Angew. Chem. 2013, 125, 2808 – 2859. [3] a) Y. Nakamura, R. Miyatake, M. Ueda, Angew. Chem. Int. Ed. 2008, 47, 7289 – 7292; Angew. Chem. 2008, 120, 7399 – 7402; b) A. Kawamura, S. Hindi, D. M. Mihai, L. James, O. Aminova, Bioorg. Med. Chem. 2008, 16, 8824 – 8829; c) K. Sakurai, R. Yamada, A. Okada, M. Tawa, S. Ozawa, M. Inoue, ChemBioChem 2013, 14, 421 – 425. [4] Recently, the use of multivalent photoaffinity probes has been reported as a promising approach to overcome the low affinity of the probe. See: a) L. Ballell, M. van Scherpenzeel, K. Buchalova, R. M. J. Liskamp, R. J. Pieters, Org. Biomol. Chem. 2006, 4, 4387 – 4394; b) T.-C. Chang, C.H. Lai, C.-W. Chien, C.-F. Liang, A. K. Adak, Y.-J. Chuang, Y.-J. Chen, C.-C. Lin, Bioconjugate Chem. 2013, 24, 1895 – 1906. [5] a) G. Dormn, G. D. Prestwich, Biochemistry 1994, 33, 5661 – 5673; b) S. A. Fleming, Tetrahedron 1995, 51, 12479 – 12520; c) A. Blencowe, W. Hayes, Soft Matter 2005, 1, 178 – 205; d) M. Hashimoto, Y. Hatanaka, Eur. J. Org. Chem. 2008, 2513 – 2523; e) J. Das, Chem. Rev. 2011, 111, 4405 – 4417; f) L. Dubinsky, B. P. Krom, M. M. Meijler, Bioorg. Med. Chem. 2012, 20, 554 – 570; g) A. Kawamura, D. M. Mihai in Methods in Molecular Biology, Vol. 803 (Eds.: G. Drewes, M. Bantscheff), Humana Press, Totowa, NJ, 2012, pp. 65 – 75. [6] M. S. Rizk, X. Shi, M. S. Platz, Biochemistry 2006, 45, 543 – 551. [7] a) P. J. Weber, A. G. Beck-Sickinger, J. Pept. Res. 1997, 49, 375 – 383; b) J. J. Tate, J. Persinger, B. Bartholomew, Nucleic Acids Res. 1998, 26, 1421 – 1426; c) J. T. Bush, L. J. Walport, J. F. McGouran, I. K. H. Leung, G. Berridge, S. S. van Berkel, A. Basak, B. M. Kessler, C. J. Schofield, Chem. Sci. 2013, 4, 4115 – 4120. [8] a) M. Wiegand, T. K. Lindhorst, Eur. J. Org. Chem. 2006, 4841 – 4851; b) C.-Y. Jiao, I. D. Alves, V. Point, S. Lavielle, S. Sagan, G. Chassaing, Bioconjugate Chem. 2010, 21, 352 – 359; c) P. P. Geurink, B. I. Florea, G. A. Van der Marel, B. M. Kessler, H. S. Overkleeft, Chem. Commun. 2010, 46, 9052 – 9054; d) Y. Nakamura, S. Inomata, M. Ebine, Y. Manabe, I. Iwakura, M. Ueda, Org. Biomol. Chem. 2011, 9, 83 – 85; e) B. Albertoni, J. S. Hannam, D. Ackermann, A. Schmitz, M. Famulok, Chem. Commun. 2012, 48, 1272 – 1274; f) H. Fuwa, Y. Takahashi, Y. Konno, N. Watanabe, H. Miyashita, M. Sasaki, H. Natsugari, T. Kan, T. Fukuyama, T. Tomita, T. Iwatsubo, ACS Chem. Biol. 2007, 2, 408 – 418.
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CHEMBIOCHEM COMMUNICATIONS [9] H. Lis, N. Sharon, Chem. Rev. 1998, 98, 637 – 674. [10] K. J. Neurohr, D. R. Bundle, N. M. Young, H. H. Mantsch, Eur. J. Biochem. 1982, 123, 305 – 310. [11] a) F. Himo, T. Lovell, R. Hilgraf, V. V. Rostovtsev, L. Noodleman, K. B. Sharpless, V. V. Fokin, J. Am. Chem. Soc. 2005, 127, 210 – 216; b) M. Meldal, C. W. Tornøe, Chem. Rev. 2008, 108, 2952 – 3015; c) J. E. Hein, V. V. Fokin, Chem. Soc. Rev. 2010, 39, 1302 – 1315. [12] A. E. Ruoho, H. Kiefer, P. E. Roeder, S. J. Singer, Proc. Natl. Acad. Sci. USA 1973, 70, 2567 – 2571. [13] In this study, we chose to conjugate BODIPY-azide with the probe-labeled PNA in gel rather than prior to gel loading based on our previous
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org findings: significant sample loss accompanied the CuAAC reaction subsequent to PAL and prior to gel, loading due to precipitation of proteins under CuAAC reaction conditions. This problem was circumvented by performing CuAAC in the gel after separating the PAL reaction mixture by SDS-PAGE. See ref. [3 c].
Received: February 21, 2014 Published online on May 27, 2014
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