Anal. Chem. 2004, 76, 211-217
The Nature of Phosphorylated Chrysin-Protein Interactions Involved in Noncovalent Complex Formation by Electrospray Ionization Mass Spectroscopy Xiao-Lan Chen,† Ling-Bo Qu,*,† Ting Zhang,† Hong-Xia Liu,† Fei Yu,† Youzhu Yu,† Xincheng Liao,† and Yu-Fen Zhao*,†,‡
Department of Chemistry, Key Laboratory of Chemical Biology, Zhengzhou University, Zhengzhou, 450052, P.R. China, and The Key Laboratory for Bioorganic Phosphorus Chemistry, Department of Chemistry School of Life Sciences and Engineering, Tsinghua University, Beijing 100084, P.R. China
In the work described in this paper, chrysin was phosphorylated by a modified Atheron-Todd reaction. The structure of phosphorylated chrysin was determined by elemental analysis, NMR, ESI-MS/MS, and X-ray data. Electrospray ionization results showed that the phosphorylated flavonoids could form noncovalent complexes with many proteins, such as lysozyme, myoglobin, bovine insulin, and cytochrome c, while noncovalent complexes were not detected in the mixed solution of the chrysin and proteins. The research shows that the phosphorylated flavonoids possess relatively stronger affinities and form noncovalent complexes with the proteins more easily than the non-phosphorylated compounds. Cellular functions are often triggered by weak noncovalent enzyme-substrate, protein-ligand, protein-protein, or antibodyantigen interactions.1 It is an established fact that esters of phosphoric acid have wide bioactivities and play a vital role in many biological processes. They appear to be synthesized and to undergo interconversion with great ease in living organisms.2-5 In recent years, flavonoids have attracted increasing interest due to their various beneficial pharmacological effects. Chrysin, a widely distributed flavonoid in nature, has been reported to have many different biological activities, such as antioxidant,6 antiviral,7 anti-diabetogenic,8 and anti-anxiolytic effects.9 Furthermore, chrysin has demonstrated anti-cancer activities.10,11 To improve the biologi†
Zhengzhou University. Tsinghua University. (1) Pramanik, B. N.; Barter, P. L.; Mirza, U. A.; Liu, Y.-h.; Ganguly, A. K. J. Mass Spectrom. 1998, 33, 911. (2) Japan Patent Kokai Tokkyo Koho 59,196897, 1984. (3) Sauers, R. F. British Patent 2,004,282, 1979. (4) E. I. du Pont de Nemours. Austrian Patent 361,246, 1981. (5) Sauers, R. F. U.S. Patent 4,228,109, 1979. (6) Chan, E. C. H.; Patchareewan, P.; Owen, L. W. J. Cardiovasc. Pharmacol. 2000, 35, 326. (7) Lee, J. H.; Kim, Y. S.; Lee, C. K.; Lee, H. K.; Han, S. S. Saengyak Hakhoechi 1999, 30, 34. (8) Shin, J. S.; Kim, Y. S.; Kim, M. B.; Jeong, J. H.; Kim, B. K. Bioorg. Med. Chem. Lett. 1999, 9, 869. (9) Zanoli, P.; Avallone, R.; Baraldi, M. Fitoterapia 2000, 71, 117. (10) Liu, Y. L.; Ho, D. K.; Cassady, J. M. J. Nat. Prod. 1992, 55, 357. (11) Hebtemariam, S. J. Nat. Prod. 1997, 60, 775. ‡
10.1021/ac035027q CCC: $27.50 Published on Web 11/25/2003
© 2004 American Chemical Society
cal activities of chrysin, a number of its derivatives have been prepared.8,12,13 In the work described herein, we selected chrysin, a representative flavone, to synthesize its phosphate ester through a simplified Atheron-Todd reaction. The binding affinities of chrysin and its phosphate ester with proteins were compared using electrospray ionization mass spectroscopy. The development of electrospray ionization and the discovery that highly charged ions of proteins are readily formed have led to dramatic growth in the application of mass spectrometry to biomolecules.14-16 Electrospray ionization is sufficiently gentle to allow the ionization and detection of intact noncovalent complexes between proteins and small molecules and of multiunit protein structures.17,18 The key to success in the study of noncovalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions. To study these noncovalent complexes, solutions of the flavonoid and its phosphate ester with different proteins, such as myoglobin, hen egg white lysozyme (HEWL), cytochrome c, and bovine insulin, were injected individually into an ion trap mass spectrometer. The results implied that phosphorylated flavonoids possess relatively stronger affinities for proteins and form noncovalent complexes with proteins more easily than their non-phosphorlyated compounds. EXPERIMENTAL SECTION Compound 1 (C23H28O10P2, CP) (Scheme 1). A 0.5-g sample of chrysin was added to a solution of 40 mL of dioxane and 10 mL of triethylamine. The mixture was stirred until chrysin was dissolved, and then a solution of diethyl phosphite (DEPH) and CCl4 (0.6 mL of DEPH + 10 mL of CCl4) was added dropwise with vigorous stirring in an ice-water bath. The reaction pro(12) (a) Flavonoids in Health and Disease; Rice-Evans, C. A., Packer, L., Eds.; Marcel Dekker: New York, 1997. (b) Bertrand, A.; Olivier, D. Helv. Chim. Acta 1999, 82, 2201. (13) Larget, R.; Lockhart, B.; Renard, P.; Largeron, M. Bioorg. Med. Chem. Lett. 2000, 10, 835. (14) Fenn, J. B.; Meng, C.k.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64. (15) Chait, B. T.; Kent, S. B. H. Science 1992, 257, 1885. (16) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882. (17) Smith, D. L.; Zhang, Z. Mass Spectrom. Rev. 1994, 13, 411. (18) Smith, R. D.; Light-Wahl, K. J. Biol. Mass Spectrom. 1993, 22, 493.
Analytical Chemistry, Vol. 76, No. 1, January 1, 2004 211
Figure 1. Crystal structure of compound 1.
Scheme 1
ceeded for 24 h at room temperature. The resulting salt of triethylamine was precipitated. The filtrate was evaporated in vacuo below 50 °C, and 10 mL of water was added. The solution was extracted with ethyl acetate. The crystalline residues (needles) were separated out. All the spectral data here are reported for the first time. For C23H28O10P2: mp 83-84 °C; 1H NMR (400 MHz, CD3Cl) δ 7.87 (dd, H-2′ and H-6′, J ) 2.0, 7.6 Hz, 2H), 7.54 (m, H-3′, H-4′ and H-5′, 3H), 7.41 (s, H-6, 1H), 7.26 (s, 1H, 8-H), 6.69 (s, 1H, 3-H), 4.33 (m, -CH2, 8H), 1.41 (m, -CH3, 12H); ESI-MS/ MS, m/z 527 [M + H]+, 499, 471, 453, 419, 391, 363, 335. Elemental analysis: found C, 52.10; H, 5.30; P, 11.35. C23H28O10P2 requires C, 52.48; H, 5.36; P, 11.77. The crystal structure of compound 1 is shown in Figure 1. Bovine insulin, myoglobin, cytochrome c, and hen egg white lysozyme (HEWL) were purchased from Sigma Chemical Co. and were used without further purification. Mass Spectrometric Conditions. Solutions of the complexes were analyzed on a Bruke-esquire 3000 fitted with an ion spray source working in the positive mode. Nitrogen was used as drying gas at a flow rate of 4 µL/min. The nebulizer pressure was 17 psi, and the dry gas was 9.00 L/min. The capillary was typically held at 4 kV. Six spectra were averaged, and the rolling average was 7. ICC was set at 30 000. The dissolved samples were continuously infused into the ESI chamber at a flow rate of 4 µL/ min using a Cole-Parmer 744900 syringe pump (Cole-Parmer Instrument Co.). RESULTS AND DISCUSSION Mass spectrometry (MS) has been an indispensable tool for biomedical research involving protein and peptide structural analysis, mainly due to the development of various gentle ionization methods. Nevertheless, the detection of noncovalent com212 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004
Figure 2. (a) Electrospray ionization mass spectrum of myoglobin. Myoglobin solution (My) was prepared by mixing 100 µL of myoglobin (0. 29 mM) with 900 µL of NH4OAc (3.47 mM) at pH 6.9. (b) Electrospray ionization mass spectrum of myoglobin-heme-CP complexes. The solution was prepared by mixing volumes of a 0.31 mM methanol solution of CP and 0.029 mM My. The orifice voltage was 121.8 V. The source temperature was maintained at 150 °C. b, multiply charged ion peaks of the myoglobin-heme complex; bO, multiply charged ion peaks of the myoglobin-heme-CP complex; bOO, multiply charged ion peaks of the myoglobin-heme-2CP complex.
plexes by MS methods has been a challenging task. To successfully perform direct MS analysis of a preformed complex, the MS ionization technique must satisfy some criteria. The control of solution pH, organic solvent, and stabilizing buffer additives is crucial to the detection of noncovalent complexes; moreover, the internal energy transfer to the macromolecule during the ionization process must be minimal to prevent complex dissociation. In this experiment, solvation conditions for myoglobin, hen egg white lysozyme (HEWL), cytochrome c, and bovine insulin were adjusted appropriately; suitable orifice voltages and capillary temperatures were applied individually to different CP-protein complexes. The corresponding ESI mass spectra are shown in Figures 2-5. Because myoglobin is readily available commercially in large quantities, it was used as a “tuning” compound to optimize experimental conditions, to maximize sensitivity for ESI, and to observe noncovalent protein complexes.19 Katta and Chait reported that positive ion ESI-MS of the heme-globin complex in myoglobin could be demonstrated at pH >3.39, while positive ion ESIMS of apomyoglobin (without heme) could be demonstrated at pH
