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β-ketoenole dyes: Synthesis and study as fluorescent sensors for protein amyloid aggregates
Dyes and Pigments 132 (2016) 274e281
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
b-ketoenole dyes: Synthesis and study as fluorescent sensors for protein amyloid aggregates Vladyslava Kovalska a, *, Svitlana Chernii a, Mykhaylo Losytskyy a, Yan Dovbii b, Iryna Tretyakova b, Rafal Czerwieniec c, Victor Chernii b, Sergiy Yarmoluk a, Sergiy Volkov b, y a b c
Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine V.I. Vernadskii Institute of General and Inorganic Chemistry NASU, 32/34 Palladin Av., 03080 Kyiv, Ukraine €tsstr. 31, 93053 Regensburg, Germany Institut für Physikalische und Theoretische Chemie, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 February 2016 Received in revised form 29 April 2016 Accepted 30 April 2016 Available online 9 May 2016
The series of the b-ketoenoles (2E,5Z,7E,9E)-2-(alkylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4ones with variation of the alkylamino tail groups was synthesized and studied as potential probes for the sensing of protein insoluble aggregates - amyloid fibrils. Depending on the structure of the alkylamino group, the dyes could increase their fluorescence intensity in the dozens of times in the presence of insulin fibrils. The compound with a 2-hydroxyethylamino substituent demonstrates the highest fluorescence response (up to 60 times) and good range of insulin fibril detection (1e50 mg/ml). In complexes with fibrils, the dyes possess fluorescence quantum yield values up to 15% and binding constant values of about 2 105 M1. The excitation and emission maxima of b-ketoenoles are located in the range 407 e427 nm and 500e554 nm correspondingly. These compounds are weakly fluorescent when free and slightly sensitive to the native proteins insulin and bovine serum albumin. Thus b-ketoenoles are considered as prospective molecules for the fluorescent detection of amyloid aggregates of proteins. © 2016 Elsevier Ltd. All rights reserved.
Keywords: b-Ketoenole dyes Fluorescent sensing Probes Amyloid fibrils Proteins
1. Introduction One of the most convenient methods for the analysis of biomolecules is the use of extrinsic fluorescent probes that noncovalently bind to them by electrostatic, van der Waals and hydrophobic interactions. A wide range of fluorescent molecules has been developed for high efficient sensing, quantification and visualization of proteins and nucleic acids in both in vitro and in vivo assays [1e4]. The detection and understanding of the spontaneous aggregation of proteins leading to formation of insoluble beta-pleated aggregates (amyloid fibrils) is among the actual targets in the biomedical researches, since these aggregates are connected with the range of harmful human diseases including neurodegenerative types. This causes an interest in the development of new appropriate analytical tools to be used in the study of this process.
* Corresponding author. E-mail address: [email protected] (V. Kovalska). y Deceased author. http://dx.doi.org/10.1016/j.dyepig.2016.04.053 0143-7208/© 2016 Elsevier Ltd. All rights reserved.
Extrinsic fluorescent probes are used for the detection and quantification of amyloid fibrils, monitoring of the kinetics of their formation and study of the factors and agents affecting these processes. For these purposes, amyloid sensitive fluorescent probes Thioflavin T and its derivatives are commonly applied. The histological dyes Congo Red and Chrysamine G are used for the staining and study of amyloid formations in tissues [5e7]. Earlier we discovered mono- and poly-methine cyanine dyes as efficient fluorescent probes for the detection of protein b-pleated aggregates [8e10]. On the base of these dyes the inhibitory assay for the search of the compounds with anti-fibrillogenic activity was developed and applied [8,11]. At the same time further research for amyloidsensitive probes with a high fluorescence response to the fibrillar protein presence is still urgent. One of the necessary requirements for the molecule to be applicable as a fluorescent probe for amyloid formations detection is high affinity of the complex formation between this molecule and the amyloid fibril. As the most probable mode of such complex formation, the insertion of the dye molecule into the groove of the amyloid fibril is suggested [12]. As a result of such binding, the fluorescence response is observed due to the quite rigid fixation of
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the dye molecule, and the polarization of the absorbed light is caused by the same orientation the bound dye molecules. Thus, in order to obtain an efficient response to the presence of the amyloid fibril, the molecule should have a shape complementary to that of the fibril groove and the size fitted to the fibril groove (about 6.5e7 Å) [13] (Fig. 1). In the present work we first studied the b-ketoenole dyes (Scheme 1) as potential fluorescent probes for the sensing of amyloid aggregates of proteins. The b-ketoenoles are the molecules of elongated shape that is suggested as preferable for fitting to the groove of the amyloid fibril [9]; besides they have a rather flexible aliphatic chromophore chain providing the low intrinsic fluorescence intensity of the unbound dye. Unlike the majority of the amyloid-sensitive dyes bearing either positive (Thioflavin T, cyanine dyes) or negative (Congo Red) charge, the molecules of bketoenoles are uncharged. With this aim the series of (2E,5Z,7E,9E)-2-(alkylamino)-6hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-one dyes with variation of alkylamyno substituents was synthesized and the fluorescent properties were characterized for the free dyes as well as in the presence of amyloidogenic proteins lysozyme and insulin in the native and aggregated form. The range of detection of the amyloid aggregates with the most efficient dye was determined. Besides, the fluorescent sensitivity of b-ketoenole dye to the serum albumin able to bind the variety of the small molecules was studied for the comparison. 2. Materials and methods 2.1. Synthesis and characterization of the dyes The general procedure of (2E,5Z,7E,9E)-2-(alkylamino)-6hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-ones synthesis is as follows. Mixture of the 4-hydroxy-6-methyl-3-((2E,4E)-5-phenylpenta2,4-dienoyl)-2H-pyran-2-one [14] (5 mmol) in DMF (10 mL) with an excess of the amine (5.5 mmol) was heated at 100 C for 30 min; evolution of carbon dioxide and changes in color from light yellow to red were observed. After cooling the reaction mixture was precipitated with water (20 mL). The resulting solid was filtered, washed with water (2 10 mL) and then crystallized from DMFEtOH mixture (70:30). The crystals were collected by vacuum filtration and washed with EtOH (2 10 mL) and then dried. All obtained compounds are yellow-orange fine-crystalline substances. Structures of investigated compounds were confirmed with IR, 1 H NMR, elemental analysis and MS. 1. (2E,5Z,7E,9E)-6-hydroxy-10-phenyl-2-(propylamino)deca2,5,7,9-tetraen-4-one Yield: 67%. M.p.: 255e259 C. IR cm1 (KBr):
Fig. 1. Arrangement of the dye molecule in the fibrillar groove [12].
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2960(w), 2932(w), 2864(w), 1632(w), 1592(s), 1540(s), 1428(s), 1316(m), 1280(s), 1132(s), 1084(m), 1036(w), 996(s), 944(m), 876(w), 836(w), 808(m), 748(m), 688(m), 556(w), 504(w), 424(w). 1 H NMR (CDCl3, 400 MHz) d 14.99 (s, 1H), 10.43 (s, 1H), 7.44 (d, J ¼ 7.4 Hz, 2H), 7.34 (dd, J ¼ 13.4, 6.2 Hz, 2H), 7.26 (dd, J ¼ 9.8, 4.7 Hz, 1H), 7.17 (dd, J ¼ 15.0, 10.8 Hz, 1H), 7.01e6.71 (m, 2H), 6.01 (d, J ¼ 15.1 Hz, 1H), 5.20 (s, 1H), 4.78 (s, 1H), 3.24 (dd, J ¼ 13.3, 6.7 Hz, 2H), 1.98 (s, 3H), 1.65 (dd, J ¼ 14.4, 7.2 Hz, 2H), 1.09e0.96 (m, 3H). Found (%): C, 76.79; H, 7.74; N, 4.64. Anal. Calcd. (%) for C19H23NO2: C, 76.73; H, 7.80; N, 4.71. MS: Found: [MþH]þ 298.186; [C6H5(CH)4CO]þ 157.163; requires: [MþH]þ 298.180; [C6H5(CH)4CO]þ 157.065. 2. (2E,5Z,7E,9E)-6-hydroxy-2-((2-hydroxyethyl)amino)-10phenyldeca-2,5,7,9-tetraen-4-one. Yield: 69%. M.p.: 273e275 C. IR cm1 (KBr): 3020(w), 2928(w), 2864(w), 1596(s), 1540(s), 1412(m), 1300(s), 1112(m), 1072(m), 996(m), 928(w), 808(m), 748(m), 688(m), 556(w), 504(m), 428(w). 1H NMR (CDCl3, 400 MHz) d 14.84 (s, 1H), 10.44 (s, 1H), 7.46 (dd, J ¼ 12.9, 7.2 Hz, 2H), 7.40e7.30 (m, 2H), 7.30e7.22 (m, 1H), 7.18 (dd, J ¼ 15.0, 10.8 Hz, 1H), 6.91 (ddd, J ¼ 23.8, 20.0, 13.2 Hz, 1H), 6.76 (d, J ¼ 15.5 Hz, 1H), 6.00 (d, J ¼ 15.0 Hz, 1H), 5.21 (s, 1H), 4.83 (s, 1H), 3.79 (dt, J ¼ 10.7, 5.2 Hz, 2H), 3.45 (dt, J ¼ 11.4, 5.6 Hz, 2H), 2.29 (s, 1H), 2.01 (s, 3H). Found (%): C, 72.14; H, 7.02; N, 4.63. Anal. Calcd. (%) for C18H21NO3: C, 72.22; H, 7.07; N, 4.68. MS: Found: [MþH]þ 300.186; [HO(CH2)2NHC(CH3)CHCO]þ 128.173; requires: [MþH]þ 300.159; [HO(CH2)2NHC(CH3)CHCO]þ 128,071. 3. (2E,5Z,7E,9E)-2-(allylamino)-6-hydroxy-10-phenyldeca2,5,7,9-tetraen-4-one. Yield: 44%. M.p.: 143e144 C. IR cm1 (KBr): 3076(w), 3020(w), 2928(w), 2852(w), 1692(m), 1552(s), 1428(m), 1284(m), 1132(w), 1092(w), 1040(w), 996(m), 944(m), 808(m), 748(m), 688(m), 564(w), 504(w), 428(w). 1H NMR (DMSO-d6, 400 MHz) d 15.13 (s, 1H), 10.30 (t, J ¼ 6.0 Hz, 1H), 7.52 (d, J ¼ 7.7 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 3H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.06 (dd, J ¼ 9.2, 4.8 Hz, 2H), 6.89 (d, J ¼ 14.2 Hz, 1H), 6.15 (dd, J ¼ 14.0, 6.4 Hz, 1H), 6.02e5.86 (m, 1H), 5.34 (s, 2H), 4.89 (s, 1H), 3.97 (t, J ¼ 5.6 Hz, 2H), 1.98 (s, 3H). Found (%): C, 77.11; H, 7.12; N, 4.70.Anal. Calcd. (%) for C19H21NO2: C, 77.26; H, 7.17; N, 4.74. MS: Found: [MþH]þ 296.161; [CH2CHCH2NHC(CH3)CHCO]þ 124.151; requires: [MþH]þ 296.165; [CH2CHCH2NHC(CH3)CHCO]þ 124.076. 4.(2E,5Z,7E,9E)-2-((3-(dimethylamino)propyl)amino)-6hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-one. Yield: 40%. M.p.: 121e123 C. IR cm1 (KBr): 2948(w), 2868(w), 2820(w), 2760(w), 1872(w), 1572(s), 1544(s), 1432(m), 1284(s), 1152(m), 1104(m), 996(m), 944(m), 836(s), 808(s), 748(s), 688(s), 564(w), 504(m), 420(m). 1H NMR (DMSO-d6, 400 MHz) d 15.19 (s, 1H), 10.32 (s, 1H), 7.52 (d, J ¼ 7.5 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.11e6.98 (m, 2H), 6.88 (dd, J ¼ 15.4, 6.4 Hz, 1H), 6.15 (d, J ¼ 14.0 Hz, 1H), 5.31 (s, 1H), 4.82 (s, 1H), 3.32 (dd, J ¼ 13.0, 6.3 Hz, 2H), 2.25 (t, J ¼ 6.8 Hz, 2H), 2.13 (s, 6H), 1.99 (s, 3H), 1.75e1.57 (m, 2H). Found (%): C, 74.02; H, 8.24; N, 8.19. Anal. Calcd. (%) for C21H28N2O2: C, 74.08; H, 8.29; N, 8.23. MS: Found: [MþH]þ 341.220; [C6H5(CH)4CO]þ 157.150; requires: [MþH]þ 341.222; þ [C6H5(CH)4CO] 157.065. 5. (2E,5Z,7E,9E)-2-(sec-butylamino)-6-hydroxy-10-phenyldeca2,5,7,9-tetraen-4-one. Yield: 36%. M.p.: 159e162 C. IR cm1 (KBr): 3024(w), 2972(w), 1864(w), 1548(s), 1416(m), 1304(s), 1132(s), 1044(m), 966(s), 932(s), 808(s), 748(s), 688(s), 556(w), 500(w), 424(w). 1H NMR (DMSO-d6, 400 MHz) d 15.05 (s, 1H), 10.30 (d, J ¼ 9.4 Hz, 1H), 7.52 (d, J ¼ 7.5 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.2 Hz, 1H), 7.04 (dt, J ¼ 14.3, 7.4 Hz, 2H), 6.88 (d, J ¼ 14.7 Hz, 1H), 6.23e6.08 (m, 1H), 5.31 (s, 1H), 4.81 (s, 1H), 3.68e3.53 (m, 1H), 2.01 (s, 3H), 1.50 (dt, J ¼ 14.0, 6.9 Hz, 2H), 1.16 (d, J ¼ 6.4 Hz, 3H), 0.89 (t, J ¼ 7.3 Hz, 3H). Found (%): C, 77.22; H, 7.97; N, 4.46.Anal. Calcd. (%) for C20H25NO2: C, 77.14; H, 8.09; N, 4.50. MS: Found: [MþH]þ 312.202; [(CH3)CH2CH(CH3)NHC(CH3)CHCO]þ 140.173; requires:
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Scheme 1. Synthesis and structures of the studied b-ketoenole dyes.
[MþH]þ 312.196; [(CH3)CH2CH(CH3)NHC(CH3)CHCO]þ 140.108. 6. (2E,5Z,7E,9E)-6-hydroxy-2-(isobutylamino)-10-phenyldeca2,5,7,9-tetraen-4-one. Yield: 45%. M.p.: 170e173 C. IR cm1 (KBr): 2966(w), 2915(w), 2871(w), 1592(s), 1540(s), 1428(s), 1284(s), 1132(s), 1080(m), 996(s), 940(m), 876(w), 836(w), 808(m), 752(m), 688(m), 504(w), 428(m). 1H NMR (DMSO-d6, 400 MHz) d 15.06 (s, 1H), 10.44 (t, J ¼ 5.9 Hz, 1H), 7.52 (d, J ¼ 7.4 Hz, 2H), 7.37 (dd, J ¼ 14.1, 6.9 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.12e6.98 (m, 2H), 6.89 (t, J ¼ 11.3 Hz, 1H), 6.27e6.03 (m, 1H), 5.32 (s, 1H), 4.85 (s, 1H), 3.15 (t, J ¼ 6.4 Hz, 2H), 1.99 (s, 3H), 1.78 (d, J ¼ 6.4 Hz, 1H), 0.94 (d, J ¼ 6.7 Hz, 6H). Found (%): C, 76.96; H, 8.01; N, 4.39. Anal. Calcd. (%) for C20H25NO2: C, 77.14; H, 8.09; N, 4.50. MS: Found: [MþH]þ 312.199; [(CH3)2CHCH2)NHC(CH3)CHCO]þ 140.157; requires: [MþH]þ 312,196; [(CH3)2CHCH2)NHC(CH3)CHCO]þ 140.108. 7. (2E,5Z,7E,9E)-6-hydroxy-2-((2-methoxyethyl)amino)-10phenyldeca-2,5,7,9-tetraen-4-one. Yield: 65%. M.p.: 153e155 C. IR cm1 (KBr): 2932(w), 2896(w), 2872(w), 1592(s), 1552(s), 1436(s), 1284(s), 1128(s), 996(s), 932(s), 808(s), 756(m), 688(m), 504(w), 424(w). 1H NMR (DMSO-d6, 400 MHz) d 15.15 (s, 1H), 10.30 (s, 1H), 7.52 (d, J ¼ 7.4 Hz, 2H), 7.37 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.14e6.96 (m, 2H), 6.98e6.80 (m, 1H), 6.16 (dd, J ¼ 13.4, 6.8 Hz, 1H), 5.32 (s, 1H), 4.85 (s, 1H), 3.49 (d, J ¼ 6.6 Hz, 4H), 3.30 (s, 3H), 2.00 (s, 3H). Found (%): C, 72.89; H, 7.34; N, 4.49.Anal. Calcd. (%) for C19H23NO3: C, 72.82; H, 7.40; N, 4.47. MS: Found: [MþH]þ 314.181; [C6H5(CH)4CO]þ 157.152; requires: [MþH]þ 314.175; [C6H5(CH)4CO]þ 157.152. 8. (2E,5Z,7E,9E)-6-hydroxy-2-((3-methoxypropyl)amino)-10phenyldeca-2,5,7,9-tetraen-4-one. Yield: 57%. M.p.: 106e107 C. IR cm1 (KBr): 2976(w), 2948(w), 2868(w), 2820(w), 1592(s), 1556(s), 1412(s), 1296(s), 1280(s), 1112(s), 996(s), 940(s), 904(m), 844(w), 808(s), 748(m), 688(m), 504(w), 416(w). 1H NMR (DMSOd6, 400 MHz) d 15.18 (s, 1H), 10.30 (t, J ¼ 5.8 Hz, 1H), 7.59e7.47 (m, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.15e6.97 (m, 2H), 6.89 (dd, J ¼ 14.3, 7.6 Hz, 1H), 6.21e6.01 (m, 1H), 5.31 (s, 1H), 4.85 (d, J ¼ 9.5 Hz, 1H), 3.38 (dd, J ¼ 10.8, 4.7 Hz, 4H), 3.25 (d, J ¼ 2.4 Hz, 3H), 1.99 (s, 3H), 1.78 (dd, J ¼ 12.7, 6.2 Hz, 2H). Found (%): C, 73.45; H, 7.65; N, 4.25. Anal. Calcd. (%) for C20H25NO3: C, 73.37; H, 7.70; N, 4.28. MS: Found: [MþH]þ 328.195; [CH3O(CH2)3NHC(CH3)CHCO]þ 156.157; requires: [MþH]þ 328.191; [CH3O(CH2)3NHC(CH3)CHCO]þ 156.102. 9. (2E,5Z,7E,9E)-2-(heptylamino)-6-hydroxy-10-phenyldeca-
2,5,7,9-tetraen-4-one. Yield: 62%. M.p.: 132e134 C. IR cm1 (KBr): 2956(m), 2928(m), 2848(m), 1592(s), 1540(s), 1416(s), 1304(s), 1128(m), 1084(m), 996(s), 940(m), 804(s), 748(m), 728(m), 688(m), 500(w), 428(w). NMR (DMSO-d6, 300 MHz) d 15.16 (s), 10.35 (s), 7.56 (d, J ¼ 7.3 Hz), 7.40 (t, J ¼ 7.4 Hz), 7.31 (t, J ¼ 7.3 Hz), 7.09 (dt, J ¼ 16.4, 11.3 Hz), 6.91 (d, J ¼ 14.4 Hz), 6.27e6.07 (m), 5.34 (s), 4.87 (s), 3.36e3.24 (m), 2.02 (s), 1.56 (s), 1.31 (d, J ¼ 4.6 Hz), 0.89 (d, J ¼ 7.1 Hz). Found (%):C, 77.95; H, 8.68; N, 3.92. Anal. Calcd. (%) for C23H31NO2: C, 78.15; H, 8.84; N, 3.96. MS: Found: [MþH]þ 354.245; [С7H15NHC(CH3)CHCO]þ 182.276; requires: [MþH]þ 354,243; [С7H15NHC(CH3)CHCO]þ 182.154. 2.2. Insulin and lysozyme aggregates formation Human insulin (Private Joint Stock Company «On the production of insulin «Indar», Ukraine) was dissolved at 340 mM concentration in 0.1 mM HCl solution (pH 2). Fibrils were formed by incubating the protein solution in a thermomixer incubator at 65 C for about 5 h. To prepare lysozyme aggregates, lysozyme of hen egg white (Sigma-Aldrich Co. USA) was dissolved at 1 mM concentration in 0.1 mM HCl solution (pH 2) and incubated in a thermomixer incubator at 65 C for 24 h. 2.3. Preparation of dye solutions and protein solutions
b-ketoenole stock solutions were prepared by dissolving the weighted amount of the dyes at 2 mM concentration in DMSO or DMF. Working solutions of free dyes were prepared by dilution of the dye stock solutions in 50 mM Tris-HCl buffer (pH 7.9) to the 2 mM concentration (if not stated otherwise). The working solutions of dye-proteins complexes were prepared by adding to the dye 2 mM (if not stated otherwise) solution the aliquot of monomer or fibrillar protein stock solution, the protein concentration in the working solution was thus 3,4 mM (if not stated otherwise) for insulin and 4 mM for lysozyme. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich Co. (USA). To prepare the dye-BSA working solution, to the 0.2 mg/ml (3 mM) BSA solution in 50 mM Tris-HCl buffer (pH 7.9) the aliquot of the dye stock solution was added, the dye concentration in the working solution was thus 2 mM.
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2.4. Photophysical measurements Absorption spectra were measured using a Specord M-40 (Germany, Carl Zeiss) spectrophotometer. Fluorescence excitation and emission spectra were registered with a CaryEclipse (Varian, Australia) or a Fluorolog 3 (Horiba Jobin Yvon) steady-state fluorescent spectrophotometer. Diluted liquid solutions were measured in standard quartz cells (1 1 cm). Thin polymer films were prepared by spin-coating a solution of poly(methyl methacrylate) (PMMA) in dichloromethane containing about 1 wt % (relative to the PMMA content) onto quartz glass substrates. Fluorescence emission of PMMA films and CH2Cl2 solutions was excited at 420 nm; for the other samples the excitation wavelength was set at the maximum of excitation spectrum of corresponding dye solution. The emission quantum yields for PMMA films and CH2Cl2 solutions were determined directly using a Hamamatsu C9920-02 system equipped with a Spectralon® integrating sphere, while for the dyes 2 and 4 (2 mM) in the presence of fibrillar insulin (13.6 mM) it was estimated by a relative method using Rhodamine 6G in ethanol as standard reference (quantum yield value 0.95) [15]. All measurements were performed at ambient temperature. 2.5. Estimation of equilibrium constants of the dye-to-fibril binding To estimate the equilibrium constant of the dyes 2 and 4 binding to fibrillar insulin, fluorescent titration of the dyes (2 mM) upon addition of 0e45 mM of fibrillar insulin was performed. Taking into account only the points for the concentrations 2 mM of protein molecules and higher, we could consider the concentration of the binding sites to be much higher than this of the dyes and thus the concentration of the free protein to be roughly equal to its total concentration. Further, the equation for the equilibrium constant K of dye-fibril binding could be written as:
Cd =Cbd ¼ 1 þ ðK CF Þ1
(1)
where Cd, Cbd and CF are total dye, fibril-bound dye and fibrillar protein concentrations respectively. Further, let us consider the totally unbound and totally bound with fibrils dye solution to have the fluorescence intensity I0 and Imax respectively. In this case the measured dye fluorescence intensity I at the fibrillar protein concentration CF could be written as I ¼ I0 (Cd e Cbd)/Cd þ Imax Cbd/Cd, that can be transformed into:
Cd =Cbd ¼ ðImax I0 Þ=ðI I0 Þ
(2)
Together with (1), (2) gives
I I0 ¼ A K CF =ð1 þ K CF Þ
(3)
A being the denotation for (ImaxI0) difference. Thus the experimental dependence of II0 on CF was approximated with the equation (3), A and K being obtained as approximation parameters. Accounting for several assumptions made, the obtained K value could be regarded as a rough estimation of the binding constant rather than its precise value. Besides it should be reminded that the estimated binding constant is only an apparent value calculated with respect to protein globule concentration and not this of the binding sites that is unknown; actually the estimation of binding constant with respect to protein globule concentration is common for the ligand-fibril binding studies. 2.6. Computer simulations of the dye 2 dimensions To estimate the dimensions of the dye 2, geometry optimization of the dye structure was first performed using the PM3 method
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from the HyperChem 6.03 program package. Further the isosurface with the total charge density 0.002 that characterizes the molecular dimensions was built; linear dimensions i.e. length, height and width of the obtained isosurface were then estimated. 3. Results and discussion 3.1. Synthesis of the b-ketoenole dyes In present work we obtained a series of new compounds by the pyran ring opening reaction of 4-hydroxy-6-methyl-3 - ((2E, 4E) -5phenylpenta-2,4-dienoyl) -2H-pyran-2-one with primary aliphatic amines (Scheme 1). The mechanism of this reaction was studied in Refs. [14,16], it occurs through the nucleophilic attack at the 6-C of the pyran cycle leading to its next opening and decarboxilation. We have found that in the first step of this reaction the interaction of 4hydroxy-6-methyl-3-((2E, 4E)-5-phenylpenta-2,4-dienoyl)-2H-pyran-2-one with amines (Scheme 1) leads to the formation of the corresponding salt. Upon heating it dissociates and at the same time the amino group attacks a carbon atom of the methyl group in the pyran ring, leading to its ring-opening and decarboxylation [14,16]. In the case of n-alkyl amines, formation of the corresponding salts, opening up of the ring and decarboxylation occurs quite easily, but iso-amines react considerably worse and require higher reaction temperature. In the case of the tert-butylamine, the reaction stopped at the stage of the salt formation, it was not followed by the decyclization of the pyran ring (Scheme 1), in the case of a-phenyl ethyl amine the reaction path was similar. Molecules of the studied b-ketoenole dyes reveal two different regions: a highly hydrophobic phenyl polyen group and a hydrophilic unit consisting of the keto-enol-amine fragment. For related compounds the existence of the set of tautomeric forms of the ketoenol fragment was established by NMR [16]. The obtained compounds also exist as mesomero-tautomeric forms, as it is suggested by rather complicated 1H NMR. The form A (Scheme 1) is considered to be the dominant one for the studied b-ketoenoles in CDCl3 solution. In the 1H NMR spectra except of the major groups of peaks (corresponding to the form A) there are additional low-intensity peaks with similar chemical shifts and morphology. These signals point towards the presence of ca. 3e6% of a minor component and are attributed to another tautomer form B. Besides in the 10e11 ppm region near the main peak of the amino group proton (form A) we observed a minor signal attributed to the proton of the form B. Recording 1H NMR spectra in different solvents including TFA showed that signals of amino groups depend on the nature of the solvent. The maximum content of the form B is observed in chloroform and it decreases in the sequence chloroform > DMSO > benzene > acetone. In TFA, the signal of only one amino group is observed that indicated the presence of a strong hydrogen bond corresponding to the form A. This suggests that the amino tautomers are observed in the spectra. According to LC/MS data the molecular ions of the main compound and the minor component have the same molecular weight that also supports the suggestion about the tautomers of the compounds (data not presented). 3.2. Rigidochromic fluorescence of free b-ketoenole dyes In order to gain basic information about the photophysical properties of the b-ketoenole dyes under study and assess their potential as fluorescent markers, emissions of free dyes were studied in diluted dichloromethane solutions and in thin poly(methyl mathacrylate) polymer films. The emission spectra are
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reproduced in Fig. 2 and the respective quantum yields and band maxima are listed in Table 1. The emission spectra are broad and partly structured. In liquid samples, the emissions are relatively weak, with quantum yields of a few percent, and are centered at lem,max of about 560 nm, as determined for the dye 1. Minor deviations from this value, e.g. lem,max ¼ 553 nm for dye 3, result probably from slightly different interactions with the dipoles of solvent molecules when the amine at the position 2 is decorated with different (hetero)aliphatic group R. In polymer films, the emission spectra are shifted to shorter wavelengths and the quantum yields (4PL of 10e15%) are significantly larger than in liquid samples. Such blue shifts of emission in media characterized by increased rigidity are frequently observed for luminescent materials undergoing large geometry changes upon electronic excitation. Such structural rearrangements lead to a smaller energy gap between the excited state and the electronic ground state and thus to a red shift of emission and efficient radiationless relaxations to the ground state [17,18]. The geometry rearrangements in the excited state occur more easily in fluid solution, but they are partly hindered in rigid polymer films. Thus, distinct increase of photoluminescence efficiency is observed in PMMA samples. Similar effects, i.e. rigidity-related enhancement of emission and hipsochromic shifts, are expected to occur for the dye molecules embedded into protein aggregates, which will allow spectral discrimination of a denatured protein from its native form.
3.3. Spectral properties of dyes in buffer and in the presence of native proteins The fluorescent characteristics of the 9 b-ketoenole compounds in the buffer solution and in the presence of insulin, lysozyme and BSA in their native form are presented in Table 2. The excitation maxima for the free form of the studied dyes are located in the range 414e427 nm; emission maxima for all the dyes are in the range 500e554 nm. The addition of insulin, lysozyme and BSA could cause the shift of the excitation and emission spectrum maxima both to longer or shorter wavelengths by up to 35 nm. Excitation maxima in the presence of proteins are in the range 405e428 nm, while the positions of the emission maxima are at 510e571 nm. The dyes both free and in the presence of proteins display broad fluorescence bands, their maximum position could shift depending presumably on tautomer/isomer conformation of the molecule. The large values of the shift between the excitation and emission maxima for the free dyes and the dyes in the presence of proteins (98e190 nm) should be mentioned. The free b-ketoenole dyes in aqueous solution show very weak fluorescence with the emission intensity of 0.8e10.9 a.u. The enhancement of the fluorescence intensity of the dyes in the presence of the native proteins does not exceed 10 times, and formed protein-dye complexes are of the quite low intensity. Generally the low sensitivity of the dyes to the serum albumin (protein that is responsible for the binding and transportation of a variety of small molecules in blood) should be mentioned. Dye 2 having the hydroxyalkyl tail gives a fluorescence response in the presence of both lysozyme and albumin. The dyes 1, 2, 4 and 8 demonstrate a quite pronounced emission increase (in 3.7e10 times) in the presence of lysozyme. The presence of native lysozyme leads to the fluorescence response for the majority of the studied dyes. This protein contains a large number of charged amino acid residues (8 negatively charged residues and 18 positively charged ones), thus pronounced binding of the dye molecules to this protein is suggested to be driven by electrostatic interaction. Although the b-ketoenole molecules are uncharged, their transition to a zwitterionic tautomer (Fig. 3) could be responsible for the dyes affinity to the highly charged protein. 3.4. Spectral properties of dyes in the presence of fibrillar proteins
Fig. 2. Ambient temperature fluorescence spectra of b-ketoenole dyes 1e9 in PMMA polymer films and in air-saturated dichloromethane solutions recorded at an excitation wavelength of lexc ¼ 420 nm.
The fluorescent characteristics of the series of 9 b-ketoenole compounds in the presence of fibrillar aggregates of insulin and lysozyme are shown in Table 3. For the majority of the studied b-ketoenole dyes, the addition of the fibrillar insulin results in the long-wavelength shift of the excitation maximum wavelength by up to 28 nm (except of the dye 9 with the 2 nm short-wavelength shift). The short-wavelength shifts of the emission maximum wavelength for up to 17 nm were also observed for the majority of the dyes except 4 (almost no change), 6 and 8 (35-nm and 19-nm long-wavelength shift respectively). The presence of fibrillar insulin leads to the enhancement of the dye fluorescence intensity (IF) as compared to that of both free dye (I0) and the dye in the presence of native insulin (IM). The highest values of the IF/IM ratio were observed for the dyes 1 and 2 containing “short” unbranched propylamino and hydroxyethylamino groups as substituents (22 and 61 times respectively) as well as for the dye 8 with methoxypropylamino substituent (26 times). The fluorescence response with IF/IM between 10 and 13 times was also observed for the dyes 3, 4 and 7 containing allylamino, dimethylaminopropylamino and methoxyethylamino substituents. The other dyes did not demonstrate significant IF/IM values. To estimate
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Table 1 Emission maxima (lem,max) and quantum yields (4PL) of b-ketoenole dyes 1e9 in PMMA polymer films and in air-saturated dichloromethane solutions. Compound
lem,max (CH2Cl2), nma
4PL(CH2Cl2), %b
lem,max (PMMA), nma
4PL(PMMA), %b
1 2 3 4 5 6 7 8 9
560 555 553 560 560 560 555 560 560
2 3 3 2 2 2 3 2 2
512 512 508 516 517 512 512 512 522
11 15 12 13 13 11 12 11 10
a b
±5 nm. ±1%.
Table 2 Spectral-luminescent properties of b-ketoenole dyes in the presence of the native proteins. Dye
1 2 3 4 5 6 7 8 9
Free dye
With insulin
With lysozyme
With BSA
lex, nm
lem, nm
I0, a.u.
lex, nm
lem, nm
IM, a.u.
lex, nm
lem, nm
IM, a.u.
lex, nm
lem, nm
IM, a.u.
427 426 415 414 417 407 415 418 413
542 539 545 523 554 500 535 518 549
1.7 1.3 2.6 0.8 5.5 4.1 3.0 3.0 10.9
413 426 415 427 415 415 427 420 416
542 539 520 526 539 553 522 536 604
3.7 1.6 4.7 2.0 6.8 6.2 4.3 3.4 0.7
427 426 419 427 415 415 423 427 410
525 540 541 526 543 571 525 525 611
17.7 10.0 1.9 7.7 10.0 9.3 2.6 17.7 3.2
427 410 415 418 417 408 415 428 435
536 540 530 523 550 555 535 531 611
1.0 7.0 5.0 0.8 3.8 5.0 4.0 2.0 15.5
lex, lem e maximum wavelength of fluorescence excitation and emission spectra. I0 (IM) e fluorescence emission intensity of the dye in free state (in the presence of native protein) at the maximum wavelength of the emission spectrum. a.u. e arbitrary units.
(Table 1); thus we can suppose that the dye binding to the fibril leads to the rigid fixation of its molecule as it also takes place in PMMA film. To characterize the fibril binding of the dyes of the studied class, for the above mentioned dyes 2 and 4 the titration with fibrillar insulin was performed; the dependence of the dye fluorescence intensity on the concentration of the added fibrils allowed us to
Fig. 3. Zwitterionic tautomer form of b-ketoenole dye.
Table 3 Spectral-luminescent properties of b-ketoenole dyes in the presence of the aggregated proteins. Dye
1 2 3 4 5 6 7 8 9
With fibrillar insulin
With fibrillar lysozyme
lex, nm
lem, nm
Stokes shift, nm
IF, a.u.
IF/IM
lex, nm
lem, nm
Stokes shift, nm
IF, a.u.
IF/IM
441 430 436 436 437 435 434 436 411
528 531 528 524 528 535 525 537 538
87 101 92 88 91 100 91 101 127
80.0 97.0 49.7 25.0 20.9 17.0 45.8 89.0 16.7
21.6 60.6 10.6 12.5 3.1 2.7 10.7 26.2 1.5
441 427 426 426 415 408 440 441 413
517 540 532 522 543 555 526 517 532
76 113 106 96 128 147 86 76 119
10.0 37.0 5.9 27.7 8.5 9.2 22.7 10.0 15.8
0.6 3.7 3.1 3.6 0.8 1.0 8.7 0.6 0.9
lex, lem e maxima of fluorescence excitation and emission spectra.
IF e emission intensity of dye in the presence of fibrillar form of the insulin and lysozyme at the maximum wavelength of the emission spectrum. IF/IM e the ratio of the dye emission intensities in the presence of fibrillar and native form of corresponding protein. a.u. e arbitrary units.
the fluorescence quantum yield that the dyes of the studied class can achieve in the presence of fibrillar proteins, we have calculated this value for the fibrillar insulin complexes of the dyes 2 possessing the highest IF and IF/IM values (Table 3) and the less sensitive dye 4 for the comparison. It was revealed that the fluorescence quantum yield value for the dyes 2 and 4 in the presence of fibrillar insulin is rather high and equals to 12 and 15% respectively. The obtained values are close to these of the dyes in PMMA film
estimate the equilibrium constant of the dye to fibril binding. This estimation gave the values of the binding constant K ¼ (2.7 ± 0.9) 105 M1 for the dye 2 and (2.5 ± 1.4) 105 M1 for the dye 4, being thus of the same order of magnitude. As for the fibrillar lysozyme, its presence leads to the longwavelength shift of the excitation spectrum maximum wavelength up to 25 nm, except for the dyes 2, 5, 6 and 9 that practically do not change the maxima positions. At the same time, for the
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emission maximum the wavelength shift was predominantly to the short-wavelength region (up to 25 nm for the dye 1), exceptions being the dyes 2, 4 and 8 (almost no change) and the dye 6 (longwavelength shifts for 55 nm). As for the emission intensity, only for the dyes 2, 3, 4 and 7 the fluorescence sensitivity to the fibrillar lysozyme overpasses that to native protein. The IF/IM values for these four dyes are not significant: the highest value is for the dye 7 (8.7 times), while for the dyes 2, 3 and 4 it is about 3.1e3.7. Other compounds possess the fluorescent sensitivity to lysozyme aggregates equal to or lower than this for the native protein. The insignificant IF/IM value shown by the dyes (that give pronounced response on insulin fibrils) on the presence of aggregated lysozyme is partially connected with the fluorescent sensitivity of these dyes to the native lysozyme (IM value). 3.5. The study of the dye fluorescence “light up” mode In order to clarify the possible mechanisms of the fluorescence intensity increase of b-ketoenoles upon their binding to fibrillar proteins, the absorption (Fig. 4, a), as well as fluorescence excitation and emission (Fig. 4, b) spectra of the dye 2 that is the most sensitive for the fibrillar insulin among the studied compounds were compared for the free dye in buffer, in ethanol and in the presence of fibrillar insulin. The absorption spectrum of the free dye in buffer has its maximum at 334 nm; at the same time the maximum of the excitation spectrum of its weak emission is situated near 390 nm. This feature indicates that the majority of the dye molecules in the buffer exist in a non-fluorescent conformation. Further, addition of the insulin fibrils to the dye buffer solution leads to the decrease in the 334-nm absorption maximum and increase in the 430 nm maximum, the latter maximum is close to the band of the excitation spectrum of the intensive emission of this sample. Thus the increase in dye emission intensity is partially explained by its transition to the fluorescent conformation (together with the possible restriction of the internal motions) upon the binding with fibrillar insulin. Finally, the band with the maximum at 412 nm dominates in the absorption spectrum of the dye 2 in ethanol, its position is close to that of the corresponding fluorescence excitation band. The fluorescence intensity of the free dye in ethanol is noticeably higher than in aqueous buffer (Fig. 4b). The absorption spectrum of the free dye in ethanol is rather close to that of the dye-fibril complex and they both strongly distinguish from that of the free dye in buffer. Thus the removal of the dye from the aqueous medium (changing of the hydrophobicity of the medium) could also
contribute to the increase of dye emission intensity upon interaction with protein fibrils. This could be attributed to the change in the dye isomer/tautomer form, or to the destruction of the possible non-fluorescent dye-water complexes (the last could be analogous to the classical case of the 1,8-ANS dye [1]). Insertion of the molecule of amyloid sensitive dye into the fibrillar groove is suggested to be the most possible mode of the dye binding to the fibril. Using HyperChem program, dimensions of dye 2 were estimated to be about 23 Å 7.8 Å 4.8 Å, that is characteristic for the dyes of the studied series. Thus the studied dyes fit to the fibrillar groove formed by the b-pleated structure of the fibril, the width of which is believed to be equal to 6.5e7Å [13]. We supposed that in the fluorescent dye-insulin fibril complex the dye molecules “prefer” to locate mainly in the groove along the long axis of the fibril (Fig 1). The nature and structure of aminoalkyl tail group is important for the “tight fitting” of the dye molecule. As it was described above, the b-ketoenoles possess less fluorescent sensitivity to fibrillar lysozyme compared to insulin. Some of the dyes responded even better to the presence of native lysozyme than to its aggregates containing grooves as “suitable binding place” for the dye molecules. This could be explained by the suggestion about electrostatic interaction as driving force of the binding sites for the dye (as zwitterionic form) to this highly charged protein. In this case the location and availability of the charged amino acids determine to a large extent the binding of the bketoenole molecules. The sensitivity to lysozyme also could be partially connected with the high content of b-sheet regions in native protein, while bovine serum albumin and insulin have predominantly a-helical motifs [19,20]. 3.6. Application of dye 2 for fibrillar insulin detection: detection limit and linear detection range To examine the applicability of the dye 2 bearing 2-hydroxyethyl group as an amyloid-sensitive probe for the quantification of amyloid insulin, we performed titration of the 2 mM dye solution with increasing amounts of the aggregated protein (Fig. 5). The lower limit for the fibrillar insulin detection by the dye 2 was determined as equal to the insulin concentration leading to the two times dye fluorescence intensity increase. The upper limit of the detection range was determined as the maximum concentration where the dependence of dye fluorescence intensity on the fibrils concentration is still linear. Thus the 2 mM concentration of the dye 2 allows fluorescent detection of the insulin amyloid fibrils in the concentration range 0.2e9 mM (1e50 mg/ml) (R ¼ 0,97703; Fig. 5); the upper limit of the fibrillar insulin detection by the dye 2 thus
Fig. 4. Absorption (a) and fluorescence excitation (b; left) and emission (b; right) spectra of the dye 2 (5 mM) in 50 mM TRIS-HCl buffer, pH 7,9 in free state (solid) and in the presence of 13.6 mM of fibrillar insulin (dash), and in ethanol (dot). Fluorescence emission was excited at the maximum wavelength of the corresponding excitation spectrum.
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form, or to the destruction of the possible non-fluorescent dyewater complexes. The fixation of the dye molecules in the groove of the amyloid fibril makes additional impact to the increase in the dye emission intensity. The b-ketoenole dyes are considered as prospective fluorescent molecules for the further design of probes for the detection of the b-pleated protein aggregates and investigation of the aggregation reaction. Acknowledgement This work was supported by the H2020-MSCA-RISE-2014-RISE645628 e METCOPH project. References
Fig. 5. Titration of dye 2 by fibrillar insulin. The linear concentration range 0.2e9 mM (1e50 mg/ml) (linear, R ¼ 0.977). The 2 mM dye solution was used.
exceeds that for the commonly used amyloid-specific dye Thioflavin T (0.5e25 mg/ml) [21]. It should be also mentioned that the large shifts between the excitation and emission maxima for the fibril-bound dye (about 100 nm), good fluorescence intensity increase, rather high quantum yield and sufficient detection range make the dye 2 promising as a probe for fluorescent detection of the fibrillar aggregates. 4. Conclusion The series of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10phenyldeca-2,5,7,9-tetraen-4-ones) b-ketoenole dyes was first synthesized and characterized, their absorption and emission properties were studied for the free dyes and in the presence of proteins. b-Ketoenoles are weakly fluorescent when free, their excitation maxima are located in region 407e427 nm and fluorescent maxima in region 500e549 nm. The dyes exhibit a very weak response to the presence of native globular proteins insulin and bovine serum albumin, while their fluorescent sensitivity to native lysozyme is more pronounced (emission intensity increase up to 10 times was observed). In the presence of amyloid fibrils of insulin a strong increase in the emission intensity from the dyes is observed (up to 60 times for the dyes bearing the 2-hydroxyethylamino groups). The fluorescent sensitivity of the dyes to fibrillar proteins is noticeably affected by their functional substituents. The dyes possess moderate equilibrium constant of binding to fibrillar insulin about (2.7 ± 0.9) 105 M1 for compound 2. Dye 2 demonstrated a good linear range (1e50 mg/ml) of insulin fibril detection, a large value of the shift between excitation and emission maxima (about 100 nm) and a quantum yield in the complex with fibrillar insulin which reaches 12%. The b-ketoenoles are less sensitive to the amyloid aggregates of lysozyme compared to that of insulin, top emission increase in 8.7 times is observed for the dye 7 which possesses a 2methoxyethylamino fragment. This feature could be explained by the Coulombic interactions as a main driving force of the dye molecules binding with this protein. The increase in the fluorescence intensity of the dyes upon binding to proteins is at least partially explained by the change of the media (removing the dye from the water). That leads either to the transition of dye to the fluorescent isomer or/and tautomer
[1] Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools protein characterization. Pharm Res 2008;25:1487e99. [2] Johnson J, Spence M. The molecular probes handbookea guide to fluorescent probes and labeling technologies. eleventh ed. 2010. [3] Tokar V, Losytskyy M, Ohulchanskyy T, Kryvorotenko D, Kovalska V, Balanda A, et al. Styryl dyes as two-photon excited fluorescent probes for DNA detection and two-photon laser scanning fluorescence microscopy of living cells. J Fluoresc 2010;20:865e72. [4] Wolfbeis O, Demchenko A. Optimized dyes for protein and nucleic acid detection advanced fluorescence reporters in chemistry and biology. III applications in sensing and imaging, chapter: optimized dyes for protein and nucleic acid detection. Springer Berlin Heidelberg; 2014. p. 161e99. [5] Naiki H, Higuchi K, Hosokawa M, Takeda T. Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal Biochem 1989;177:244e9. [6] Klunk W, Wang Y, Huang G, Debnath M, Holt D, Mathis C. Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain. Life Sci 2001;69:1471e84. [7] Schmidt M, Schuck T, Sheridan S, Kung M, Kung H, Zhuang Z, et al. The fluorescent congo red derivative,trans,trans)-1-bromo-2,5-bis-(3hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), labels diverse beta-pleated sheet structures in postmortem human neurodegenerative disease brains. Am J Pathol 2001;159:937e43. [8] Volkova K, Kovalska V, Inshin D, Slominskii Y, Tolmachev O, Yarmoluk S. Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay. Biotech Histochem 2011;86:188e91. [9] Kovalska V, Losytskyy M, Tolmachev O, Slominskii Y, Segers-Nolten G, Subramaniam V, et al. Tri- and pentamethine cyanine dyes for fluorescent detection of a-synuclein oligomeric aggregates. J Fluoresc 2012;22:1441e8. [10] Volkova K, Losytskyy M, Fal K, Derevyanko N, Slominskii Y, Tolmachov O, et al. Hydroxy and methoxy substituted thiacarbocyanines for fluorescent detection of amyloid formation. J Fluoresc 2011;21:775e84. [11] Kovalska V, Losytskyy M, Chernii V, Volkova K, Tretyakova I, Cherepanov V, et al. Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands. Bioorg Med Chem 2012;20:330e4. [12] Volkova K, Kovalska V, Balanda A, Losytskyy M, Golub A, Vermeij R, et al. Specific fluorescent detection of fibrillar alpha-synuclein using mono- and trimethine cyanine dyes. Bioorg Med Chem 2008;16:1452e9. [13] Backlund F, Wigenius J, Westerlund F, Inganas O, Solin N. Amyloid fibrils as dispersing agents for oligothiophenes: control of photophysical properties through nanoscale templating and flow induced fibril alignment. J. Mater Chem 2014;2:7811e22. [14] Aït-Baziz N, Rachedi Y, Hamdi M, Silva A, Balegroune F, Thierry R, et al. 4hydroxy-6-methyl-3-(5-phenyl-2E,4E-pentadien-1-oyl)-2H-pyran-2-one: synthesis and reactivity with amines. J. Heterocycl Chem 2004;41:587e91. [15] Kubin R, Fletcher A. Fluorescence quantum yields of some rhodamine dyes. J. Luminesc 1982;27:455e62. ne M, Spe ziale V, Lattes A. Re activite des [16] Chergui D, Hamdi M, Baboule cinnamoyl-3 pyrones-2 vis a vis des amines primaires. J. Heterocycl Chem 1986;23:1721e4. [17] Yersin H, Rausch AF, Czerwieniec R, Hofbeck T, Fischer T. The triplet state of organo-transition metal compounds. Triplet harvesting and singlet harvesting for efficient OLEDs. Coord Chem Rev 2011;255:2622e52. [18] Czerwieniec R, Kowalski K, Yersin H. Highly efficient thermally activated fluorescence of a new rigid Cu(I) complex [Cu(dmp)(phanephos)]þ. Dalton Trans. 2013;42:9826e30. [19] Ye F, An Y, Qin D, Yang L, She L, Xing R. Spectroscopic study on the effect of crystallization of the hydroxyapatite on the secondary structure of bovine serum albumin. Guang Pu Xue Yu Guang Pu Fen Xi 2007;27:321e4. [20] http://www.uniprot.org/uniprot/P00698-secondarystructure. [21] Kuperman M, Chernii S, Losytskyy M, Kryvorotenko D, Derevyanko N, Slominskii Y, et al. Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: the effect of N,N’-substituent. Anal Biochem 2015;484:9e17.
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
b-ketoenole dyes: Synthesis and study as fluorescent sensors for protein amyloid aggregates Vladyslava Kovalska a, *, Svitlana Chernii a, Mykhaylo Losytskyy a, Yan Dovbii b, Iryna Tretyakova b, Rafal Czerwieniec c, Victor Chernii b, Sergiy Yarmoluk a, Sergiy Volkov b, y a b c
Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine V.I. Vernadskii Institute of General and Inorganic Chemistry NASU, 32/34 Palladin Av., 03080 Kyiv, Ukraine €tsstr. 31, 93053 Regensburg, Germany Institut für Physikalische und Theoretische Chemie, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 February 2016 Received in revised form 29 April 2016 Accepted 30 April 2016 Available online 9 May 2016
The series of the b-ketoenoles (2E,5Z,7E,9E)-2-(alkylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4ones with variation of the alkylamino tail groups was synthesized and studied as potential probes for the sensing of protein insoluble aggregates - amyloid fibrils. Depending on the structure of the alkylamino group, the dyes could increase their fluorescence intensity in the dozens of times in the presence of insulin fibrils. The compound with a 2-hydroxyethylamino substituent demonstrates the highest fluorescence response (up to 60 times) and good range of insulin fibril detection (1e50 mg/ml). In complexes with fibrils, the dyes possess fluorescence quantum yield values up to 15% and binding constant values of about 2 105 M1. The excitation and emission maxima of b-ketoenoles are located in the range 407 e427 nm and 500e554 nm correspondingly. These compounds are weakly fluorescent when free and slightly sensitive to the native proteins insulin and bovine serum albumin. Thus b-ketoenoles are considered as prospective molecules for the fluorescent detection of amyloid aggregates of proteins. © 2016 Elsevier Ltd. All rights reserved.
Keywords: b-Ketoenole dyes Fluorescent sensing Probes Amyloid fibrils Proteins
1. Introduction One of the most convenient methods for the analysis of biomolecules is the use of extrinsic fluorescent probes that noncovalently bind to them by electrostatic, van der Waals and hydrophobic interactions. A wide range of fluorescent molecules has been developed for high efficient sensing, quantification and visualization of proteins and nucleic acids in both in vitro and in vivo assays [1e4]. The detection and understanding of the spontaneous aggregation of proteins leading to formation of insoluble beta-pleated aggregates (amyloid fibrils) is among the actual targets in the biomedical researches, since these aggregates are connected with the range of harmful human diseases including neurodegenerative types. This causes an interest in the development of new appropriate analytical tools to be used in the study of this process.
* Corresponding author. E-mail address: [email protected] (V. Kovalska). y Deceased author. http://dx.doi.org/10.1016/j.dyepig.2016.04.053 0143-7208/© 2016 Elsevier Ltd. All rights reserved.
Extrinsic fluorescent probes are used for the detection and quantification of amyloid fibrils, monitoring of the kinetics of their formation and study of the factors and agents affecting these processes. For these purposes, amyloid sensitive fluorescent probes Thioflavin T and its derivatives are commonly applied. The histological dyes Congo Red and Chrysamine G are used for the staining and study of amyloid formations in tissues [5e7]. Earlier we discovered mono- and poly-methine cyanine dyes as efficient fluorescent probes for the detection of protein b-pleated aggregates [8e10]. On the base of these dyes the inhibitory assay for the search of the compounds with anti-fibrillogenic activity was developed and applied [8,11]. At the same time further research for amyloidsensitive probes with a high fluorescence response to the fibrillar protein presence is still urgent. One of the necessary requirements for the molecule to be applicable as a fluorescent probe for amyloid formations detection is high affinity of the complex formation between this molecule and the amyloid fibril. As the most probable mode of such complex formation, the insertion of the dye molecule into the groove of the amyloid fibril is suggested [12]. As a result of such binding, the fluorescence response is observed due to the quite rigid fixation of
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the dye molecule, and the polarization of the absorbed light is caused by the same orientation the bound dye molecules. Thus, in order to obtain an efficient response to the presence of the amyloid fibril, the molecule should have a shape complementary to that of the fibril groove and the size fitted to the fibril groove (about 6.5e7 Å) [13] (Fig. 1). In the present work we first studied the b-ketoenole dyes (Scheme 1) as potential fluorescent probes for the sensing of amyloid aggregates of proteins. The b-ketoenoles are the molecules of elongated shape that is suggested as preferable for fitting to the groove of the amyloid fibril [9]; besides they have a rather flexible aliphatic chromophore chain providing the low intrinsic fluorescence intensity of the unbound dye. Unlike the majority of the amyloid-sensitive dyes bearing either positive (Thioflavin T, cyanine dyes) or negative (Congo Red) charge, the molecules of bketoenoles are uncharged. With this aim the series of (2E,5Z,7E,9E)-2-(alkylamino)-6hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-one dyes with variation of alkylamyno substituents was synthesized and the fluorescent properties were characterized for the free dyes as well as in the presence of amyloidogenic proteins lysozyme and insulin in the native and aggregated form. The range of detection of the amyloid aggregates with the most efficient dye was determined. Besides, the fluorescent sensitivity of b-ketoenole dye to the serum albumin able to bind the variety of the small molecules was studied for the comparison. 2. Materials and methods 2.1. Synthesis and characterization of the dyes The general procedure of (2E,5Z,7E,9E)-2-(alkylamino)-6hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-ones synthesis is as follows. Mixture of the 4-hydroxy-6-methyl-3-((2E,4E)-5-phenylpenta2,4-dienoyl)-2H-pyran-2-one [14] (5 mmol) in DMF (10 mL) with an excess of the amine (5.5 mmol) was heated at 100 C for 30 min; evolution of carbon dioxide and changes in color from light yellow to red were observed. After cooling the reaction mixture was precipitated with water (20 mL). The resulting solid was filtered, washed with water (2 10 mL) and then crystallized from DMFEtOH mixture (70:30). The crystals were collected by vacuum filtration and washed with EtOH (2 10 mL) and then dried. All obtained compounds are yellow-orange fine-crystalline substances. Structures of investigated compounds were confirmed with IR, 1 H NMR, elemental analysis and MS. 1. (2E,5Z,7E,9E)-6-hydroxy-10-phenyl-2-(propylamino)deca2,5,7,9-tetraen-4-one Yield: 67%. M.p.: 255e259 C. IR cm1 (KBr):
Fig. 1. Arrangement of the dye molecule in the fibrillar groove [12].
275
2960(w), 2932(w), 2864(w), 1632(w), 1592(s), 1540(s), 1428(s), 1316(m), 1280(s), 1132(s), 1084(m), 1036(w), 996(s), 944(m), 876(w), 836(w), 808(m), 748(m), 688(m), 556(w), 504(w), 424(w). 1 H NMR (CDCl3, 400 MHz) d 14.99 (s, 1H), 10.43 (s, 1H), 7.44 (d, J ¼ 7.4 Hz, 2H), 7.34 (dd, J ¼ 13.4, 6.2 Hz, 2H), 7.26 (dd, J ¼ 9.8, 4.7 Hz, 1H), 7.17 (dd, J ¼ 15.0, 10.8 Hz, 1H), 7.01e6.71 (m, 2H), 6.01 (d, J ¼ 15.1 Hz, 1H), 5.20 (s, 1H), 4.78 (s, 1H), 3.24 (dd, J ¼ 13.3, 6.7 Hz, 2H), 1.98 (s, 3H), 1.65 (dd, J ¼ 14.4, 7.2 Hz, 2H), 1.09e0.96 (m, 3H). Found (%): C, 76.79; H, 7.74; N, 4.64. Anal. Calcd. (%) for C19H23NO2: C, 76.73; H, 7.80; N, 4.71. MS: Found: [MþH]þ 298.186; [C6H5(CH)4CO]þ 157.163; requires: [MþH]þ 298.180; [C6H5(CH)4CO]þ 157.065. 2. (2E,5Z,7E,9E)-6-hydroxy-2-((2-hydroxyethyl)amino)-10phenyldeca-2,5,7,9-tetraen-4-one. Yield: 69%. M.p.: 273e275 C. IR cm1 (KBr): 3020(w), 2928(w), 2864(w), 1596(s), 1540(s), 1412(m), 1300(s), 1112(m), 1072(m), 996(m), 928(w), 808(m), 748(m), 688(m), 556(w), 504(m), 428(w). 1H NMR (CDCl3, 400 MHz) d 14.84 (s, 1H), 10.44 (s, 1H), 7.46 (dd, J ¼ 12.9, 7.2 Hz, 2H), 7.40e7.30 (m, 2H), 7.30e7.22 (m, 1H), 7.18 (dd, J ¼ 15.0, 10.8 Hz, 1H), 6.91 (ddd, J ¼ 23.8, 20.0, 13.2 Hz, 1H), 6.76 (d, J ¼ 15.5 Hz, 1H), 6.00 (d, J ¼ 15.0 Hz, 1H), 5.21 (s, 1H), 4.83 (s, 1H), 3.79 (dt, J ¼ 10.7, 5.2 Hz, 2H), 3.45 (dt, J ¼ 11.4, 5.6 Hz, 2H), 2.29 (s, 1H), 2.01 (s, 3H). Found (%): C, 72.14; H, 7.02; N, 4.63. Anal. Calcd. (%) for C18H21NO3: C, 72.22; H, 7.07; N, 4.68. MS: Found: [MþH]þ 300.186; [HO(CH2)2NHC(CH3)CHCO]þ 128.173; requires: [MþH]þ 300.159; [HO(CH2)2NHC(CH3)CHCO]þ 128,071. 3. (2E,5Z,7E,9E)-2-(allylamino)-6-hydroxy-10-phenyldeca2,5,7,9-tetraen-4-one. Yield: 44%. M.p.: 143e144 C. IR cm1 (KBr): 3076(w), 3020(w), 2928(w), 2852(w), 1692(m), 1552(s), 1428(m), 1284(m), 1132(w), 1092(w), 1040(w), 996(m), 944(m), 808(m), 748(m), 688(m), 564(w), 504(w), 428(w). 1H NMR (DMSO-d6, 400 MHz) d 15.13 (s, 1H), 10.30 (t, J ¼ 6.0 Hz, 1H), 7.52 (d, J ¼ 7.7 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 3H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.06 (dd, J ¼ 9.2, 4.8 Hz, 2H), 6.89 (d, J ¼ 14.2 Hz, 1H), 6.15 (dd, J ¼ 14.0, 6.4 Hz, 1H), 6.02e5.86 (m, 1H), 5.34 (s, 2H), 4.89 (s, 1H), 3.97 (t, J ¼ 5.6 Hz, 2H), 1.98 (s, 3H). Found (%): C, 77.11; H, 7.12; N, 4.70.Anal. Calcd. (%) for C19H21NO2: C, 77.26; H, 7.17; N, 4.74. MS: Found: [MþH]þ 296.161; [CH2CHCH2NHC(CH3)CHCO]þ 124.151; requires: [MþH]þ 296.165; [CH2CHCH2NHC(CH3)CHCO]þ 124.076. 4.(2E,5Z,7E,9E)-2-((3-(dimethylamino)propyl)amino)-6hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-one. Yield: 40%. M.p.: 121e123 C. IR cm1 (KBr): 2948(w), 2868(w), 2820(w), 2760(w), 1872(w), 1572(s), 1544(s), 1432(m), 1284(s), 1152(m), 1104(m), 996(m), 944(m), 836(s), 808(s), 748(s), 688(s), 564(w), 504(m), 420(m). 1H NMR (DMSO-d6, 400 MHz) d 15.19 (s, 1H), 10.32 (s, 1H), 7.52 (d, J ¼ 7.5 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.11e6.98 (m, 2H), 6.88 (dd, J ¼ 15.4, 6.4 Hz, 1H), 6.15 (d, J ¼ 14.0 Hz, 1H), 5.31 (s, 1H), 4.82 (s, 1H), 3.32 (dd, J ¼ 13.0, 6.3 Hz, 2H), 2.25 (t, J ¼ 6.8 Hz, 2H), 2.13 (s, 6H), 1.99 (s, 3H), 1.75e1.57 (m, 2H). Found (%): C, 74.02; H, 8.24; N, 8.19. Anal. Calcd. (%) for C21H28N2O2: C, 74.08; H, 8.29; N, 8.23. MS: Found: [MþH]þ 341.220; [C6H5(CH)4CO]þ 157.150; requires: [MþH]þ 341.222; þ [C6H5(CH)4CO] 157.065. 5. (2E,5Z,7E,9E)-2-(sec-butylamino)-6-hydroxy-10-phenyldeca2,5,7,9-tetraen-4-one. Yield: 36%. M.p.: 159e162 C. IR cm1 (KBr): 3024(w), 2972(w), 1864(w), 1548(s), 1416(m), 1304(s), 1132(s), 1044(m), 966(s), 932(s), 808(s), 748(s), 688(s), 556(w), 500(w), 424(w). 1H NMR (DMSO-d6, 400 MHz) d 15.05 (s, 1H), 10.30 (d, J ¼ 9.4 Hz, 1H), 7.52 (d, J ¼ 7.5 Hz, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.2 Hz, 1H), 7.04 (dt, J ¼ 14.3, 7.4 Hz, 2H), 6.88 (d, J ¼ 14.7 Hz, 1H), 6.23e6.08 (m, 1H), 5.31 (s, 1H), 4.81 (s, 1H), 3.68e3.53 (m, 1H), 2.01 (s, 3H), 1.50 (dt, J ¼ 14.0, 6.9 Hz, 2H), 1.16 (d, J ¼ 6.4 Hz, 3H), 0.89 (t, J ¼ 7.3 Hz, 3H). Found (%): C, 77.22; H, 7.97; N, 4.46.Anal. Calcd. (%) for C20H25NO2: C, 77.14; H, 8.09; N, 4.50. MS: Found: [MþH]þ 312.202; [(CH3)CH2CH(CH3)NHC(CH3)CHCO]þ 140.173; requires:
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Scheme 1. Synthesis and structures of the studied b-ketoenole dyes.
[MþH]þ 312.196; [(CH3)CH2CH(CH3)NHC(CH3)CHCO]þ 140.108. 6. (2E,5Z,7E,9E)-6-hydroxy-2-(isobutylamino)-10-phenyldeca2,5,7,9-tetraen-4-one. Yield: 45%. M.p.: 170e173 C. IR cm1 (KBr): 2966(w), 2915(w), 2871(w), 1592(s), 1540(s), 1428(s), 1284(s), 1132(s), 1080(m), 996(s), 940(m), 876(w), 836(w), 808(m), 752(m), 688(m), 504(w), 428(m). 1H NMR (DMSO-d6, 400 MHz) d 15.06 (s, 1H), 10.44 (t, J ¼ 5.9 Hz, 1H), 7.52 (d, J ¼ 7.4 Hz, 2H), 7.37 (dd, J ¼ 14.1, 6.9 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.12e6.98 (m, 2H), 6.89 (t, J ¼ 11.3 Hz, 1H), 6.27e6.03 (m, 1H), 5.32 (s, 1H), 4.85 (s, 1H), 3.15 (t, J ¼ 6.4 Hz, 2H), 1.99 (s, 3H), 1.78 (d, J ¼ 6.4 Hz, 1H), 0.94 (d, J ¼ 6.7 Hz, 6H). Found (%): C, 76.96; H, 8.01; N, 4.39. Anal. Calcd. (%) for C20H25NO2: C, 77.14; H, 8.09; N, 4.50. MS: Found: [MþH]þ 312.199; [(CH3)2CHCH2)NHC(CH3)CHCO]þ 140.157; requires: [MþH]þ 312,196; [(CH3)2CHCH2)NHC(CH3)CHCO]þ 140.108. 7. (2E,5Z,7E,9E)-6-hydroxy-2-((2-methoxyethyl)amino)-10phenyldeca-2,5,7,9-tetraen-4-one. Yield: 65%. M.p.: 153e155 C. IR cm1 (KBr): 2932(w), 2896(w), 2872(w), 1592(s), 1552(s), 1436(s), 1284(s), 1128(s), 996(s), 932(s), 808(s), 756(m), 688(m), 504(w), 424(w). 1H NMR (DMSO-d6, 400 MHz) d 15.15 (s, 1H), 10.30 (s, 1H), 7.52 (d, J ¼ 7.4 Hz, 2H), 7.37 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.14e6.96 (m, 2H), 6.98e6.80 (m, 1H), 6.16 (dd, J ¼ 13.4, 6.8 Hz, 1H), 5.32 (s, 1H), 4.85 (s, 1H), 3.49 (d, J ¼ 6.6 Hz, 4H), 3.30 (s, 3H), 2.00 (s, 3H). Found (%): C, 72.89; H, 7.34; N, 4.49.Anal. Calcd. (%) for C19H23NO3: C, 72.82; H, 7.40; N, 4.47. MS: Found: [MþH]þ 314.181; [C6H5(CH)4CO]þ 157.152; requires: [MþH]þ 314.175; [C6H5(CH)4CO]þ 157.152. 8. (2E,5Z,7E,9E)-6-hydroxy-2-((3-methoxypropyl)amino)-10phenyldeca-2,5,7,9-tetraen-4-one. Yield: 57%. M.p.: 106e107 C. IR cm1 (KBr): 2976(w), 2948(w), 2868(w), 2820(w), 1592(s), 1556(s), 1412(s), 1296(s), 1280(s), 1112(s), 996(s), 940(s), 904(m), 844(w), 808(s), 748(m), 688(m), 504(w), 416(w). 1H NMR (DMSOd6, 400 MHz) d 15.18 (s, 1H), 10.30 (t, J ¼ 5.8 Hz, 1H), 7.59e7.47 (m, 2H), 7.36 (t, J ¼ 7.5 Hz, 2H), 7.28 (t, J ¼ 7.3 Hz, 1H), 7.15e6.97 (m, 2H), 6.89 (dd, J ¼ 14.3, 7.6 Hz, 1H), 6.21e6.01 (m, 1H), 5.31 (s, 1H), 4.85 (d, J ¼ 9.5 Hz, 1H), 3.38 (dd, J ¼ 10.8, 4.7 Hz, 4H), 3.25 (d, J ¼ 2.4 Hz, 3H), 1.99 (s, 3H), 1.78 (dd, J ¼ 12.7, 6.2 Hz, 2H). Found (%): C, 73.45; H, 7.65; N, 4.25. Anal. Calcd. (%) for C20H25NO3: C, 73.37; H, 7.70; N, 4.28. MS: Found: [MþH]þ 328.195; [CH3O(CH2)3NHC(CH3)CHCO]þ 156.157; requires: [MþH]þ 328.191; [CH3O(CH2)3NHC(CH3)CHCO]þ 156.102. 9. (2E,5Z,7E,9E)-2-(heptylamino)-6-hydroxy-10-phenyldeca-
2,5,7,9-tetraen-4-one. Yield: 62%. M.p.: 132e134 C. IR cm1 (KBr): 2956(m), 2928(m), 2848(m), 1592(s), 1540(s), 1416(s), 1304(s), 1128(m), 1084(m), 996(s), 940(m), 804(s), 748(m), 728(m), 688(m), 500(w), 428(w). NMR (DMSO-d6, 300 MHz) d 15.16 (s), 10.35 (s), 7.56 (d, J ¼ 7.3 Hz), 7.40 (t, J ¼ 7.4 Hz), 7.31 (t, J ¼ 7.3 Hz), 7.09 (dt, J ¼ 16.4, 11.3 Hz), 6.91 (d, J ¼ 14.4 Hz), 6.27e6.07 (m), 5.34 (s), 4.87 (s), 3.36e3.24 (m), 2.02 (s), 1.56 (s), 1.31 (d, J ¼ 4.6 Hz), 0.89 (d, J ¼ 7.1 Hz). Found (%):C, 77.95; H, 8.68; N, 3.92. Anal. Calcd. (%) for C23H31NO2: C, 78.15; H, 8.84; N, 3.96. MS: Found: [MþH]þ 354.245; [С7H15NHC(CH3)CHCO]þ 182.276; requires: [MþH]þ 354,243; [С7H15NHC(CH3)CHCO]þ 182.154. 2.2. Insulin and lysozyme aggregates formation Human insulin (Private Joint Stock Company «On the production of insulin «Indar», Ukraine) was dissolved at 340 mM concentration in 0.1 mM HCl solution (pH 2). Fibrils were formed by incubating the protein solution in a thermomixer incubator at 65 C for about 5 h. To prepare lysozyme aggregates, lysozyme of hen egg white (Sigma-Aldrich Co. USA) was dissolved at 1 mM concentration in 0.1 mM HCl solution (pH 2) and incubated in a thermomixer incubator at 65 C for 24 h. 2.3. Preparation of dye solutions and protein solutions
b-ketoenole stock solutions were prepared by dissolving the weighted amount of the dyes at 2 mM concentration in DMSO or DMF. Working solutions of free dyes were prepared by dilution of the dye stock solutions in 50 mM Tris-HCl buffer (pH 7.9) to the 2 mM concentration (if not stated otherwise). The working solutions of dye-proteins complexes were prepared by adding to the dye 2 mM (if not stated otherwise) solution the aliquot of monomer or fibrillar protein stock solution, the protein concentration in the working solution was thus 3,4 mM (if not stated otherwise) for insulin and 4 mM for lysozyme. Bovine serum albumin (BSA) was obtained from Sigma-Aldrich Co. (USA). To prepare the dye-BSA working solution, to the 0.2 mg/ml (3 mM) BSA solution in 50 mM Tris-HCl buffer (pH 7.9) the aliquot of the dye stock solution was added, the dye concentration in the working solution was thus 2 mM.
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2.4. Photophysical measurements Absorption spectra were measured using a Specord M-40 (Germany, Carl Zeiss) spectrophotometer. Fluorescence excitation and emission spectra were registered with a CaryEclipse (Varian, Australia) or a Fluorolog 3 (Horiba Jobin Yvon) steady-state fluorescent spectrophotometer. Diluted liquid solutions were measured in standard quartz cells (1 1 cm). Thin polymer films were prepared by spin-coating a solution of poly(methyl methacrylate) (PMMA) in dichloromethane containing about 1 wt % (relative to the PMMA content) onto quartz glass substrates. Fluorescence emission of PMMA films and CH2Cl2 solutions was excited at 420 nm; for the other samples the excitation wavelength was set at the maximum of excitation spectrum of corresponding dye solution. The emission quantum yields for PMMA films and CH2Cl2 solutions were determined directly using a Hamamatsu C9920-02 system equipped with a Spectralon® integrating sphere, while for the dyes 2 and 4 (2 mM) in the presence of fibrillar insulin (13.6 mM) it was estimated by a relative method using Rhodamine 6G in ethanol as standard reference (quantum yield value 0.95) [15]. All measurements were performed at ambient temperature. 2.5. Estimation of equilibrium constants of the dye-to-fibril binding To estimate the equilibrium constant of the dyes 2 and 4 binding to fibrillar insulin, fluorescent titration of the dyes (2 mM) upon addition of 0e45 mM of fibrillar insulin was performed. Taking into account only the points for the concentrations 2 mM of protein molecules and higher, we could consider the concentration of the binding sites to be much higher than this of the dyes and thus the concentration of the free protein to be roughly equal to its total concentration. Further, the equation for the equilibrium constant K of dye-fibril binding could be written as:
Cd =Cbd ¼ 1 þ ðK CF Þ1
(1)
where Cd, Cbd and CF are total dye, fibril-bound dye and fibrillar protein concentrations respectively. Further, let us consider the totally unbound and totally bound with fibrils dye solution to have the fluorescence intensity I0 and Imax respectively. In this case the measured dye fluorescence intensity I at the fibrillar protein concentration CF could be written as I ¼ I0 (Cd e Cbd)/Cd þ Imax Cbd/Cd, that can be transformed into:
Cd =Cbd ¼ ðImax I0 Þ=ðI I0 Þ
(2)
Together with (1), (2) gives
I I0 ¼ A K CF =ð1 þ K CF Þ
(3)
A being the denotation for (ImaxI0) difference. Thus the experimental dependence of II0 on CF was approximated with the equation (3), A and K being obtained as approximation parameters. Accounting for several assumptions made, the obtained K value could be regarded as a rough estimation of the binding constant rather than its precise value. Besides it should be reminded that the estimated binding constant is only an apparent value calculated with respect to protein globule concentration and not this of the binding sites that is unknown; actually the estimation of binding constant with respect to protein globule concentration is common for the ligand-fibril binding studies. 2.6. Computer simulations of the dye 2 dimensions To estimate the dimensions of the dye 2, geometry optimization of the dye structure was first performed using the PM3 method
277
from the HyperChem 6.03 program package. Further the isosurface with the total charge density 0.002 that characterizes the molecular dimensions was built; linear dimensions i.e. length, height and width of the obtained isosurface were then estimated. 3. Results and discussion 3.1. Synthesis of the b-ketoenole dyes In present work we obtained a series of new compounds by the pyran ring opening reaction of 4-hydroxy-6-methyl-3 - ((2E, 4E) -5phenylpenta-2,4-dienoyl) -2H-pyran-2-one with primary aliphatic amines (Scheme 1). The mechanism of this reaction was studied in Refs. [14,16], it occurs through the nucleophilic attack at the 6-C of the pyran cycle leading to its next opening and decarboxilation. We have found that in the first step of this reaction the interaction of 4hydroxy-6-methyl-3-((2E, 4E)-5-phenylpenta-2,4-dienoyl)-2H-pyran-2-one with amines (Scheme 1) leads to the formation of the corresponding salt. Upon heating it dissociates and at the same time the amino group attacks a carbon atom of the methyl group in the pyran ring, leading to its ring-opening and decarboxylation [14,16]. In the case of n-alkyl amines, formation of the corresponding salts, opening up of the ring and decarboxylation occurs quite easily, but iso-amines react considerably worse and require higher reaction temperature. In the case of the tert-butylamine, the reaction stopped at the stage of the salt formation, it was not followed by the decyclization of the pyran ring (Scheme 1), in the case of a-phenyl ethyl amine the reaction path was similar. Molecules of the studied b-ketoenole dyes reveal two different regions: a highly hydrophobic phenyl polyen group and a hydrophilic unit consisting of the keto-enol-amine fragment. For related compounds the existence of the set of tautomeric forms of the ketoenol fragment was established by NMR [16]. The obtained compounds also exist as mesomero-tautomeric forms, as it is suggested by rather complicated 1H NMR. The form A (Scheme 1) is considered to be the dominant one for the studied b-ketoenoles in CDCl3 solution. In the 1H NMR spectra except of the major groups of peaks (corresponding to the form A) there are additional low-intensity peaks with similar chemical shifts and morphology. These signals point towards the presence of ca. 3e6% of a minor component and are attributed to another tautomer form B. Besides in the 10e11 ppm region near the main peak of the amino group proton (form A) we observed a minor signal attributed to the proton of the form B. Recording 1H NMR spectra in different solvents including TFA showed that signals of amino groups depend on the nature of the solvent. The maximum content of the form B is observed in chloroform and it decreases in the sequence chloroform > DMSO > benzene > acetone. In TFA, the signal of only one amino group is observed that indicated the presence of a strong hydrogen bond corresponding to the form A. This suggests that the amino tautomers are observed in the spectra. According to LC/MS data the molecular ions of the main compound and the minor component have the same molecular weight that also supports the suggestion about the tautomers of the compounds (data not presented). 3.2. Rigidochromic fluorescence of free b-ketoenole dyes In order to gain basic information about the photophysical properties of the b-ketoenole dyes under study and assess their potential as fluorescent markers, emissions of free dyes were studied in diluted dichloromethane solutions and in thin poly(methyl mathacrylate) polymer films. The emission spectra are
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reproduced in Fig. 2 and the respective quantum yields and band maxima are listed in Table 1. The emission spectra are broad and partly structured. In liquid samples, the emissions are relatively weak, with quantum yields of a few percent, and are centered at lem,max of about 560 nm, as determined for the dye 1. Minor deviations from this value, e.g. lem,max ¼ 553 nm for dye 3, result probably from slightly different interactions with the dipoles of solvent molecules when the amine at the position 2 is decorated with different (hetero)aliphatic group R. In polymer films, the emission spectra are shifted to shorter wavelengths and the quantum yields (4PL of 10e15%) are significantly larger than in liquid samples. Such blue shifts of emission in media characterized by increased rigidity are frequently observed for luminescent materials undergoing large geometry changes upon electronic excitation. Such structural rearrangements lead to a smaller energy gap between the excited state and the electronic ground state and thus to a red shift of emission and efficient radiationless relaxations to the ground state [17,18]. The geometry rearrangements in the excited state occur more easily in fluid solution, but they are partly hindered in rigid polymer films. Thus, distinct increase of photoluminescence efficiency is observed in PMMA samples. Similar effects, i.e. rigidity-related enhancement of emission and hipsochromic shifts, are expected to occur for the dye molecules embedded into protein aggregates, which will allow spectral discrimination of a denatured protein from its native form.
3.3. Spectral properties of dyes in buffer and in the presence of native proteins The fluorescent characteristics of the 9 b-ketoenole compounds in the buffer solution and in the presence of insulin, lysozyme and BSA in their native form are presented in Table 2. The excitation maxima for the free form of the studied dyes are located in the range 414e427 nm; emission maxima for all the dyes are in the range 500e554 nm. The addition of insulin, lysozyme and BSA could cause the shift of the excitation and emission spectrum maxima both to longer or shorter wavelengths by up to 35 nm. Excitation maxima in the presence of proteins are in the range 405e428 nm, while the positions of the emission maxima are at 510e571 nm. The dyes both free and in the presence of proteins display broad fluorescence bands, their maximum position could shift depending presumably on tautomer/isomer conformation of the molecule. The large values of the shift between the excitation and emission maxima for the free dyes and the dyes in the presence of proteins (98e190 nm) should be mentioned. The free b-ketoenole dyes in aqueous solution show very weak fluorescence with the emission intensity of 0.8e10.9 a.u. The enhancement of the fluorescence intensity of the dyes in the presence of the native proteins does not exceed 10 times, and formed protein-dye complexes are of the quite low intensity. Generally the low sensitivity of the dyes to the serum albumin (protein that is responsible for the binding and transportation of a variety of small molecules in blood) should be mentioned. Dye 2 having the hydroxyalkyl tail gives a fluorescence response in the presence of both lysozyme and albumin. The dyes 1, 2, 4 and 8 demonstrate a quite pronounced emission increase (in 3.7e10 times) in the presence of lysozyme. The presence of native lysozyme leads to the fluorescence response for the majority of the studied dyes. This protein contains a large number of charged amino acid residues (8 negatively charged residues and 18 positively charged ones), thus pronounced binding of the dye molecules to this protein is suggested to be driven by electrostatic interaction. Although the b-ketoenole molecules are uncharged, their transition to a zwitterionic tautomer (Fig. 3) could be responsible for the dyes affinity to the highly charged protein. 3.4. Spectral properties of dyes in the presence of fibrillar proteins
Fig. 2. Ambient temperature fluorescence spectra of b-ketoenole dyes 1e9 in PMMA polymer films and in air-saturated dichloromethane solutions recorded at an excitation wavelength of lexc ¼ 420 nm.
The fluorescent characteristics of the series of 9 b-ketoenole compounds in the presence of fibrillar aggregates of insulin and lysozyme are shown in Table 3. For the majority of the studied b-ketoenole dyes, the addition of the fibrillar insulin results in the long-wavelength shift of the excitation maximum wavelength by up to 28 nm (except of the dye 9 with the 2 nm short-wavelength shift). The short-wavelength shifts of the emission maximum wavelength for up to 17 nm were also observed for the majority of the dyes except 4 (almost no change), 6 and 8 (35-nm and 19-nm long-wavelength shift respectively). The presence of fibrillar insulin leads to the enhancement of the dye fluorescence intensity (IF) as compared to that of both free dye (I0) and the dye in the presence of native insulin (IM). The highest values of the IF/IM ratio were observed for the dyes 1 and 2 containing “short” unbranched propylamino and hydroxyethylamino groups as substituents (22 and 61 times respectively) as well as for the dye 8 with methoxypropylamino substituent (26 times). The fluorescence response with IF/IM between 10 and 13 times was also observed for the dyes 3, 4 and 7 containing allylamino, dimethylaminopropylamino and methoxyethylamino substituents. The other dyes did not demonstrate significant IF/IM values. To estimate
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Table 1 Emission maxima (lem,max) and quantum yields (4PL) of b-ketoenole dyes 1e9 in PMMA polymer films and in air-saturated dichloromethane solutions. Compound
lem,max (CH2Cl2), nma
4PL(CH2Cl2), %b
lem,max (PMMA), nma
4PL(PMMA), %b
1 2 3 4 5 6 7 8 9
560 555 553 560 560 560 555 560 560
2 3 3 2 2 2 3 2 2
512 512 508 516 517 512 512 512 522
11 15 12 13 13 11 12 11 10
a b
±5 nm. ±1%.
Table 2 Spectral-luminescent properties of b-ketoenole dyes in the presence of the native proteins. Dye
1 2 3 4 5 6 7 8 9
Free dye
With insulin
With lysozyme
With BSA
lex, nm
lem, nm
I0, a.u.
lex, nm
lem, nm
IM, a.u.
lex, nm
lem, nm
IM, a.u.
lex, nm
lem, nm
IM, a.u.
427 426 415 414 417 407 415 418 413
542 539 545 523 554 500 535 518 549
1.7 1.3 2.6 0.8 5.5 4.1 3.0 3.0 10.9
413 426 415 427 415 415 427 420 416
542 539 520 526 539 553 522 536 604
3.7 1.6 4.7 2.0 6.8 6.2 4.3 3.4 0.7
427 426 419 427 415 415 423 427 410
525 540 541 526 543 571 525 525 611
17.7 10.0 1.9 7.7 10.0 9.3 2.6 17.7 3.2
427 410 415 418 417 408 415 428 435
536 540 530 523 550 555 535 531 611
1.0 7.0 5.0 0.8 3.8 5.0 4.0 2.0 15.5
lex, lem e maximum wavelength of fluorescence excitation and emission spectra. I0 (IM) e fluorescence emission intensity of the dye in free state (in the presence of native protein) at the maximum wavelength of the emission spectrum. a.u. e arbitrary units.
(Table 1); thus we can suppose that the dye binding to the fibril leads to the rigid fixation of its molecule as it also takes place in PMMA film. To characterize the fibril binding of the dyes of the studied class, for the above mentioned dyes 2 and 4 the titration with fibrillar insulin was performed; the dependence of the dye fluorescence intensity on the concentration of the added fibrils allowed us to
Fig. 3. Zwitterionic tautomer form of b-ketoenole dye.
Table 3 Spectral-luminescent properties of b-ketoenole dyes in the presence of the aggregated proteins. Dye
1 2 3 4 5 6 7 8 9
With fibrillar insulin
With fibrillar lysozyme
lex, nm
lem, nm
Stokes shift, nm
IF, a.u.
IF/IM
lex, nm
lem, nm
Stokes shift, nm
IF, a.u.
IF/IM
441 430 436 436 437 435 434 436 411
528 531 528 524 528 535 525 537 538
87 101 92 88 91 100 91 101 127
80.0 97.0 49.7 25.0 20.9 17.0 45.8 89.0 16.7
21.6 60.6 10.6 12.5 3.1 2.7 10.7 26.2 1.5
441 427 426 426 415 408 440 441 413
517 540 532 522 543 555 526 517 532
76 113 106 96 128 147 86 76 119
10.0 37.0 5.9 27.7 8.5 9.2 22.7 10.0 15.8
0.6 3.7 3.1 3.6 0.8 1.0 8.7 0.6 0.9
lex, lem e maxima of fluorescence excitation and emission spectra.
IF e emission intensity of dye in the presence of fibrillar form of the insulin and lysozyme at the maximum wavelength of the emission spectrum. IF/IM e the ratio of the dye emission intensities in the presence of fibrillar and native form of corresponding protein. a.u. e arbitrary units.
the fluorescence quantum yield that the dyes of the studied class can achieve in the presence of fibrillar proteins, we have calculated this value for the fibrillar insulin complexes of the dyes 2 possessing the highest IF and IF/IM values (Table 3) and the less sensitive dye 4 for the comparison. It was revealed that the fluorescence quantum yield value for the dyes 2 and 4 in the presence of fibrillar insulin is rather high and equals to 12 and 15% respectively. The obtained values are close to these of the dyes in PMMA film
estimate the equilibrium constant of the dye to fibril binding. This estimation gave the values of the binding constant K ¼ (2.7 ± 0.9) 105 M1 for the dye 2 and (2.5 ± 1.4) 105 M1 for the dye 4, being thus of the same order of magnitude. As for the fibrillar lysozyme, its presence leads to the longwavelength shift of the excitation spectrum maximum wavelength up to 25 nm, except for the dyes 2, 5, 6 and 9 that practically do not change the maxima positions. At the same time, for the
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emission maximum the wavelength shift was predominantly to the short-wavelength region (up to 25 nm for the dye 1), exceptions being the dyes 2, 4 and 8 (almost no change) and the dye 6 (longwavelength shifts for 55 nm). As for the emission intensity, only for the dyes 2, 3, 4 and 7 the fluorescence sensitivity to the fibrillar lysozyme overpasses that to native protein. The IF/IM values for these four dyes are not significant: the highest value is for the dye 7 (8.7 times), while for the dyes 2, 3 and 4 it is about 3.1e3.7. Other compounds possess the fluorescent sensitivity to lysozyme aggregates equal to or lower than this for the native protein. The insignificant IF/IM value shown by the dyes (that give pronounced response on insulin fibrils) on the presence of aggregated lysozyme is partially connected with the fluorescent sensitivity of these dyes to the native lysozyme (IM value). 3.5. The study of the dye fluorescence “light up” mode In order to clarify the possible mechanisms of the fluorescence intensity increase of b-ketoenoles upon their binding to fibrillar proteins, the absorption (Fig. 4, a), as well as fluorescence excitation and emission (Fig. 4, b) spectra of the dye 2 that is the most sensitive for the fibrillar insulin among the studied compounds were compared for the free dye in buffer, in ethanol and in the presence of fibrillar insulin. The absorption spectrum of the free dye in buffer has its maximum at 334 nm; at the same time the maximum of the excitation spectrum of its weak emission is situated near 390 nm. This feature indicates that the majority of the dye molecules in the buffer exist in a non-fluorescent conformation. Further, addition of the insulin fibrils to the dye buffer solution leads to the decrease in the 334-nm absorption maximum and increase in the 430 nm maximum, the latter maximum is close to the band of the excitation spectrum of the intensive emission of this sample. Thus the increase in dye emission intensity is partially explained by its transition to the fluorescent conformation (together with the possible restriction of the internal motions) upon the binding with fibrillar insulin. Finally, the band with the maximum at 412 nm dominates in the absorption spectrum of the dye 2 in ethanol, its position is close to that of the corresponding fluorescence excitation band. The fluorescence intensity of the free dye in ethanol is noticeably higher than in aqueous buffer (Fig. 4b). The absorption spectrum of the free dye in ethanol is rather close to that of the dye-fibril complex and they both strongly distinguish from that of the free dye in buffer. Thus the removal of the dye from the aqueous medium (changing of the hydrophobicity of the medium) could also
contribute to the increase of dye emission intensity upon interaction with protein fibrils. This could be attributed to the change in the dye isomer/tautomer form, or to the destruction of the possible non-fluorescent dye-water complexes (the last could be analogous to the classical case of the 1,8-ANS dye [1]). Insertion of the molecule of amyloid sensitive dye into the fibrillar groove is suggested to be the most possible mode of the dye binding to the fibril. Using HyperChem program, dimensions of dye 2 were estimated to be about 23 Å 7.8 Å 4.8 Å, that is characteristic for the dyes of the studied series. Thus the studied dyes fit to the fibrillar groove formed by the b-pleated structure of the fibril, the width of which is believed to be equal to 6.5e7Å [13]. We supposed that in the fluorescent dye-insulin fibril complex the dye molecules “prefer” to locate mainly in the groove along the long axis of the fibril (Fig 1). The nature and structure of aminoalkyl tail group is important for the “tight fitting” of the dye molecule. As it was described above, the b-ketoenoles possess less fluorescent sensitivity to fibrillar lysozyme compared to insulin. Some of the dyes responded even better to the presence of native lysozyme than to its aggregates containing grooves as “suitable binding place” for the dye molecules. This could be explained by the suggestion about electrostatic interaction as driving force of the binding sites for the dye (as zwitterionic form) to this highly charged protein. In this case the location and availability of the charged amino acids determine to a large extent the binding of the bketoenole molecules. The sensitivity to lysozyme also could be partially connected with the high content of b-sheet regions in native protein, while bovine serum albumin and insulin have predominantly a-helical motifs [19,20]. 3.6. Application of dye 2 for fibrillar insulin detection: detection limit and linear detection range To examine the applicability of the dye 2 bearing 2-hydroxyethyl group as an amyloid-sensitive probe for the quantification of amyloid insulin, we performed titration of the 2 mM dye solution with increasing amounts of the aggregated protein (Fig. 5). The lower limit for the fibrillar insulin detection by the dye 2 was determined as equal to the insulin concentration leading to the two times dye fluorescence intensity increase. The upper limit of the detection range was determined as the maximum concentration where the dependence of dye fluorescence intensity on the fibrils concentration is still linear. Thus the 2 mM concentration of the dye 2 allows fluorescent detection of the insulin amyloid fibrils in the concentration range 0.2e9 mM (1e50 mg/ml) (R ¼ 0,97703; Fig. 5); the upper limit of the fibrillar insulin detection by the dye 2 thus
Fig. 4. Absorption (a) and fluorescence excitation (b; left) and emission (b; right) spectra of the dye 2 (5 mM) in 50 mM TRIS-HCl buffer, pH 7,9 in free state (solid) and in the presence of 13.6 mM of fibrillar insulin (dash), and in ethanol (dot). Fluorescence emission was excited at the maximum wavelength of the corresponding excitation spectrum.
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form, or to the destruction of the possible non-fluorescent dyewater complexes. The fixation of the dye molecules in the groove of the amyloid fibril makes additional impact to the increase in the dye emission intensity. The b-ketoenole dyes are considered as prospective fluorescent molecules for the further design of probes for the detection of the b-pleated protein aggregates and investigation of the aggregation reaction. Acknowledgement This work was supported by the H2020-MSCA-RISE-2014-RISE645628 e METCOPH project. References
Fig. 5. Titration of dye 2 by fibrillar insulin. The linear concentration range 0.2e9 mM (1e50 mg/ml) (linear, R ¼ 0.977). The 2 mM dye solution was used.
exceeds that for the commonly used amyloid-specific dye Thioflavin T (0.5e25 mg/ml) [21]. It should be also mentioned that the large shifts between the excitation and emission maxima for the fibril-bound dye (about 100 nm), good fluorescence intensity increase, rather high quantum yield and sufficient detection range make the dye 2 promising as a probe for fluorescent detection of the fibrillar aggregates. 4. Conclusion The series of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10phenyldeca-2,5,7,9-tetraen-4-ones) b-ketoenole dyes was first synthesized and characterized, their absorption and emission properties were studied for the free dyes and in the presence of proteins. b-Ketoenoles are weakly fluorescent when free, their excitation maxima are located in region 407e427 nm and fluorescent maxima in region 500e549 nm. The dyes exhibit a very weak response to the presence of native globular proteins insulin and bovine serum albumin, while their fluorescent sensitivity to native lysozyme is more pronounced (emission intensity increase up to 10 times was observed). In the presence of amyloid fibrils of insulin a strong increase in the emission intensity from the dyes is observed (up to 60 times for the dyes bearing the 2-hydroxyethylamino groups). The fluorescent sensitivity of the dyes to fibrillar proteins is noticeably affected by their functional substituents. The dyes possess moderate equilibrium constant of binding to fibrillar insulin about (2.7 ± 0.9) 105 M1 for compound 2. Dye 2 demonstrated a good linear range (1e50 mg/ml) of insulin fibril detection, a large value of the shift between excitation and emission maxima (about 100 nm) and a quantum yield in the complex with fibrillar insulin which reaches 12%. The b-ketoenoles are less sensitive to the amyloid aggregates of lysozyme compared to that of insulin, top emission increase in 8.7 times is observed for the dye 7 which possesses a 2methoxyethylamino fragment. This feature could be explained by the Coulombic interactions as a main driving force of the dye molecules binding with this protein. The increase in the fluorescence intensity of the dyes upon binding to proteins is at least partially explained by the change of the media (removing the dye from the water). That leads either to the transition of dye to the fluorescent isomer or/and tautomer
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