Research Article Received: 25 May 2013,
Revised: 4 December 2013,
Accepted: 14 December 2013,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2351
NMR screening of new carbocyanine dyes as ligands for affinity chromatography Carla Cruz*, Renato E. F. Boto, Anna K. Drzazga, Paulo Almeida and João A. Queiroz Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; proteins; cyanine ligands; molecular interactions
INTRODUCTION
J. Mol. Recognit. 2014; 27: 197–204
* Correspondence to: Carla Cruz, CICS-UBI—Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. E-mail: [email protected] C. Cruz, R. E. F. Boto, A. K. Drzazga, P. Almeida, J. A. Queiroz CICS-UBI—Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal
Copyright © 2014 John Wiley & Sons, Ltd.
197
Synthetic dyes have been used in affinity chromatography, known as dye-affinity chromatography (dye-AC) (Denizli and Piskin 2001), allowing to purify several types of proteins along with various enzymes and, in some cases, in a remarkably specific manner biomimeting natural ligands (Clonis et al. 1987; Lowe et al. 1986). As representative example, triazine dyes mimic the binding of natural anionic heterocyclic substrates, such as nucleic acids, nucleotides, coenzymes, and vitamins, likely representing the first example of combinatorial screening for protein binding (Clonis et al. 2000). The triazine dye Cibacron blue F3G-A was used to separate alcohol dehydrogenase from horse liver (Labrou et al. 1996). Also, anthraquinonemoiety-containing aromatic sulfonated dyes, such as Procion Blue H-B, MX-R, and Vilmafix Blue A-R, bind preferentially the nucleotide-binding site of several proteins and mimic the binding of naturally occurring anionic coenzymes (NADH, FAD) (Clonis and Lowe 1980). Other dye ligands have also been employed to remove prion proteins, human immunodeficiency virus-1, and hepatitis B viral particles from biological fluids (Caspi et al. 1998; Hattori et al. 1997; Brown and Combridge 1986). The use of dye-AC for protein purification requires selection of one or more immobilized dyes, because, in some cases, these ligands established nonspecific interactions with other parts of the proteins by a combination of electrostatic, hydrophobic, hydrogen bonding, and charge-transfer interactions, depending on the structure of the dyes. Several techniques including NMR, electron spin resonance, circular dichroism, and computational methods have been used to explain dye–protein interactions (Skotland 1981; Federici et al. 1985). NMR-based screening, despite of its low intrinsic sensitivity, offers the largest dynamic range and is capable of capturing very weak interactions. STD-NMR experiments use small quantities of nonlabeled
biomolecules, focusing on the signals of the ligand without any need of processing NMR information about the biomolecule (Viegas et al. 2011). Cyanines have been used for a large number of applications, in particular, as functional dyes; nevertheless, their ability as ligands for dye-AC has been recently explored (Boto et al. 2009; Boto et al. 2008; Cruz et al. 2011). In these studies, a thiacarbocyanine has been postgrafted onto beaded cellulose by a curing method and showed a selective interaction allowing separation of three proteins from an artificial mixture (Boto et al. 2009). The adsorption of lysozyme onto this immobilized dye was further investigated, presenting a maximum dynamic binding capacity value of 8.6 mg/ml for lysozyme, achieved at 30 °C and pH 9, and a dissociation constant (KD) of 2.61 ± 0.36 × 10–5 M (Boto et al. 2008). Following these studies and looking for the development of new cyanine for dye-AC, four indocarbocyanines were synthesized (Figure 1). These indocarbocyanines have carboxyalkyl chains, apart from acting as spacer arms that potentially enhance the efficiency of the purification process, that offer a convenient way to couple the dye to the matrices of cellulose and agarose. Further, some structural variations were introduced, such as, sulfonic difunctionalization, symmetric, and asymmetric groups to provide a desirable interaction with the standard proteins. Saturation transfer difference-NMR experiments have been used to screen the potential binding sites within cyanines that
C. CRUZ ET AL.
Figure 1. Indocarbocyanines 1–4.
specifically interact with BSA, α-chymotrypsin, lysozyme, RNase, and gamma globulin.
EXPERIMENTAL General All reagents were of the highest purity available, purchased from Sigma-Aldrich Company, and used as received. Solvents were of analytical grade and were dried over 3 Ǻ molecular sieves prior to use. Standard BSA (A-6793), α-chymotrypsin (from bovine pancreas, C-4129), lysozyme (from chicken egg white, Biochemika 62970), and gamma globulin (from bovine blood, G5009) were also purchased from Sigma-Aldrich Co. RNase (from Bovine Pancreas, 27033002) and was purchased from USB Corp. All other chemicals were of analytical grade or higher and used as received. All reactions were monitored by thin-layer chromatography on aluminum plates precoated with Merck silica gel 60 F254 (0.25 mm) using dichloromethane or dichloromethane/methanol (9.5:0.5 or 9:1), and the spots have been examined under 254, 312, and 365 nm UV light. Melting points (M.p.) were measured in open capillary tubes in a Buchi 530 M.p. apparatus and were uncorrected. Infrared spectra (IR) were recorded on Thermo Scientific Nicolet iS10 FT-IR spectrophotometer. All samples were prepared by mixing FT-IR grade KBr (Sigma-Aldrich) with 1% (w/w) dye and grinding to a fine powder. Spectra were recorded over the 600–4000 cm 1 range without baseline corrections. Characteristic absorptions are given in cm 1. 1 H and 13C NMR spectra were recorded in DMSO-d6 solutions at a temperature of 298 K on a Bruker Avance III 600-MHz spectrometer operating at 14.09 T, observing 1H at 600.13, and
13
C at 150.91 MHz. The spectrometer was equipped with a cryoprobe, and all spectra were processed with the software topspin 3.1. Chemical shifts are reported in ppm using tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are given in Hz. High resolution mass spectra (HRMS) were performed on an microTOF (focus) mass spectrometer (Bruker Daltonics, Bremen, Germany). Ions were generated using an ApolloII (ESI) source. Ionization was achieved by electrospray, using a voltage of 4500 V applied to the needle, and a counter voltage between 100 and 150 V applied to the capillary. Samples were prepared by adding a spray solution of 70:30 (v/v) methanol/water with 0.1% of formic acid to a solution of the sample in CH2Cl2 or another solvent at a v/v ratio of 1% to 5% to give the best signal-to-noise ratio. Data acquisition was performed using the microTOF control software version 2.1, and data processing was performed using the data analysis software, version 3.4 both from Bruker Daltonics. Synthesis of the quaternarium ammonium salts 6a–c Sulphonated indole 5b was synthesized and has been already described (Jiang et al. 2007). The quaternary ammonium salts 6a–c were synthesized according to our previous work (Boto et al. 2007) by reacting 15.7 mmol of the trimethylindole derivatives 5a–c with 17.3 mmol of 11-bromoundecanoic acid at 150 ºC. The crude product was washed several times with diethyl ether and purified by recrystallization from acetonitrile (Scheme 1). 1-(10-carboxydecyl)-2,3,3-trimethyl-3H-indolium bromide (6a) Yield: 79%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.21–1.26 (8H, m, CH2), 1.28–1.33 (2H, m, CH2), 1.38–1.42 (2H, m,
198
Scheme 1. Quaternary ammonium salts and indocarbocyanine synthesis.
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
NMR SCREENING OF NEW CARBOCYANINE DYES CH2), 1.49 (2H, q, J = 7.1 Hz, CH2), 1.53 (6H, s, 3-CH3), 1.82 (2H, q, J = 7.6 Hz, NCH2CH2), 2.17 (2H, t, J = 7.4 Hz, CH2COO), 2.85 (3H, s, 2-CH3), 4.45 (2H, t, J = 7.7 Hz, NCH2), 7.61–7.63 (2H, m, 5-CH,6CH), 7.84–7.86 (1H, m, 7-CH), 7.98–8.00 (1H, m, 4-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 14.0 (2-CH3), 22.0 (3-CH3), 24.5 (CH2), 25.9 (CH2), 27.2 (CH2), 28.4 (CH2), 28.5 (CH2), 28.6 (CH2), 28.6 (CH2), 28.7 (CH2), 33.7 (CH2COO), 47.6 (NCH2), 54.2 (3-C), 115.5 (4-CH), 123.5 (7-CH), 129.0 (6-CH), 129.4 (5-CH), 141.1 (7aC), 141.9 (3a-C), 174.5 (CO), 196.4 (2-C). IR (KBr) υ (cm 1): 3420, 2928, 2855, 1731 (C = O), 1625 (C = C), 1461, 1198, 767. HRMS (ESI-TOF) C22H34NO2: Calcd.: 344.25841; Found: 344.25823. 2.2.2. 1-(10-carboxydecyl)-2,3,3-trimethyl-5-sulfo-3H-indolium bromide (6b) Yield: 66%. M.p. 197–200 ºC. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.22–1.27 (8H, m, CH2), 1.29–1.33 (2H, m, CH2), 1.37–1.42 (2H, m, CH2), 1.45–1.50 (2H, m, CH2), 1.53 (6H, s, 3-CH3), 1.81 (2H, q, J = 7.0 Hz, NCH2CH2), 2.18 (2H, t, J = 7.2 Hz, CH2COO), 2.82 (3H, s, 2CH3), 4.43 (2H, t, J = 7.2 Hz, NCH2), 7.80 (1H, d, J = 8.2 Hz, 6-CH), 7.90 (1H, d, J = 8.2 Hz, 7-CH), 8.01 (1H, s, 4-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 13.9 (2-CH3), 21.9 (3-CH3), 24.4 (CH2), 25.9 (CH2), 27.2 (CH2), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.7 (CH2), 28.8 (CH2), 33.6 (CH2COO), 47.6 (NCH2), 55.5 (3-C), 114.9 (7-CH), 120.7 (4-CH), 126.3 (6-CH), 140.9 (7a-C), 141.5 (3aC), 149.5 (5-CH), 174.5 (CO), 197.2 (2-C). IR (KBr) υ (cm-1): 3448, 2926, 2852, 1731 (C = O), 1624 (C = C), 1467, 1182, 686. HRMS (ESI-TOF) C22H34NO5S: Calcd.: 424.21522; Found: 424.21502. 2.2.3. 3-(10-carboxydecyl)-1,1,2-trimethyl-1H-benzo[e]indolium bromide (6c) Yield: 52%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.20–1.26 (8H, m, CH2), 1.32 (2H, q, J = 7.0 Hz, CH2), 1.41–1.46 (4H, m, CH2), 1.76 (6H, s, 1-CH3), 1.88 (2H, q, J = 7.6 Hz, NCH2CH2), 2.16 (2H, t, J = 7.3 Hz, CH2COO), 2.95 (3H, s, 2-CH3), 4.58 (2H, t, J = 7.7 Hz, NCH2), 7.72 (1H, t, J = 7.9 Hz, 7-CH), 7.78 (1H, t, J = 8.0 Hz, 8-CH), 8.16 (1H, d, J = 8.9 Hz, 4-CH), 8.21 (1H, d, J = 8.2 Hz, 6-CH), 8.29 (1H, d, J = 8.9 Hz, 5-CH), 8.37 (1H, d, J = 8.3 Hz, 9-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 13.9 (2-CH3), 21.6 (1-CH3), 24.5 (CH2), 25.9 (CH2), 27.5 (CH2), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.8 (CH2), 28.8 (CH2), 33.7 (CH2COO), 47.9 (NCH2), 55.5 (1-C), 113.4 (4-CH), 123.5 (9-CH), 127.3 (7-CH), 127.3 (9a-C), 128.4 (8-CH), 129.8 (6-CH), 130.7 (5-CH), 133.1 (5a-C), 137.0 (9b-C), 138.5 (3a-C), 174.5 (CO), 196.3 (2-C). IR (KBr) υ (cm-1): 3417, 2926, 2853, 1723 (C = O), 1630 (C = C), 1581, 1465, 1174, 814, 753. HRMS (ESI-TOF) C26H36NO2: Calcd.: 394.27406; Found: 394.27379. Synthesis of indocarbocyanines 1–4 Symmetric indocarbocyanines 1–3
J. Mol. Recognit. 2014; 27: 197–204
1-(10-carboxydecyl)-2-((1E,3Z)-3-(1-(10-carboxydecyl)-3,3-dime thylindolin-2-ylidene)prop-1-enyl)-3,3-dimethyl-3H-indolium iodide (1) Yield: 70%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.21–1.26 (16H, m, CH2), 1.31–1.34 (4H, m, CH2), 1.38–1.41 (4H, m, CH2), 1.44–1.48 (4H, m, CH2), 1.70 (12H, s, 3-CH3), 1.73 (4H, q, J = 7.0 Hz, NCH2CH2, N″CH2CH2), 2.11 (4H, t, J = 7.3 Hz, CH2COO), 4.12 (4H, t, J = 6.7 Hz, NCH2, N″CH2), 6.51 (2H, d, J = 13.4 Hz, 1ʹ-CH, 3ʹ-CH), 7.30 (2H, t, J = 7.0 Hz, 5-CH, 5″-CH), 7.43–7.47 (4H, m, 6-CH, 6″-CH, 7CH, 7″-CH), 7.64 (2H, d, J = 7.3 Hz, 4-CH, 4″-CH), 8.35 (1H, t, J = 13.4 Hz, 2ʹ-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 24.5 (CH2), 24.7 (CH2), 26.1 (CH2), 27.0 (CH2), 27.4 (3-CH3), 28.4 (CH2), 28.6 (CH2), 28.7 (CH2), 28.7 (CH2), 28.8 (CH2), 34.4 (CH2COO), 43.8 (NCH2,N″ CH2), 102.5 (1ʹ-CH, 3ʹ-CH), 111.6 (7-CH, 7″-CH), 122.5 (4-CH, 4″-CH), 125.2 (5-CH, 5″-CH), 128.7 (6-CH, 6″-CH), 140.6 (3a-C, 3a″-C), 141.9 (7a-C, 7a ″-C), 149.8 (2ʹ-CH), 173.8 (2-C, 2″-C), 174.8 (CO). IR (KBr) υ (cm 1): 3448, 2926, 2852, 1730 (C = O), 1557 (C = C), 1429, 1138, 929, 757. HRMS (ESI-TOF) C45H65N2O4: Calcd.: 697.49389; Found: 697.49346. 1-(10-carboxydecyl)-2-{3-[1-(10-carboxydecyl)-3,3-dimethyl-5sulfoindolin-2-ylidene] prop-1-enyl}-3,3-dimethyl-5-sulfo-3H-indolium hydrogenosulfate (2) Yield: 75%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.21–1.25 (16H, m, CH2), 1.28–1.32 (4H, m, CH2), 1.34–1.39 (4H, m, CH2), 1.42–1.48 (4H, m, CH2), 1.69 (12H, s, 3-CH3), 1.71–1.74 (4H, m, NCH2CH2, N″CH2CH2), 2.15 (4H, t, J = 7.3 Hz, CH2COO), 4.12 (4H, t, J = 6.9 Hz, NCH2, N″CH2), 6.52 (2H, d, J = 13.4 Hz, 1ʹ-CH, 3ʹ-CH), 7.39 (2H, d, J = 8.3 Hz, 7-CH, 7″-CH), 7.62 (2H, d, J = 8.2 Hz, 6-CH, 6″-CH), 7.80 (2H, s, 4-CH, 4″-CH), 8.35 (1H, t, J = 13.4 Hz, 2ʹ-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 24.4 (CH2), 26.0 (CH2), 27.0 (CH2), 27.4 (3-CH3), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.7 (CH2), 28.8 (CH2), 33.6 (CH2COO), 43.9 (NCH2, N″CH2), 48.9 (3C, 3″C), 103.0 (1ʹCH, 3ʹ-CH), 110.8 (7-CH, 7″-CH), 119.8 (4-CH, 4″-CH), 126.3 (6-CH, 6″-CH), 140.1 (3a-C, 3a″-C), 141.9 (7a-C, 7a″-C), 145.4 (5-CH, 5″-CH), 149.9 (2ʹ-CH), 174.3 (2-C, 2″-C), 174.5 (CO).). IR (KBr) υ (cm 1): 3423, 2927, 2854, 1717 (C = O), 1557 (C = C), 1440, 1113, 931, 689. HRMS (ESI-TOF) C45H65N2O10S2: Calcd.: 857.40751; Found: 857.40724. 3-(10-carboxydecyl)-2-{3-[3-(10-carboxydecyl)-1,1-dimethyl1H-benzo[e]indol-2(3H)-ylidene]prop-1-enyl}-1,1-dimethyl-1H-benzo [e]indolium iodide (3) Yield: 71%. M.p. 107 ºC–109 ºC. 1H NMR (600.13 MHz, DMSOd6) δ (ppm): 1.16–1.24 (16H, m, CH2), 1.29–1.34 (4H, m, CH2), 1.39–1.44 (8H, m, CH2), 1.76–1.81 (4H, m, NCH2CH2, N″CH2CH2), 1.99 (12H, s, 1-CH3), 2.13 (4H, t, J = 7.3 Hz, CH2COO, ), 4.26 (4H, t, J = 7.1 Hz, NCH2, N″CH2), 6.60 (2H, d, J = 13.4 Hz, 1ʹ-CH, 3ʹ-CH), 7.53 (2H, t, J = 7.4 Hz, 7-CH, 7″-CH), 7.68 (2H, t, J = 7.5 Hz, 8-CH, 8″-CH), 7.77 (2H, d, J = 8.7 Hz, 4-CH, 4″-CH), 8.07 (2H, d, J = 8.2 Hz, 6-CH, 6″-CH), 8.10 (2H, d, J = 8.8 Hz, 5-CH, 5″-CH), 8.29 (2H, d, J = 8.5 Hz, 9-CH, 9″-CH), 8.58 (1H, t, J = 13.5 Hz, 2ʹ-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 24.6 (CH2), 26.1 (CH2), 27.2 (1CH3), 27.4 (CH2), 28.6 (CH2), 28.8 (CH2), 28.9 (CH2), 28.9 (CH2), 29.0 (CH2), 33.8 (CH2COO), 44.0 (NCH2, N″CH2), 50.7 (1C, 1C″), 102.2 (1ʹ-CH, 3ʹ-CH), 111.9 (4-CH, 4″-CH), 122.3 (9-CH, 9″-CH), 125.2 (7-CH, 7″-CH), 127.5 (9a-CH, 9a″-CH), 128.1 (8-CH, 8″-CH), 130.1 (6-CH, 6″-CH), 130.6 (5-CH, 5″-CH), 131.6 (5a-CH, 5a″-CH), 133.3 (9b-C, 9b″-C), 139.7 (3a-C, 3a″-C), 148.6 (2ʹ-CH), 174.7 (2-C, 2″-C), 175.2 (CO). ). IR (KBr) υ (cm 1): 3447, 2925, 2852, 1729
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
199
The condensation of indole quaternary salts was carried out by refluxing for 18 h and 3.5 mmol of salts with 7.22 mmol of triethylortoformate in 10 ml of pyridine per gram of salt. After cooling to a room temperature, diethyl ether was added, and the resulting mixture was refrigerated to allow complete precipitation. The dye was counter-ion exchanged to hydrogen sulfate (for sulfoindocarbocyanine) or iodide (others) by dissolution in a minimum amount of acetonitrile, followed by an addition of 5% (v/v) aqueous H2SO4 or 14% (w/v) aqueous KI, respectively. The solution or suspension obtained was heated until reflux and then cooled to a room temperature and refrigerated overnight. The resulting crystalline product was collected
by filtration under reduced pressure, washed with water and diethyl ether and recrystallized from dry acetonitrile.
C. CRUZ ET AL. (C = O), 1557 (C = C), 1428, 1172, 936, 653. HRMS (ESI-TOF) C53H69N2O4: Calcd.: 797.52519; Found: 797.52513. Asymmetric thioindocarbocyanine (4) A solution of 2.30 g (4.93 mmol) of quaternary ammonium salt 6c and 1.95 g (4.47 mmol) of 2-[2-(acetylphenylamino)vinyl]-3-ethyl benzothiazol-3-ium iodide, prepared as previously described (Boto et al. 2007), in 10 ml of dry pyridine was refluxed for 1 h. After cooling to a room temperature, diethyl ether was added, and the resulting mixture was refrigerated to allow complete precipitation. The crystalline product obtained was collected by filtration under reduced pressure, washed with diethyl ether and recrystallized from dry acetonitrile. 3-(10-carboxydecyl)-2-((1E,3E)-3-(3-ethylbenzo[d]thiazol-2(3H)ylidene)prop-1-enyl)-1,1-dimethyl-1H-benzo[e]indolium iodide (4) Yield: 14%. M.p. 105 ºC–108 ºC. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.19–1.24 (8H, m, CH2), 1.31–1.34 (2H, m, CH2), 1.38–1.47
(7H, m, N″CH2CH3, CH2), 1.73 (2H, quint, J = 7.3 Hz, NCH2CH2), 1.92 (6H, s, 1-CH3), 2.14 (2H, t, J = 7.3 Hz, CH2COO), 4.16 (2H, t, J = 7.0 Hz, NCH2), 4.50 (2H, q, J = 7.1 Hz, N″CH2), 6.41 (1H, d, J = 13.2 Hz, 1ʹCH), 6.86 (1H, d, J = 13.0 Hz, 3ʹ-CH), 7.47–7.53 (2H, m, 7-CH, 6″-CH), 7.63–7.66 (2H, m, 8-CH + 5″-CH), 7.70 (1H, d, J = 8.6 Hz, 4-CH), 7.88 (1H, d, J = 8.3 Hz, 4″-CH), 8.04 (1H, d, J = 8.4 Hz, 6-CH), 8.06 (1H, d, J = 9.0 Hz, 5-CH), 8.09 (1H, d, J = 7.9 Hz, 7″-CH), 8.14 (1H, t, J = 13.1 Hz, 2ʹ-CH), 8.25 (1H, d, J = 8.5 Hz, 9-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 13.0 (CH3), 24.4 (CH2), 26.1 (CH2), 27.0 (CH2), 28.0 (1-CH3), 28.4 (CH2), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.8 (CH2), 33.3 (CH2COO), 42.1 (N″CH2), 43.8 (NCH2), 50.2 (1C), 99.3 (1ʹ-CH), 101.7 (3ʹ-CH), 111.5 (4-CH), 114.1 (4″-CH), 122.0 (9-CH), 123.4 (7″-CH), 124.6 (7-CH), 125.6 (7a″-CH), 126.0 (6″-CH), 127.6 (8-CH), 127.7 (5″-CH), 128.5 (6-CH), 129.9 (9a-C), 130.3 (5-CH), 131.1 (5a-C), 132.4 (9b-C), 139.9 (3a-C),140.9 (3a″-C), 147.1 (2ʹ-C), 166.5 (2″-C), 172.9 (2-C), 174.5 (CO). IR (KBr) υ (cm 1): 3440, 2925, 2852, 1731 (C = O), 1555 (C = C), 1424, 1131, 939, 751. HRMS (ESI-TOF) C37H45N2O2S: Calcd.: 581.31963; Found: 581.31945.
200
1
Figure 2. H NMR reference spectra of the cyanine 1 and STD-NMR spectra of the complexes cyanine 1-lysozyme, cyanine 1-α-chymotrypsin, cyanine 1-RNase and cyanine 1-BSA. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
NMR SCREENING OF NEW CARBOCYANINE DYES STD-NMR spectroscopy Preparation of the samples A 100 μM protein stock solution was prepared in 100 mM potassium phosphate buffer in H2O: D2O (9:1) at pH 8.0. For all cyanine dyes, 1 mM stock solutions in DMSO-d6 were prepared and stored in the dark at 4 ºC. Samples for STD-NMR build-up analysis were prepared as follow: 240 μl of cyanine was added to 250 μl of protein stock solution and 10 μl of trimethylsilyl propionate. Method The quaternary ammonium salts and cyanine dyes resonances were assigned using 1H, 13C, DEPT90, DEPT135, 1H-1H correlation spectroscopy, 1H-1H total correlation spectroscopy, 1H-13C heteronuclear single-quantum correlation and 1H-13C heteronuclear multiplebond correlation. The STD-NMR spectra were recorded with 512 scans and selective saturation of protein resonances at 0 to 1 ppm and 10 to 11 ppm (on resonance) and 30 ppm (off
resonance) using Eburp2.1000, a shaped pulse (length for the protein saturation 50 ms, 1-ms delay between pulses) for a saturation time of 2.0 s. The number of averages (L4) was set to 16, and a relaxation delay was 3 s. Also, eight dummy scans were employed to reduce subtraction artifacts. The rows of the pseudo-2D spectrum obtained correspond to the different saturation frequencies (on and off resonance). The difference spectrum is obtained by direct subtraction of the individual rows (off resonance–on resonance) (Mayer and Meyer 1999). The STD build-up data processing was obtained by dividing STD signal intensities by the intensities of the reference spectrum [(I0-ISTD)/I0] (Mayer and Meyer 2001). The intensity of the largest STD effect was set to 100%, and the others percentages are calculated accordingly. Reference experiments using the free cyanines themselves were performed under the same experimental conditions to verify true ligand binding. No signal was present in the difference spectra, indicating that the effects observed in the presence of the proteins were because of true saturation transfer.
J. Mol. Recognit. 2014; 27: 197–204
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
201
1
Figure 3. H NMR reference spectra of the cyanine 2 and STD-NMR spectra of the complexes cyanine 2-lysozyme, cyanine 2-α-chymotrypsin, cyanine 2-BSA and cyanine 2-gamma globulin. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
C. CRUZ ET AL.
RESULTS AND DISCUSSION The binding affinity properties of the four cyanines to BSA, αchymotrypsin, lysozyme, RNase, and gamma globulin were achieved by STD-NMR, ranked according to their STD intensities. The STD-NMR spectra obtained for cyanine–protein complexes, and the reference spectrum of each cyanine are shown in Figures 2, 3, 4, and 5. The cyanines that differ in their symmetry, heterocyclic moieties, functional groups, and carboxyalkyl chains showed different kinds of interaction with these proteins. Almost all cyanines showed a response in the STD spectra, except cyanines 1, 3, and 4 with gamma globulin, cyanines 3 and 4 with BSA, and cyanine 2 with RNase. The results can be interpreted as explained hereafter. Cyanine 1 The largest STD effects are observed for the heterocyclic moiety of cyanine 1 with lysozyme, α-chymotrypsin, and RNase, whereas with BSA, the alkyl chain showed the largest STD effect. No STD responses were found for gamma globulin. The 1H NMR spectrum of 1 and the STD spectra of complex cyanine 1-proteins are presented in Figure ure 2, as well as its protons chemical shifts assignments. Accordingly, methyl groups should be the ones more directly involved in binding and closer to lysozyme and α-chymotrypsin, followed by protons H-2ʹ, H-5, H-4, H-7, and H-6. No STD
response was found for the alkyl chain indicating that they should be the ones more distant from the lysozyme and αchymotrypsin. The binding epitope of cyanine 1 to RNase indicates that the major interactions involve protons H-5, H-6, and H-7. The methyl group has a lower relative STD value indicating that it should be distant from the protein surface. In the presence of BSA, lower STD contacts were found in the low-field region of the STD spectrum corresponding to the heterocyclic moiety, and stronger STD signals were found with the carboxyalkyl chain of 1, suggesting close proximity of this ligand region to the protein surface. Cyanine 2 The comparison between the spectra pointed out that the cyanine 2 establishes more contacts with large proteins such as BSA and gamma globulin. The 1H NMR spectrum of cyanine 2 and the STD spectra are presented in Figure ure 3. The epitope mapping is similar for these two proteins. The largest STD effect is observed for alkyl chain, and weaker STD contacts are observed with heterocyclic protons H-1ʹ, H-6, H-4, H-7 and methyl groups. On contrary for lysozyme and α-chymotrypsin, the major interactions of cyanine 2 involve the methyl groups (100% STD absolute intensities) in the high-field region of the STD spectra. The other heterocyclic protons do not seem to be in close contact with these proteins. No STD signals were detected for RNase.
202
1
Figure 4. H NMR reference spectra of the cyanine 3 and STD-NMR spectra of the complexes cyanine 3-lysozyme, cyanine 3-α-chymotrypsin and cyanine 3-RNase. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
NMR SCREENING OF NEW CARBOCYANINE DYES
1
Figure 5. H NMR reference spectra of the cyanine 4 and STD-NMR spectra of the complexes cyanine 4-lysozyme, cyanine 4-α-chymotrypsin and cyanine 4-RNase. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
Cyanine 3 The STD spectra of cyanine 3 show only STD signals for RNase, α-chymotrypsin, and lysozyme indicating binding preference to these proteins instead of BSA and gamma globulin (Figure 4). The binding epitope for α-chymotrypsin and lysozyme are quite similar; proton H-5 (100% of saturation) followed by H-6 leads (~90% of saturation) to the most prominent STD signals, indicating that are in close contact with these proteins surface. The remaining protons of the heterocyclic moiety, H-4, H-7, H-8, H-9, and methyl groups (12%–29% of saturation) give weaker signals. For RNase, the STD-NMR spectrum of cyanine 3 showing the involvement of H-7 (100% of saturation) in the binding to the protein because of the highest amount of saturation received, followed by protons H-8 and H-4 (84% and 75% of saturation, respectively). The other protons of the heterocyclic moiety establish weak STD contacts with the protein through H-5 (29% of saturation), H-6 (30% of saturation) H-9, and CH3 (14% of saturation).
Cyanine 4
J. Mol. Recognit. 2014; 27: 197–204
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
203
The binding profiles of asymmetric cyanine 4 are similar to cyanine 3. Indeed, this ligand did not show STD signals with gamma globulin and BSA as for cyanine 3 (Figure ure 5). The binding epitope of cyanine 4 with lysozyme and α-chymotrypsin are similar; however, different interaction patterns were obtained with RNase.
The analysis of the STD signals indicates that the major interactions of 4 with lysozyme and α-chymotrypsin are through H-7″ (100% of saturation) and H-2ʹ (76% of saturation for lysozyme and 57% of saturation for α-chymotrypsin), indicating that the benzothiazole unit is in close contact with these proteins during the complex formation. While relative weaker responses are observed for protons of dibenzopyrrole, indicating that this unit should be more distant from the protein surface. On the contrary for RNase, protons of dibenzopyrrole, H-7 (100% of saturation), followed by H-4 (94% of saturation) and H-8 (62% of saturation), receive the higher magnetization, suggesting that are more involve in the binding to RNase. However, protons of benzothiazole unit are less intimate contact with RNase since receiving the lower amount of magnetization (H-7″ 12% of saturation and H-4″ 32% of saturation). These results show that the four cyanines, which differ in symmetry, aromatic units, sulfonic functionalization, and Nalkyl chains, present different binding profiles representing specific molecular recognition with the different proteins. In the case of cyanines 1 and 3, the benzo and dibenzo units contributed to ligand binding through π–π interactions with lysozyme, α-chymotrypsin, and RNase, whereas the alkyl chain of cyanine 2 favored the binding to BSA and gamma globulin through hydrophobic interactions. This ligand has a clear preference to interact with BSA, and gamma globulin, while cyanines 3 and 4 interacted preferentially with lysozyme, αchymotrypsin, and RNase.
C. CRUZ ET AL. Interesting binding profiles were obtained for the asymmetric cyanine 4; the dibenzopyrrole and benzothiazole units have different binding preferences according with the type of proteins. Cyanine 4 interacted with lysozyme and α-chymotrypsin by stacking interaction through contact of benzothiazole and through dibenzopyrrole with RNase.
CONCLUSIONS Four novel cyanines have been synthetized, and their interactions with lysozyme, α-chymotrypsin, RNase, BSA, and gamma globulin were evaluated by STD-NMR. STD experiments allowed the identification of the epitope, pinpointing the interactions between parts of each cyanine with the proteins. Cyanines 1 and 2, which differ only in their SO3H substitution at carbons 5 and 5″, showed different binding preferences with these proteins. Whereas cyanine 2 interacted preferentially through its alkyl chain with BSA and gamma globulin, cyanine 1 bounded preferentially lysozyme, α-chymotrypsin, and RNase through its benzopyrrole moiety.
Cyanines 3 and 4 did not interact with BSA and gamma globulin. In the case of cyanine 3, the main interactions involved the dibenzopyrrole unit with lysozyme, α-chymotrypsin, and RNase, reflecting π–π interactions. Cyanine 4 interacted with lysozyme and α-chymotrypsin through the benzothiazole and with RNase through dibenzopyrrole. In summary, our approach allowed screening the different binding modes of the four cyanines, bringing important structural information about molecular dye–protein interactions that can be used to select a desirable cyanine for protein purification.
Acknowledgements Carla Cruz acknowledges the post-doctoral grant from FCT (SFRH/BPD/46934/2008). This work was supported by the Portuguese Foundation for Science and Technology, (PTDC/QUI-QUI/ 100896/2008, PTDC/EBB-BIO/114320/2009 and PEst-C/SAU/ UI0709/2011 COMPETE). The NMR spectrometers are localized at Health Sciences Research Centre, University of Beira Interior and were purchased by the project POVT-0439-FEDER-00001.
REFERENCES Boto REF, El-Shishtawy RM, Santos PF, Reis LV, Almeida P. 2007. Synthesis and characterization of novel mono- and dicarboxy alkylthiacarbocyanines and their ester derivatives. Dyes Pigments 73: 195–205. Boto REF, Almeida P, Queiroz JA. 2008. Thiacarbocyanine as ligand in dyeaffinity chromatography for protein purification. Biomed. Chromatogr. 22: 278–288. Boto REF, Anyanwu U, Sousa F, Almeida P, Queiroz JA. 2009. Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model. Biomed. Chromatogr. 23: 987–993. Brown RA, Combridge BS. 1986. Binding of hepatitis virus particles to immobilized Procion Blue-HB and Cibacron Blue 3GA. J. Virol. Methods 14: 267–274. Caspi S, Halimi M, Yanai A, Sasson SB, Taraboulos A, Gabizon R. 1998. The antiprion activity of Congo Red. Putative mechanism, J. Biol. Chem. 273: 3484–3489. Clonis YD, Lowe CR. 1980. Triazine dyes: a new class of affinity labels for nucleotide-dependent enzymes. Biochem. J. 191: 247–251. Clonis YD, Stead CV, Lowe CR. 1987. Novel cationic dyes for protein purification. Biotechnol. Bioeng. 30: 621–627. Clonis YD, Labrou NE, Kotsira VP, Mazitsos C, Melissis S, Gogolas G. 2000. Biomimetic dyes as affinity chromatography tools in enzyme purification. J. Chromatogr. A 891: 33–44. Cruz C, Boto REF, Almeida P, Queiroz JA. 2011. Study of specific interaction between nucleotides and dye support by nuclear magnetic resonance. J. Mol. Recognit. 24: 975–980. Denizli A, Piskin E. 2001. Dye-ligand affinity systems. J. Biochem. Biophys. Methods 49: 391–416.
Federici MM, Chock PB, Stadtman ER. 1985. Interaction of Cibacron Blue F3GA with glutamine synthetase: use of the dye as a conformational probe: 1. Studies using unfractionated dye samples. Biochemistry 24: 647–660. Hattori T, Zhang X, Weiss C, Xu Y, Kubo T, Sato Y, Nishikawa S, Sakaida H, Uchiyama T. 1997. Triazine dyes inhibit HIV-1 entry by binding to envelope glycoproteins. Microbiol. Immunol. 41: 717–724. Jiang L-L, Dou L-F, Li B-L. 2007. An efficient approach to the synthesis of water-soluble cyanine dyes using poly(ethylene glycol) as a soluble support. Tetrahedron Lett. 48: 5825–5829. Labrou NE, Eliopoulos E, Clonis YD. 1996. Dye affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site. Biochem. J. 315: 687–694. Lowe CR, Burton SJ, Pearson J, Clonis YD, Stead CV. 1986. Design and application of biomimetic dyes in biotechnology. J. Chromatogr. 376: 121–126. Mayer M, Meyer B. 1999. Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy. Angew. Chem. Int. Ed. 38: 1784–1788. Mayer M, Meyer B. 2001. Group Epitope Mapping by Saturation Transfer Difference NMR to Identify Segments of a Ligand in Direct Contact with a Protein Receptor. J. Am. Chem. Soc. 123: 6108–6117. Skotland T. 1981. Studies on the interaction of Cibacron Blue and Procion Red with dopamine betamonooxygenase. Biochim. Biophys. Acta 659: 312–325. Viegas A, Manso J, Nobrega FL, Cabrita EJ. 2011. Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding. J. Chem. Educ. 88: 990–994.
204 wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
Revised: 4 December 2013,
Accepted: 14 December 2013,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2351
NMR screening of new carbocyanine dyes as ligands for affinity chromatography Carla Cruz*, Renato E. F. Boto, Anna K. Drzazga, Paulo Almeida and João A. Queiroz Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; proteins; cyanine ligands; molecular interactions
INTRODUCTION
J. Mol. Recognit. 2014; 27: 197–204
* Correspondence to: Carla Cruz, CICS-UBI—Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. E-mail: [email protected] C. Cruz, R. E. F. Boto, A. K. Drzazga, P. Almeida, J. A. Queiroz CICS-UBI—Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal
Copyright © 2014 John Wiley & Sons, Ltd.
197
Synthetic dyes have been used in affinity chromatography, known as dye-affinity chromatography (dye-AC) (Denizli and Piskin 2001), allowing to purify several types of proteins along with various enzymes and, in some cases, in a remarkably specific manner biomimeting natural ligands (Clonis et al. 1987; Lowe et al. 1986). As representative example, triazine dyes mimic the binding of natural anionic heterocyclic substrates, such as nucleic acids, nucleotides, coenzymes, and vitamins, likely representing the first example of combinatorial screening for protein binding (Clonis et al. 2000). The triazine dye Cibacron blue F3G-A was used to separate alcohol dehydrogenase from horse liver (Labrou et al. 1996). Also, anthraquinonemoiety-containing aromatic sulfonated dyes, such as Procion Blue H-B, MX-R, and Vilmafix Blue A-R, bind preferentially the nucleotide-binding site of several proteins and mimic the binding of naturally occurring anionic coenzymes (NADH, FAD) (Clonis and Lowe 1980). Other dye ligands have also been employed to remove prion proteins, human immunodeficiency virus-1, and hepatitis B viral particles from biological fluids (Caspi et al. 1998; Hattori et al. 1997; Brown and Combridge 1986). The use of dye-AC for protein purification requires selection of one or more immobilized dyes, because, in some cases, these ligands established nonspecific interactions with other parts of the proteins by a combination of electrostatic, hydrophobic, hydrogen bonding, and charge-transfer interactions, depending on the structure of the dyes. Several techniques including NMR, electron spin resonance, circular dichroism, and computational methods have been used to explain dye–protein interactions (Skotland 1981; Federici et al. 1985). NMR-based screening, despite of its low intrinsic sensitivity, offers the largest dynamic range and is capable of capturing very weak interactions. STD-NMR experiments use small quantities of nonlabeled
biomolecules, focusing on the signals of the ligand without any need of processing NMR information about the biomolecule (Viegas et al. 2011). Cyanines have been used for a large number of applications, in particular, as functional dyes; nevertheless, their ability as ligands for dye-AC has been recently explored (Boto et al. 2009; Boto et al. 2008; Cruz et al. 2011). In these studies, a thiacarbocyanine has been postgrafted onto beaded cellulose by a curing method and showed a selective interaction allowing separation of three proteins from an artificial mixture (Boto et al. 2009). The adsorption of lysozyme onto this immobilized dye was further investigated, presenting a maximum dynamic binding capacity value of 8.6 mg/ml for lysozyme, achieved at 30 °C and pH 9, and a dissociation constant (KD) of 2.61 ± 0.36 × 10–5 M (Boto et al. 2008). Following these studies and looking for the development of new cyanine for dye-AC, four indocarbocyanines were synthesized (Figure 1). These indocarbocyanines have carboxyalkyl chains, apart from acting as spacer arms that potentially enhance the efficiency of the purification process, that offer a convenient way to couple the dye to the matrices of cellulose and agarose. Further, some structural variations were introduced, such as, sulfonic difunctionalization, symmetric, and asymmetric groups to provide a desirable interaction with the standard proteins. Saturation transfer difference-NMR experiments have been used to screen the potential binding sites within cyanines that
C. CRUZ ET AL.
Figure 1. Indocarbocyanines 1–4.
specifically interact with BSA, α-chymotrypsin, lysozyme, RNase, and gamma globulin.
EXPERIMENTAL General All reagents were of the highest purity available, purchased from Sigma-Aldrich Company, and used as received. Solvents were of analytical grade and were dried over 3 Ǻ molecular sieves prior to use. Standard BSA (A-6793), α-chymotrypsin (from bovine pancreas, C-4129), lysozyme (from chicken egg white, Biochemika 62970), and gamma globulin (from bovine blood, G5009) were also purchased from Sigma-Aldrich Co. RNase (from Bovine Pancreas, 27033002) and was purchased from USB Corp. All other chemicals were of analytical grade or higher and used as received. All reactions were monitored by thin-layer chromatography on aluminum plates precoated with Merck silica gel 60 F254 (0.25 mm) using dichloromethane or dichloromethane/methanol (9.5:0.5 or 9:1), and the spots have been examined under 254, 312, and 365 nm UV light. Melting points (M.p.) were measured in open capillary tubes in a Buchi 530 M.p. apparatus and were uncorrected. Infrared spectra (IR) were recorded on Thermo Scientific Nicolet iS10 FT-IR spectrophotometer. All samples were prepared by mixing FT-IR grade KBr (Sigma-Aldrich) with 1% (w/w) dye and grinding to a fine powder. Spectra were recorded over the 600–4000 cm 1 range without baseline corrections. Characteristic absorptions are given in cm 1. 1 H and 13C NMR spectra were recorded in DMSO-d6 solutions at a temperature of 298 K on a Bruker Avance III 600-MHz spectrometer operating at 14.09 T, observing 1H at 600.13, and
13
C at 150.91 MHz. The spectrometer was equipped with a cryoprobe, and all spectra were processed with the software topspin 3.1. Chemical shifts are reported in ppm using tetramethylsilane (TMS) as an internal standard. Coupling constants (J) are given in Hz. High resolution mass spectra (HRMS) were performed on an microTOF (focus) mass spectrometer (Bruker Daltonics, Bremen, Germany). Ions were generated using an ApolloII (ESI) source. Ionization was achieved by electrospray, using a voltage of 4500 V applied to the needle, and a counter voltage between 100 and 150 V applied to the capillary. Samples were prepared by adding a spray solution of 70:30 (v/v) methanol/water with 0.1% of formic acid to a solution of the sample in CH2Cl2 or another solvent at a v/v ratio of 1% to 5% to give the best signal-to-noise ratio. Data acquisition was performed using the microTOF control software version 2.1, and data processing was performed using the data analysis software, version 3.4 both from Bruker Daltonics. Synthesis of the quaternarium ammonium salts 6a–c Sulphonated indole 5b was synthesized and has been already described (Jiang et al. 2007). The quaternary ammonium salts 6a–c were synthesized according to our previous work (Boto et al. 2007) by reacting 15.7 mmol of the trimethylindole derivatives 5a–c with 17.3 mmol of 11-bromoundecanoic acid at 150 ºC. The crude product was washed several times with diethyl ether and purified by recrystallization from acetonitrile (Scheme 1). 1-(10-carboxydecyl)-2,3,3-trimethyl-3H-indolium bromide (6a) Yield: 79%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.21–1.26 (8H, m, CH2), 1.28–1.33 (2H, m, CH2), 1.38–1.42 (2H, m,
198
Scheme 1. Quaternary ammonium salts and indocarbocyanine synthesis.
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
NMR SCREENING OF NEW CARBOCYANINE DYES CH2), 1.49 (2H, q, J = 7.1 Hz, CH2), 1.53 (6H, s, 3-CH3), 1.82 (2H, q, J = 7.6 Hz, NCH2CH2), 2.17 (2H, t, J = 7.4 Hz, CH2COO), 2.85 (3H, s, 2-CH3), 4.45 (2H, t, J = 7.7 Hz, NCH2), 7.61–7.63 (2H, m, 5-CH,6CH), 7.84–7.86 (1H, m, 7-CH), 7.98–8.00 (1H, m, 4-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 14.0 (2-CH3), 22.0 (3-CH3), 24.5 (CH2), 25.9 (CH2), 27.2 (CH2), 28.4 (CH2), 28.5 (CH2), 28.6 (CH2), 28.6 (CH2), 28.7 (CH2), 33.7 (CH2COO), 47.6 (NCH2), 54.2 (3-C), 115.5 (4-CH), 123.5 (7-CH), 129.0 (6-CH), 129.4 (5-CH), 141.1 (7aC), 141.9 (3a-C), 174.5 (CO), 196.4 (2-C). IR (KBr) υ (cm 1): 3420, 2928, 2855, 1731 (C = O), 1625 (C = C), 1461, 1198, 767. HRMS (ESI-TOF) C22H34NO2: Calcd.: 344.25841; Found: 344.25823. 2.2.2. 1-(10-carboxydecyl)-2,3,3-trimethyl-5-sulfo-3H-indolium bromide (6b) Yield: 66%. M.p. 197–200 ºC. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.22–1.27 (8H, m, CH2), 1.29–1.33 (2H, m, CH2), 1.37–1.42 (2H, m, CH2), 1.45–1.50 (2H, m, CH2), 1.53 (6H, s, 3-CH3), 1.81 (2H, q, J = 7.0 Hz, NCH2CH2), 2.18 (2H, t, J = 7.2 Hz, CH2COO), 2.82 (3H, s, 2CH3), 4.43 (2H, t, J = 7.2 Hz, NCH2), 7.80 (1H, d, J = 8.2 Hz, 6-CH), 7.90 (1H, d, J = 8.2 Hz, 7-CH), 8.01 (1H, s, 4-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 13.9 (2-CH3), 21.9 (3-CH3), 24.4 (CH2), 25.9 (CH2), 27.2 (CH2), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.7 (CH2), 28.8 (CH2), 33.6 (CH2COO), 47.6 (NCH2), 55.5 (3-C), 114.9 (7-CH), 120.7 (4-CH), 126.3 (6-CH), 140.9 (7a-C), 141.5 (3aC), 149.5 (5-CH), 174.5 (CO), 197.2 (2-C). IR (KBr) υ (cm-1): 3448, 2926, 2852, 1731 (C = O), 1624 (C = C), 1467, 1182, 686. HRMS (ESI-TOF) C22H34NO5S: Calcd.: 424.21522; Found: 424.21502. 2.2.3. 3-(10-carboxydecyl)-1,1,2-trimethyl-1H-benzo[e]indolium bromide (6c) Yield: 52%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.20–1.26 (8H, m, CH2), 1.32 (2H, q, J = 7.0 Hz, CH2), 1.41–1.46 (4H, m, CH2), 1.76 (6H, s, 1-CH3), 1.88 (2H, q, J = 7.6 Hz, NCH2CH2), 2.16 (2H, t, J = 7.3 Hz, CH2COO), 2.95 (3H, s, 2-CH3), 4.58 (2H, t, J = 7.7 Hz, NCH2), 7.72 (1H, t, J = 7.9 Hz, 7-CH), 7.78 (1H, t, J = 8.0 Hz, 8-CH), 8.16 (1H, d, J = 8.9 Hz, 4-CH), 8.21 (1H, d, J = 8.2 Hz, 6-CH), 8.29 (1H, d, J = 8.9 Hz, 5-CH), 8.37 (1H, d, J = 8.3 Hz, 9-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 13.9 (2-CH3), 21.6 (1-CH3), 24.5 (CH2), 25.9 (CH2), 27.5 (CH2), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.8 (CH2), 28.8 (CH2), 33.7 (CH2COO), 47.9 (NCH2), 55.5 (1-C), 113.4 (4-CH), 123.5 (9-CH), 127.3 (7-CH), 127.3 (9a-C), 128.4 (8-CH), 129.8 (6-CH), 130.7 (5-CH), 133.1 (5a-C), 137.0 (9b-C), 138.5 (3a-C), 174.5 (CO), 196.3 (2-C). IR (KBr) υ (cm-1): 3417, 2926, 2853, 1723 (C = O), 1630 (C = C), 1581, 1465, 1174, 814, 753. HRMS (ESI-TOF) C26H36NO2: Calcd.: 394.27406; Found: 394.27379. Synthesis of indocarbocyanines 1–4 Symmetric indocarbocyanines 1–3
J. Mol. Recognit. 2014; 27: 197–204
1-(10-carboxydecyl)-2-((1E,3Z)-3-(1-(10-carboxydecyl)-3,3-dime thylindolin-2-ylidene)prop-1-enyl)-3,3-dimethyl-3H-indolium iodide (1) Yield: 70%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.21–1.26 (16H, m, CH2), 1.31–1.34 (4H, m, CH2), 1.38–1.41 (4H, m, CH2), 1.44–1.48 (4H, m, CH2), 1.70 (12H, s, 3-CH3), 1.73 (4H, q, J = 7.0 Hz, NCH2CH2, N″CH2CH2), 2.11 (4H, t, J = 7.3 Hz, CH2COO), 4.12 (4H, t, J = 6.7 Hz, NCH2, N″CH2), 6.51 (2H, d, J = 13.4 Hz, 1ʹ-CH, 3ʹ-CH), 7.30 (2H, t, J = 7.0 Hz, 5-CH, 5″-CH), 7.43–7.47 (4H, m, 6-CH, 6″-CH, 7CH, 7″-CH), 7.64 (2H, d, J = 7.3 Hz, 4-CH, 4″-CH), 8.35 (1H, t, J = 13.4 Hz, 2ʹ-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 24.5 (CH2), 24.7 (CH2), 26.1 (CH2), 27.0 (CH2), 27.4 (3-CH3), 28.4 (CH2), 28.6 (CH2), 28.7 (CH2), 28.7 (CH2), 28.8 (CH2), 34.4 (CH2COO), 43.8 (NCH2,N″ CH2), 102.5 (1ʹ-CH, 3ʹ-CH), 111.6 (7-CH, 7″-CH), 122.5 (4-CH, 4″-CH), 125.2 (5-CH, 5″-CH), 128.7 (6-CH, 6″-CH), 140.6 (3a-C, 3a″-C), 141.9 (7a-C, 7a ″-C), 149.8 (2ʹ-CH), 173.8 (2-C, 2″-C), 174.8 (CO). IR (KBr) υ (cm 1): 3448, 2926, 2852, 1730 (C = O), 1557 (C = C), 1429, 1138, 929, 757. HRMS (ESI-TOF) C45H65N2O4: Calcd.: 697.49389; Found: 697.49346. 1-(10-carboxydecyl)-2-{3-[1-(10-carboxydecyl)-3,3-dimethyl-5sulfoindolin-2-ylidene] prop-1-enyl}-3,3-dimethyl-5-sulfo-3H-indolium hydrogenosulfate (2) Yield: 75%. Oleum. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.21–1.25 (16H, m, CH2), 1.28–1.32 (4H, m, CH2), 1.34–1.39 (4H, m, CH2), 1.42–1.48 (4H, m, CH2), 1.69 (12H, s, 3-CH3), 1.71–1.74 (4H, m, NCH2CH2, N″CH2CH2), 2.15 (4H, t, J = 7.3 Hz, CH2COO), 4.12 (4H, t, J = 6.9 Hz, NCH2, N″CH2), 6.52 (2H, d, J = 13.4 Hz, 1ʹ-CH, 3ʹ-CH), 7.39 (2H, d, J = 8.3 Hz, 7-CH, 7″-CH), 7.62 (2H, d, J = 8.2 Hz, 6-CH, 6″-CH), 7.80 (2H, s, 4-CH, 4″-CH), 8.35 (1H, t, J = 13.4 Hz, 2ʹ-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 24.4 (CH2), 26.0 (CH2), 27.0 (CH2), 27.4 (3-CH3), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.7 (CH2), 28.8 (CH2), 33.6 (CH2COO), 43.9 (NCH2, N″CH2), 48.9 (3C, 3″C), 103.0 (1ʹCH, 3ʹ-CH), 110.8 (7-CH, 7″-CH), 119.8 (4-CH, 4″-CH), 126.3 (6-CH, 6″-CH), 140.1 (3a-C, 3a″-C), 141.9 (7a-C, 7a″-C), 145.4 (5-CH, 5″-CH), 149.9 (2ʹ-CH), 174.3 (2-C, 2″-C), 174.5 (CO).). IR (KBr) υ (cm 1): 3423, 2927, 2854, 1717 (C = O), 1557 (C = C), 1440, 1113, 931, 689. HRMS (ESI-TOF) C45H65N2O10S2: Calcd.: 857.40751; Found: 857.40724. 3-(10-carboxydecyl)-2-{3-[3-(10-carboxydecyl)-1,1-dimethyl1H-benzo[e]indol-2(3H)-ylidene]prop-1-enyl}-1,1-dimethyl-1H-benzo [e]indolium iodide (3) Yield: 71%. M.p. 107 ºC–109 ºC. 1H NMR (600.13 MHz, DMSOd6) δ (ppm): 1.16–1.24 (16H, m, CH2), 1.29–1.34 (4H, m, CH2), 1.39–1.44 (8H, m, CH2), 1.76–1.81 (4H, m, NCH2CH2, N″CH2CH2), 1.99 (12H, s, 1-CH3), 2.13 (4H, t, J = 7.3 Hz, CH2COO, ), 4.26 (4H, t, J = 7.1 Hz, NCH2, N″CH2), 6.60 (2H, d, J = 13.4 Hz, 1ʹ-CH, 3ʹ-CH), 7.53 (2H, t, J = 7.4 Hz, 7-CH, 7″-CH), 7.68 (2H, t, J = 7.5 Hz, 8-CH, 8″-CH), 7.77 (2H, d, J = 8.7 Hz, 4-CH, 4″-CH), 8.07 (2H, d, J = 8.2 Hz, 6-CH, 6″-CH), 8.10 (2H, d, J = 8.8 Hz, 5-CH, 5″-CH), 8.29 (2H, d, J = 8.5 Hz, 9-CH, 9″-CH), 8.58 (1H, t, J = 13.5 Hz, 2ʹ-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 24.6 (CH2), 26.1 (CH2), 27.2 (1CH3), 27.4 (CH2), 28.6 (CH2), 28.8 (CH2), 28.9 (CH2), 28.9 (CH2), 29.0 (CH2), 33.8 (CH2COO), 44.0 (NCH2, N″CH2), 50.7 (1C, 1C″), 102.2 (1ʹ-CH, 3ʹ-CH), 111.9 (4-CH, 4″-CH), 122.3 (9-CH, 9″-CH), 125.2 (7-CH, 7″-CH), 127.5 (9a-CH, 9a″-CH), 128.1 (8-CH, 8″-CH), 130.1 (6-CH, 6″-CH), 130.6 (5-CH, 5″-CH), 131.6 (5a-CH, 5a″-CH), 133.3 (9b-C, 9b″-C), 139.7 (3a-C, 3a″-C), 148.6 (2ʹ-CH), 174.7 (2-C, 2″-C), 175.2 (CO). ). IR (KBr) υ (cm 1): 3447, 2925, 2852, 1729
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
199
The condensation of indole quaternary salts was carried out by refluxing for 18 h and 3.5 mmol of salts with 7.22 mmol of triethylortoformate in 10 ml of pyridine per gram of salt. After cooling to a room temperature, diethyl ether was added, and the resulting mixture was refrigerated to allow complete precipitation. The dye was counter-ion exchanged to hydrogen sulfate (for sulfoindocarbocyanine) or iodide (others) by dissolution in a minimum amount of acetonitrile, followed by an addition of 5% (v/v) aqueous H2SO4 or 14% (w/v) aqueous KI, respectively. The solution or suspension obtained was heated until reflux and then cooled to a room temperature and refrigerated overnight. The resulting crystalline product was collected
by filtration under reduced pressure, washed with water and diethyl ether and recrystallized from dry acetonitrile.
C. CRUZ ET AL. (C = O), 1557 (C = C), 1428, 1172, 936, 653. HRMS (ESI-TOF) C53H69N2O4: Calcd.: 797.52519; Found: 797.52513. Asymmetric thioindocarbocyanine (4) A solution of 2.30 g (4.93 mmol) of quaternary ammonium salt 6c and 1.95 g (4.47 mmol) of 2-[2-(acetylphenylamino)vinyl]-3-ethyl benzothiazol-3-ium iodide, prepared as previously described (Boto et al. 2007), in 10 ml of dry pyridine was refluxed for 1 h. After cooling to a room temperature, diethyl ether was added, and the resulting mixture was refrigerated to allow complete precipitation. The crystalline product obtained was collected by filtration under reduced pressure, washed with diethyl ether and recrystallized from dry acetonitrile. 3-(10-carboxydecyl)-2-((1E,3E)-3-(3-ethylbenzo[d]thiazol-2(3H)ylidene)prop-1-enyl)-1,1-dimethyl-1H-benzo[e]indolium iodide (4) Yield: 14%. M.p. 105 ºC–108 ºC. 1H NMR (600.13 MHz, DMSO-d6) δ (ppm): 1.19–1.24 (8H, m, CH2), 1.31–1.34 (2H, m, CH2), 1.38–1.47
(7H, m, N″CH2CH3, CH2), 1.73 (2H, quint, J = 7.3 Hz, NCH2CH2), 1.92 (6H, s, 1-CH3), 2.14 (2H, t, J = 7.3 Hz, CH2COO), 4.16 (2H, t, J = 7.0 Hz, NCH2), 4.50 (2H, q, J = 7.1 Hz, N″CH2), 6.41 (1H, d, J = 13.2 Hz, 1ʹCH), 6.86 (1H, d, J = 13.0 Hz, 3ʹ-CH), 7.47–7.53 (2H, m, 7-CH, 6″-CH), 7.63–7.66 (2H, m, 8-CH + 5″-CH), 7.70 (1H, d, J = 8.6 Hz, 4-CH), 7.88 (1H, d, J = 8.3 Hz, 4″-CH), 8.04 (1H, d, J = 8.4 Hz, 6-CH), 8.06 (1H, d, J = 9.0 Hz, 5-CH), 8.09 (1H, d, J = 7.9 Hz, 7″-CH), 8.14 (1H, t, J = 13.1 Hz, 2ʹ-CH), 8.25 (1H, d, J = 8.5 Hz, 9-CH). 13C NMR (150.91 MHz, DMSO-d6) δ (ppm): 13.0 (CH3), 24.4 (CH2), 26.1 (CH2), 27.0 (CH2), 28.0 (1-CH3), 28.4 (CH2), 28.5 (CH2), 28.6 (CH2), 28.7 (CH2), 28.8 (CH2), 33.3 (CH2COO), 42.1 (N″CH2), 43.8 (NCH2), 50.2 (1C), 99.3 (1ʹ-CH), 101.7 (3ʹ-CH), 111.5 (4-CH), 114.1 (4″-CH), 122.0 (9-CH), 123.4 (7″-CH), 124.6 (7-CH), 125.6 (7a″-CH), 126.0 (6″-CH), 127.6 (8-CH), 127.7 (5″-CH), 128.5 (6-CH), 129.9 (9a-C), 130.3 (5-CH), 131.1 (5a-C), 132.4 (9b-C), 139.9 (3a-C),140.9 (3a″-C), 147.1 (2ʹ-C), 166.5 (2″-C), 172.9 (2-C), 174.5 (CO). IR (KBr) υ (cm 1): 3440, 2925, 2852, 1731 (C = O), 1555 (C = C), 1424, 1131, 939, 751. HRMS (ESI-TOF) C37H45N2O2S: Calcd.: 581.31963; Found: 581.31945.
200
1
Figure 2. H NMR reference spectra of the cyanine 1 and STD-NMR spectra of the complexes cyanine 1-lysozyme, cyanine 1-α-chymotrypsin, cyanine 1-RNase and cyanine 1-BSA. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
NMR SCREENING OF NEW CARBOCYANINE DYES STD-NMR spectroscopy Preparation of the samples A 100 μM protein stock solution was prepared in 100 mM potassium phosphate buffer in H2O: D2O (9:1) at pH 8.0. For all cyanine dyes, 1 mM stock solutions in DMSO-d6 were prepared and stored in the dark at 4 ºC. Samples for STD-NMR build-up analysis were prepared as follow: 240 μl of cyanine was added to 250 μl of protein stock solution and 10 μl of trimethylsilyl propionate. Method The quaternary ammonium salts and cyanine dyes resonances were assigned using 1H, 13C, DEPT90, DEPT135, 1H-1H correlation spectroscopy, 1H-1H total correlation spectroscopy, 1H-13C heteronuclear single-quantum correlation and 1H-13C heteronuclear multiplebond correlation. The STD-NMR spectra were recorded with 512 scans and selective saturation of protein resonances at 0 to 1 ppm and 10 to 11 ppm (on resonance) and 30 ppm (off
resonance) using Eburp2.1000, a shaped pulse (length for the protein saturation 50 ms, 1-ms delay between pulses) for a saturation time of 2.0 s. The number of averages (L4) was set to 16, and a relaxation delay was 3 s. Also, eight dummy scans were employed to reduce subtraction artifacts. The rows of the pseudo-2D spectrum obtained correspond to the different saturation frequencies (on and off resonance). The difference spectrum is obtained by direct subtraction of the individual rows (off resonance–on resonance) (Mayer and Meyer 1999). The STD build-up data processing was obtained by dividing STD signal intensities by the intensities of the reference spectrum [(I0-ISTD)/I0] (Mayer and Meyer 2001). The intensity of the largest STD effect was set to 100%, and the others percentages are calculated accordingly. Reference experiments using the free cyanines themselves were performed under the same experimental conditions to verify true ligand binding. No signal was present in the difference spectra, indicating that the effects observed in the presence of the proteins were because of true saturation transfer.
J. Mol. Recognit. 2014; 27: 197–204
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
201
1
Figure 3. H NMR reference spectra of the cyanine 2 and STD-NMR spectra of the complexes cyanine 2-lysozyme, cyanine 2-α-chymotrypsin, cyanine 2-BSA and cyanine 2-gamma globulin. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
C. CRUZ ET AL.
RESULTS AND DISCUSSION The binding affinity properties of the four cyanines to BSA, αchymotrypsin, lysozyme, RNase, and gamma globulin were achieved by STD-NMR, ranked according to their STD intensities. The STD-NMR spectra obtained for cyanine–protein complexes, and the reference spectrum of each cyanine are shown in Figures 2, 3, 4, and 5. The cyanines that differ in their symmetry, heterocyclic moieties, functional groups, and carboxyalkyl chains showed different kinds of interaction with these proteins. Almost all cyanines showed a response in the STD spectra, except cyanines 1, 3, and 4 with gamma globulin, cyanines 3 and 4 with BSA, and cyanine 2 with RNase. The results can be interpreted as explained hereafter. Cyanine 1 The largest STD effects are observed for the heterocyclic moiety of cyanine 1 with lysozyme, α-chymotrypsin, and RNase, whereas with BSA, the alkyl chain showed the largest STD effect. No STD responses were found for gamma globulin. The 1H NMR spectrum of 1 and the STD spectra of complex cyanine 1-proteins are presented in Figure ure 2, as well as its protons chemical shifts assignments. Accordingly, methyl groups should be the ones more directly involved in binding and closer to lysozyme and α-chymotrypsin, followed by protons H-2ʹ, H-5, H-4, H-7, and H-6. No STD
response was found for the alkyl chain indicating that they should be the ones more distant from the lysozyme and αchymotrypsin. The binding epitope of cyanine 1 to RNase indicates that the major interactions involve protons H-5, H-6, and H-7. The methyl group has a lower relative STD value indicating that it should be distant from the protein surface. In the presence of BSA, lower STD contacts were found in the low-field region of the STD spectrum corresponding to the heterocyclic moiety, and stronger STD signals were found with the carboxyalkyl chain of 1, suggesting close proximity of this ligand region to the protein surface. Cyanine 2 The comparison between the spectra pointed out that the cyanine 2 establishes more contacts with large proteins such as BSA and gamma globulin. The 1H NMR spectrum of cyanine 2 and the STD spectra are presented in Figure ure 3. The epitope mapping is similar for these two proteins. The largest STD effect is observed for alkyl chain, and weaker STD contacts are observed with heterocyclic protons H-1ʹ, H-6, H-4, H-7 and methyl groups. On contrary for lysozyme and α-chymotrypsin, the major interactions of cyanine 2 involve the methyl groups (100% STD absolute intensities) in the high-field region of the STD spectra. The other heterocyclic protons do not seem to be in close contact with these proteins. No STD signals were detected for RNase.
202
1
Figure 4. H NMR reference spectra of the cyanine 3 and STD-NMR spectra of the complexes cyanine 3-lysozyme, cyanine 3-α-chymotrypsin and cyanine 3-RNase. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
NMR SCREENING OF NEW CARBOCYANINE DYES
1
Figure 5. H NMR reference spectra of the cyanine 4 and STD-NMR spectra of the complexes cyanine 4-lysozyme, cyanine 4-α-chymotrypsin and cyanine 4-RNase. The ratios of the intensities ISTD/I0 were normalized using the largest STD effect 100% as a reference.
Cyanine 3 The STD spectra of cyanine 3 show only STD signals for RNase, α-chymotrypsin, and lysozyme indicating binding preference to these proteins instead of BSA and gamma globulin (Figure 4). The binding epitope for α-chymotrypsin and lysozyme are quite similar; proton H-5 (100% of saturation) followed by H-6 leads (~90% of saturation) to the most prominent STD signals, indicating that are in close contact with these proteins surface. The remaining protons of the heterocyclic moiety, H-4, H-7, H-8, H-9, and methyl groups (12%–29% of saturation) give weaker signals. For RNase, the STD-NMR spectrum of cyanine 3 showing the involvement of H-7 (100% of saturation) in the binding to the protein because of the highest amount of saturation received, followed by protons H-8 and H-4 (84% and 75% of saturation, respectively). The other protons of the heterocyclic moiety establish weak STD contacts with the protein through H-5 (29% of saturation), H-6 (30% of saturation) H-9, and CH3 (14% of saturation).
Cyanine 4
J. Mol. Recognit. 2014; 27: 197–204
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr
203
The binding profiles of asymmetric cyanine 4 are similar to cyanine 3. Indeed, this ligand did not show STD signals with gamma globulin and BSA as for cyanine 3 (Figure ure 5). The binding epitope of cyanine 4 with lysozyme and α-chymotrypsin are similar; however, different interaction patterns were obtained with RNase.
The analysis of the STD signals indicates that the major interactions of 4 with lysozyme and α-chymotrypsin are through H-7″ (100% of saturation) and H-2ʹ (76% of saturation for lysozyme and 57% of saturation for α-chymotrypsin), indicating that the benzothiazole unit is in close contact with these proteins during the complex formation. While relative weaker responses are observed for protons of dibenzopyrrole, indicating that this unit should be more distant from the protein surface. On the contrary for RNase, protons of dibenzopyrrole, H-7 (100% of saturation), followed by H-4 (94% of saturation) and H-8 (62% of saturation), receive the higher magnetization, suggesting that are more involve in the binding to RNase. However, protons of benzothiazole unit are less intimate contact with RNase since receiving the lower amount of magnetization (H-7″ 12% of saturation and H-4″ 32% of saturation). These results show that the four cyanines, which differ in symmetry, aromatic units, sulfonic functionalization, and Nalkyl chains, present different binding profiles representing specific molecular recognition with the different proteins. In the case of cyanines 1 and 3, the benzo and dibenzo units contributed to ligand binding through π–π interactions with lysozyme, α-chymotrypsin, and RNase, whereas the alkyl chain of cyanine 2 favored the binding to BSA and gamma globulin through hydrophobic interactions. This ligand has a clear preference to interact with BSA, and gamma globulin, while cyanines 3 and 4 interacted preferentially with lysozyme, αchymotrypsin, and RNase.
C. CRUZ ET AL. Interesting binding profiles were obtained for the asymmetric cyanine 4; the dibenzopyrrole and benzothiazole units have different binding preferences according with the type of proteins. Cyanine 4 interacted with lysozyme and α-chymotrypsin by stacking interaction through contact of benzothiazole and through dibenzopyrrole with RNase.
CONCLUSIONS Four novel cyanines have been synthetized, and their interactions with lysozyme, α-chymotrypsin, RNase, BSA, and gamma globulin were evaluated by STD-NMR. STD experiments allowed the identification of the epitope, pinpointing the interactions between parts of each cyanine with the proteins. Cyanines 1 and 2, which differ only in their SO3H substitution at carbons 5 and 5″, showed different binding preferences with these proteins. Whereas cyanine 2 interacted preferentially through its alkyl chain with BSA and gamma globulin, cyanine 1 bounded preferentially lysozyme, α-chymotrypsin, and RNase through its benzopyrrole moiety.
Cyanines 3 and 4 did not interact with BSA and gamma globulin. In the case of cyanine 3, the main interactions involved the dibenzopyrrole unit with lysozyme, α-chymotrypsin, and RNase, reflecting π–π interactions. Cyanine 4 interacted with lysozyme and α-chymotrypsin through the benzothiazole and with RNase through dibenzopyrrole. In summary, our approach allowed screening the different binding modes of the four cyanines, bringing important structural information about molecular dye–protein interactions that can be used to select a desirable cyanine for protein purification.
Acknowledgements Carla Cruz acknowledges the post-doctoral grant from FCT (SFRH/BPD/46934/2008). This work was supported by the Portuguese Foundation for Science and Technology, (PTDC/QUI-QUI/ 100896/2008, PTDC/EBB-BIO/114320/2009 and PEst-C/SAU/ UI0709/2011 COMPETE). The NMR spectrometers are localized at Health Sciences Research Centre, University of Beira Interior and were purchased by the project POVT-0439-FEDER-00001.
REFERENCES Boto REF, El-Shishtawy RM, Santos PF, Reis LV, Almeida P. 2007. Synthesis and characterization of novel mono- and dicarboxy alkylthiacarbocyanines and their ester derivatives. Dyes Pigments 73: 195–205. Boto REF, Almeida P, Queiroz JA. 2008. Thiacarbocyanine as ligand in dyeaffinity chromatography for protein purification. Biomed. Chromatogr. 22: 278–288. Boto REF, Anyanwu U, Sousa F, Almeida P, Queiroz JA. 2009. Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model. Biomed. Chromatogr. 23: 987–993. Brown RA, Combridge BS. 1986. Binding of hepatitis virus particles to immobilized Procion Blue-HB and Cibacron Blue 3GA. J. Virol. Methods 14: 267–274. Caspi S, Halimi M, Yanai A, Sasson SB, Taraboulos A, Gabizon R. 1998. The antiprion activity of Congo Red. Putative mechanism, J. Biol. Chem. 273: 3484–3489. Clonis YD, Lowe CR. 1980. Triazine dyes: a new class of affinity labels for nucleotide-dependent enzymes. Biochem. J. 191: 247–251. Clonis YD, Stead CV, Lowe CR. 1987. Novel cationic dyes for protein purification. Biotechnol. Bioeng. 30: 621–627. Clonis YD, Labrou NE, Kotsira VP, Mazitsos C, Melissis S, Gogolas G. 2000. Biomimetic dyes as affinity chromatography tools in enzyme purification. J. Chromatogr. A 891: 33–44. Cruz C, Boto REF, Almeida P, Queiroz JA. 2011. Study of specific interaction between nucleotides and dye support by nuclear magnetic resonance. J. Mol. Recognit. 24: 975–980. Denizli A, Piskin E. 2001. Dye-ligand affinity systems. J. Biochem. Biophys. Methods 49: 391–416.
Federici MM, Chock PB, Stadtman ER. 1985. Interaction of Cibacron Blue F3GA with glutamine synthetase: use of the dye as a conformational probe: 1. Studies using unfractionated dye samples. Biochemistry 24: 647–660. Hattori T, Zhang X, Weiss C, Xu Y, Kubo T, Sato Y, Nishikawa S, Sakaida H, Uchiyama T. 1997. Triazine dyes inhibit HIV-1 entry by binding to envelope glycoproteins. Microbiol. Immunol. 41: 717–724. Jiang L-L, Dou L-F, Li B-L. 2007. An efficient approach to the synthesis of water-soluble cyanine dyes using poly(ethylene glycol) as a soluble support. Tetrahedron Lett. 48: 5825–5829. Labrou NE, Eliopoulos E, Clonis YD. 1996. Dye affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site. Biochem. J. 315: 687–694. Lowe CR, Burton SJ, Pearson J, Clonis YD, Stead CV. 1986. Design and application of biomimetic dyes in biotechnology. J. Chromatogr. 376: 121–126. Mayer M, Meyer B. 1999. Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy. Angew. Chem. Int. Ed. 38: 1784–1788. Mayer M, Meyer B. 2001. Group Epitope Mapping by Saturation Transfer Difference NMR to Identify Segments of a Ligand in Direct Contact with a Protein Receptor. J. Am. Chem. Soc. 123: 6108–6117. Skotland T. 1981. Studies on the interaction of Cibacron Blue and Procion Red with dopamine betamonooxygenase. Biochim. Biophys. Acta 659: 312–325. Viegas A, Manso J, Nobrega FL, Cabrita EJ. 2011. Saturation-Transfer Difference (STD) NMR: A Simple and Fast Method for Ligand Screening and Characterization of Protein Binding. J. Chem. Educ. 88: 990–994.
204 wileyonlinelibrary.com/journal/jmr
Copyright © 2014 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2014; 27: 197–204
