Synthesis and Properties of Phosphonic Acid Containing Cyanine and

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Bioconjugate Chem. 2007, 18, 2178–2190

TECHNICAL NOTES Synthesis and Properties of Phosphonic Acid Containing Cyanine and Squaraine Dyes for Use as Fluorescent Labels Mark V. Reddington† Biosearch Technologies, Inc., 81 Digital Drive, Novato, California 94949. Received March 14, 2007; Revised Manuscript Received August 24, 2007

Cyanine and squaraine dyes that contain one or two phosphonate groups attached directly to the aromatic residues of the dyes were synthesized. Absorption and fluorescence properties of the dyes were determined. Succinimidyl active esters were prepared from the dyes and were used to label proteins and oligonucleotides. Some of the dyes permit the preparation of brighter conjugates than do commercially available analogues such as Cy3 and Cy5.

INTRODUCTION Fluorescent reporters are widely used as labels in biology and medicine (1, 2). As researchers seek to interrogate smaller sample volumes and fewer molecules of analyte, the requirements for the reporter become more demanding. In the case of fluorescent dyes, certain properties have been found to be advantageous. These include (1) high extinction coefficients, (2) high fluorescence quantum yields, (3) low nonspecific binding, (4) high photostability, and (5) ease of chemical modification to permit the ready incorporation of different reactive functionalities and the tuning of absorption and emission wavelengths. For many applications, good water solubility and resistance to the formation of dimers and higher aggregates are essential to prevent adverse effects upon substrate solubility and to prevent quenching of fluorescence through dye–dye groundstate interactions (1–5). Generally, these latter properties are achieved by attaching charged or neutral water-solubilizing residues to the dye. Subtle differences in the position, the hydrophilicity, and the charge of the water-solubilizing group often result in vastly different performance of a dye as a label (1). The sulfonate group is one of the most commonly used water solubilizing groups for commercial fluorescent labels (e.g., Alexa and Cy dyes) in applications where high labeling densities are required because electronic repulsion of the negatively charged dyes inhibits dye–dye contact and quenching interactions (3–6). To a lesser degree, quaternary ammonium groups (7), carbohydrates (8), oligonucleotides (9), polyethylene glycol residues (10), and phosphonate monoesters (11) have also been considered for this purpose but have yet to find wide-scale commercial acceptance. Except in the cases of porphyrins (12) and cyanines (13), where aliphatic phosphonates were used, the phosphonic acid group has remained largely ignored as a means to provide water solubility, resistance to aggregation, and other useful properties for fluorescent labels. Since the phosphonate group may carry two negative charges at some biologically relevant pH values, compared to one charge for the sulfonate group, it offers the possibility of water-soluble labels with enhanced resistance to aggregation and fluorescence quenching † Telephone: 415-883-8400. Fax: 415-883-8488. E-mail mark@ biosearchtech.com.

and therefore to more brightly labeled substrates. Herein, the synthesis, the photophysical properties, and the protein and oligonucleotide labeling capabilities of some representative examples of phosphonic acid containing cyanine and squarane dyes are presented and discussed. These new dyes have phosphonic acid groups attached directly to the aromatic residues of the dyes rather than through aliphatic linkers because this approach was proven to be more advantageous in the case of analogous sulfonated cyanine dyes (4, 5).

EXPERIMENTAL PROCEDURES General Methods. All reagents and solvents were either ACS reagent grade or HPLC/spectroscopy grade and were obtained from Sigma-Aldrich, Acros, TCI, or Burdick & Jackson unless otherwise noted. Cy3 monofunctional and Cy5 monofunctional active esters were obtained from Amersham Pharmacia Biotech. Quasar 570 amidite and Quasar 670 amidite were obtained from Biosearch Technologies, Inc. Analytical TLC was performed on EM Biosciences 5534-3 aluminum-backed silica-254 plates or Analtech RPS-F hydrocarbon impregnated silica gel plates. Column chromatography was performed on either Merck silica gel 60 or Analtech bonded C18 reversed-phase (RP) silica gel. For RP column chromatography compressed air was applied to the top of the column to achieve convenient flow rates. For RP column chromatography of dyes, movement of dyes through the column was monitored qualitatively by eye. Deionized (DI) water was used in the preparation of all solvent mixtures used for RP and ion exchange chromatography. Preparative HPLC was run on a Waters HPLC system with a 600E controller, a 600 pump, and a 490E detector using either a Dionex DNAPac (9 × 250 mm) PA-100 column or a Hamilton PRP-1 column. Analytical HPLC was performed on a Waters 2795 system with a 996 photodiode array detector using either a Dionex DNAPac (4 × 250 mm) PA-100 column or a Higgins Haisil HL C18 column. Buffers used for preparative and analytical HPLC were AXA (15% acetonitrile, 0.038 M Tris), AXB (15% acetonitrile, 0.038 M Tris, 1.0 M sodium bromide), RPA (5% acetonitrile, 0.05 M triethylammonium acetate), and RPB (acetonitrile). MALDI mass spectrometry was performed on a Bruker Biflex III MALDI-TOF instrument. Electrospray mass spectrometry was performed on a Finnigan LC Q, and LC–MS was

10.1021/bc070090y CCC: $37.00  2007 American Chemical Society Published on Web 10/11/2007

Technical Notes

performed on a Micromass LCT ESI mass spectrometer coupled with a Waters 2790 HPLC and a 996 PDA detector. NMR analysis (1H, 400 Mz; 13C, 100 Mz) was performed by Acorn NMR (Livermore, CA). 13C NMR spectra were acquired with broadband proton–carbon decoupling. Coupling constants reported for 13C NMR spectra are for phosphorus–carbon coupling. Elemental analysis was performed by Desert Analytics (Tucson, AZ). UV–vis spectra were recorded on a HewlettPackard 8453 spectrophotometer. Fluorescence spectra were recorded on a PTI spectrofluorometer and were corrected for instrument response characteristics. DNA synthesis was performed on a Biosearch 8700 DNA synthesizer using fastdeprotecting phosphoramidites from Pierce (Milwaukee, WI). Phosphate buffered saline (PBS), pH 7.3 (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 · 7H2O, 1.4 mM KH2PO4), borate buffered saline (BBS), pH 8.1 (12.5 mM Na2B4O7 · 10H2O, 0.25 mM HCl(aq)), carbonate–bicarbonate buffer, pH 9.3 (0.1 M Na2CO3, 0.1 M NaHCO3), and PCR buffer (50 mM KCl, 10 mM TrisHCl, 1.5 mM MgCl2) were prepared according to standard methods. p-Bromophenylhydrazine Hydrochloride (3). To a 1 L flask were added finely powdered p-bromoaniline (69 g, 0.4 mol), water (200 mL), and concentrated hydrochloric acid (100 mL, 1.2 mol). The mixture was cooled in an ice bath to less than 5 °C. A solution of sodium nitrite (30 g, 0.43 mol) in water (90 mL) was added dropwise to the aniline suspension over approximately 1 h while maintaining the temperature at less than 5 °C. The mixture was stirred for a further 0.5 h and then was gravity-filtered through a no. 1 paper into a chilled flask. The solution was added in small portions to a solution of 92% ammonium sulfite (118 g, 0.8 mol) in water (250 mL), which had been chilled to 0 °C, at a rate such that the temperature remained at less than 5 °C (approximately 1 h). During the addition, a yellow solid formed in the mixture. After the addition was completed, stirring was continued for 1 h at 0 °C and then the mixture was allowed to warm to room temperature. The solid was filtered off, and the filtrate was treated with concentrated hydrochloric acid (120 mL). The solution was heated to reflux, during which time a solid formed and then mostly dissolved. The hot solution was filtered and then allowed to cool to room temperature before being chilled in a refrigerator overnight. The crystals that formed were filtered off, washed with 1.0 M hydrochloric acid (60 mL), and dried under vacuum to afford 3 (54.3 g, 60%). 5-Bromo-2,3,3-trimethyl-3H-indole (4). To a 500 mL flask were added 3 (40.0 g, 0.179 mol), glacial acetic acid (250 mL), and 3-methylbutanone (50 mL, 0.36 mol). The mixture was heated at reflux for 3 h and then was cooled to room temperature. The volatile components were removed under vacuum, and the residue was partitioned between ether (350 mL) and water (100 mL). The aqueous phase was washed again with petroleum ether (2 × 100 mL), and then the combined organic solutions were dried (MgSO4), filtered, and evaporated under vacuum to afford 4 (35 g, 82%). The bulk of the material was used without further purification. A sample of the oil was distilled on a Kugelrohr to yield a colorless oil: bp, 75 °C at 0.20 mmHg; Rf ) 0.62 (SiO2, dichloromethane (DCM)/ methanol, 19:1); 1H NMR (CDCl3) δ 1.29 (s, 6H), 2.26 (s, 3H), and 7.36–7.44 (m, 3H); 13C NMR δ 15.5, 23.0, 54.2, 118.9, 121.4, 124.9, 130.7, 147.9, 152.7, 188.5; ESI-MS obsd (+) 238.0 (M + H)+, calcd 237.02 (M ) C11H12NBr). 5-Bromo-1-ethyl-2,3,3-trimethyl-3H-indolium Iodide (5). To a 250 mL flask were added 4 (12 g, 50.4 mmol), acetonitrile (25 mL), and ethyl iodide (20 mL). The mixture was heated at reflux under argon for 40 h, and then most of the volatile components were removed by distillation. The residue was triturated with ether (100 mL) to give a powder. The solid was

Bioconjugate Chem., Vol. 18, No. 6, 2007 2179

filtered off, washed with ether (2 × 150 mL), and dried under high vacuum to afford 5 as a solid (17.3 g, 87%): Rf ) 0.70 (SiO2, DCM/methanol, 9:1); 1H NMR (DMSO-d6) δ 1.42 (t, J ) 7.3 Hz, 3H), 1.55 (s, 6H), 2.84 (s, 3H), 4.48 (quartet, J ) 7.3 Hz, 2H), 7.84 (dd, J ) 1.9 Hz and J ) 8.6 Hz, 1H), 7.96 (d, J ) 8.6 Hz, 1H) and 8.19 (d, J ) 1.9 Hz, 1H); 13C NMR δ 12.8, 14.3, 21.8, 43.5, 54.6, 117.5, 122.9, 127.1, 132.0, 140.2, 144.4, 196.8; ESI-MS obsd (+) 266.1 (M – I)+, calcd 392.96 (M ) C13H17NBrI). Anal. Calcd for C13H17NBrI: C, 39.62; H, 4.35; N, 3.55. Found: C, 39.56; H, 4.28; N, 3.57. 5-Bromo-1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium iodide (6). To a 250 mL flask were added 4 (5.8 g, 24 mmol), acetonitrile (25 mL), and 6-iodohexanoic acid (5.1 g, 21 mmol). The mixture was heated at reflux under argon for 40 h. Most of the volatile components were removed by distillation, and then the mixture was heated at 140 °C for 2 h. The mixture was cooled and was triturated with DCM (100 mL) to give a powder. The solid was filtered off, washed with DCM (2 × 50 mL), and dried under high vacuum to afford 6 (8.7 g, 87 %): Rf ) 0.38 (SiO2, DCM/methanol, 9:1); 1H NMR (DMSO-d6) δ 1.43–1.48 (m, 2H), 1.50–1.55 (m, 8H), 1.82 (quintet, J ) 7.5 Hz, 2H), 2.22 (t, J ) 7.1 Hz, 2H), 2.83 (s, 3H), 4.44 (t, J ) 7.6 Hz, 2H), 7.82–7.85 (m, 1H), 7.95 (d, J ) 8.5 Hz, 1H) and 8.19 (d, J ) 1.7 Hz, 1H); 13C NMR δ 14.4, 22.0, 24.2, 25.6, 27.1, 33.5, 47.8, 54.6, 117.7, 123.0, 127.1, 132.0, 140.6, 144.3, 174.5, 197.62; ESI-MS obsd (+) 352.2 (M – I)+, calcd 479.00 (M ) C17H23NO2BrI). Anal. Calcd for C19H27NO2BrI: C, 42.52; H, 4.83; N, 2.92. Found: C, 42.34; H, 4.78; N, 2.94. 5-Bromo-1-(5-ethoxycarbonylpentyl)-2,3,3-trimethyl-3H-indolium iodide (7). To a 250 mL flask were added 6 (2.00 g, 4.17 mmol), ethanol (60 mL), and concentrated sulfuric acid (0.2 mL). The mixture was heated at reflux for 2 h and then was cooled to room temperature. The ethanol was removed under vacuum, and the residue was partitioned between water (40 mL) and DCM (80 mL). The organic layer was washed with 1 M HCl (40 mL), water (40 mL), and a saturated solution of sodium iodide (40 mL). It was dried (MgSO4) and was evaporated under vacuum to afford 7 as a brown solid (1.74 g, 82 %): Rf ) 0.79 (SiO2, DCM/methanol, 9:1); 1H NMR (DMSO-d6) δ 1.15 (t, J ) 7.1 Hz, 3H), 1.43–1.48 (m, 2H), 1.53–1.60 (m, 8H), 1.82 (quintet, J ) 7.5 Hz, 2H), 2.29 (t, J ) 7.3 Hz, 2H), 2.84 (s, 3H), 4.02 (quartet, J ) 7.1 Hz, 2H), 4.44 (t, J ) 7.5 Hz, 2H), 7.83–7.86 (m, 1H), 7.95 (d, J ) 8.6 Hz, 1H), and 8.19 (d, J ) 1.7 Hz, 1H); 13C NMR δ 14.3, 14.4, 22.0, 24.2, 25.4, 27.0, 33.4, 47.8, 54.6, 59.9, 117.6, 123.0, 127.1, 132.0, 140.6, 144.3, 172.9, 197.2; ESI-MS obsd (+) 380.3 (M – I)+, calcd 507.03 (M ) C19H27NO2BrI). (1-Ethyl-3,3-dimethyl-2-methylene-2,3-dihydro-1H-indol-5-yl)phosphonic Acid Diethyl Ester (8). To a dry three-neck 500 mL flask were added 5 (9.6 g, 20 mmol) and nickel chloride (0.52 g, 4.0 mmol). The flask was fitted with a dropping funnel and a condenser and then was lowered into an oil bath. The mixture was heated to 175 °C. At approximately 165 °C triethyl phosphite (15 mL, 86 mmol) was added dropwise over 0.5 h. The mixture fizzed and became dark at first before turning a dark straw color. After the mixture was heated at 175 °C for 0.5 h, a second portion of nickel chloride (0.52 g, 4.0 mmol) was added. The mixture fizzed vigorously, gave off much vapor, and became dark gray-green in color. Heating was continued for 0.5 h, and then the mixture was cooled to room temperature. The mixture was treated with methanol (100 mL) and was filtered through a bed of Celite. The methanol was evaporated under vacuum to leave an oil that was distilled on a Kugelrohr to afford 8 as a colorless liquid that was stored under argon (4.0 g, 62%): bp 155–165 °C at 0.4 mmHg; Rf ) 0.67 (SiO2,

2180 Bioconjugate Chem., Vol. 18, No. 6, 2007

DCM/methanol, 9:1); ESI-MS obsd (+) 324.2 (M + H)+, calcd 323.17 (M ) C17H26NO3P). (1-(5-Ethoxycarbonylpentyl)-3,3-dimethyl-2-methylene-2,3-dihydro-1H-indol-5-yl)phosphonic Acid Diethyl Ester (9). To a dry three-neck 500 mL flask were added 7 (11.2 g, 22.1 mmol) and nickel chloride (0.72 g, 5.6 mmol). The flask was fitted with a dropping funnel and a condenser. The mixture was heated to 170 °C, and then triethyl phosphite (30 mL, 173 mmol) was added dropwise over 0.5 h. The temperature was raised to 180 °C, and then a second portion of nickel chloride (0.72 g, 5.6 mmol) was added. After about 20 s, rapid fizzing occurred and the mixture turned grey-green in color. After heating for a further 0.5 h, the mixture was cooled to room temperature and was treated with DCM (200 mL). The mixture was filtered through a fiberglass filter pad, and then the DCM was removed under vacuum. The excess triethyl phosphite and volatile components were removed using a Kugelrohr (110 °C at 0.4 mmHg), and the residue was subjected to column chromatography on silica gel (6 cm × 14 cm) using 2.5–10 % (v/v) methanol in DCM as an eluent. Fractions containing the required compound had a tendency to turn pink upon exposure to air. Evaporation of the fractions afforded 9 as a pink oil that was stored under argon (8.0 g, 86 %): Rf ) 0.68 (SiO2, DCM/methanol, 9:1); ESI-MS obsd (+) 438.2 (M + H)+, calcd 437.23 (M ) C23H36NO5P). 1-Ethyl-2,3,3-trimethyl-5-phosphono-3H-indolium (1). To a 250 mL flask were added 8 (4.8 g, 14.8 mmol) and 6 M hydrochloric acid (60 mL). The mixture was stirred at room temperature until the oil dissolved and then was heated at reflux overnight. After cooling, the mixture was filtered and was evaporated to dryness under vacuum. The residue was treated with water (100 mL) and, after the mixture was stirred for 30 min, was filtered. The aqueous solution was washed with DCM (2 × 60 mL), filtered, and concentrated under vacuum to approximately 4–6 mL. The solution was loaded onto a column of RP silica gel (7 cm × 15 cm) in water, and then the column was eluted with water. Pure fractions (by RP HPLC) were evaporated under vacuum to afford 1 as an off-white solid that was dried over phosphorous pentoxide and stored under argon (3.67 g, 80%): Rf ) 0.50 (C18-SiO2, water/methanol, 3:1); 1H NMR (D2O) δ 1.54 (t, J ) 7.4 Hz, 3H), 1.60 (s, 6H), 4.52 (quartet, J ) 7.4 Hz, 2H), 7.86–7.90 (m, 1H), 7.94–8.01 (m, 1H), and 8.03–8.08 (m, 1H). 13C NMR δ 15.1, 15.9, 24.4, 46.5, 57.6, 118.1 (d, J ) 15 Hz), 128.2 (d, J ) 12 Hz), 134.4 (d, J ) 11 Hz), 138.5 (d, J ) 178 Hz), 144.9 (d, J ) 15 Hz), 145.6 (d, J ) 3 Hz), 200.6; ESI-MS obsd (+) 268.2 (M + H)+, 534.7 (2M + H)+, (–) 532.9 (2M – H)-, 799.5 (3M – H)- 266.1 (M – H)-, calcd 267.10 (M ) C13H18NO3P). 1-(5-Carboxypentyl)-2,3,3-trimethyl-5-phosphono-3H-indolium (2). Procedure is as for 1 using 9 (8.0 g, 18.3 mmol) and 6 M hydrochloric acid (280 mL) to afford 2 (5.3 g, 76%): Rf ) 0.54 (C18-SiO2, water/methanol, 3:1); 1H NMR (D2O) δ 1.42–1.51 (m, 2H), 1.58–1.72 (m, 8H), 2.04 (quintet, J ) 7.8 Hz, 2H), 2.38 (t, J ) 7.2 Hz, 2H), 4.52 (t, J ) 7.4 Hz, 2H), 7.83–7.87 (m, 2H), 7.93–7.98 (m, 1H), and 8.02–8.06 (m, 1H); 13C NMR δ 15.9, 24.6, 26.7, 28.2, 29.8, 36.4, 50.9, 57.6, 118.1 (d, J ) 15 Hz), 128.1 (d, J ) 11Hz), 134.2 (d, J ) 12 Hz), 140.3 (d, J ) 176 Hz), 144.7 (d, J ) 15 Hz), 145.4 (d, J ) 4 Hz), 181.6, 200.7; ESI-MS obsd (+) 354.3 (M + H)+, 706.9 (2M + H)+, (–) 705.0 (2M – H)-, 352.2 (M – H)-, calcd 353.14 (M ) C17H24NO5P). 1-Ethyl-5-formylaminomethyl-2,3,3-trimethyl-3H-indolium iodide (13). 4-Aminomethylindole (18) (21.4 g, 0.114 mol) was dissolved in methyl formate (60 mL), and the mixture was heated at reflux for 2 days. The volatile components were evaporated under vacuum to leave the formamide as a paleyellow oil. The bulk of the material was used without further purification. A sample of the oil was distilled on a Kugelrohr

Reddington

to afford the formamide as a colorless oil: bp 182 °C at 0.15 mmHg: Rf ) 0.28 (SiO2, DCM/methanol, 19:1); IR (film, νmax cm-1) 1668, 3256; 1H (DMSO-d6) 1.20 (s, 6H), 2.18 (s, 3H), 4.37 and 4.42 (2 × d, J ) 6 Hz, 2H), 6.81 and 6.48 (2 × s, 1H), 7.11–7.16 (m, 2H), 7.32–7.40 (m, 1H), and 8.18 (s, 1H); 13 C NMR δ 15.4, 23.1, 42.1, 53.7, 119.8, 121.1, 127.3, 134.7, 146.4, 153.1, 161.3, 188.7; ESI-MS obsd (+) 217.1 (M + H)+, calcd 216.13 (M ) C13H16N2O). The amide (10.8 g, 50 mmol) was dissolved in acetonitrile (25 mL), and ethyl iodide (25 mL) was added. The mixture was heated at reflux for 40 h, and then the volatile components were removed under vacuum. The residue was dissolved in methanol (20 mL), and this solution was added dropwise to stirred ether (700 mL). After the mixture was stirred for 1.5 h, the solid was filtered off and was washed with ether to afford 13 as a pale-pink solid (17.8 g, 96%): Rf ) 0.25 (SiO2, DCM/methanol, 9:1); 1H (DMSO-d6) δ 1.43 (t, J ) 7.3 Hz, 3H), 1.52 (s, 6H), 2.84 (s, 3H), 4.42 (d, J ) 6.1 Hz, 2H), 4.48 (quartet, J ) 7.0 Hz, 2H), 7.48–7.51 (m, 1H), 7.72 (d, J ) 1.1 Hz, 1H), 7.94 (d, J ) 8.4 Hz, 1H), 8.17 (m, 1H), and 8.62 (t, J ) 5.3 Hz, 1H); 13C NMR δ 12.9, 14.1 22.3, 40.3, 43.7, 54.2, 115.8, 122.5, 127.9, 139.8, 141.1, 142.4, 161.4, 195.8; ESI-MS obsd (+) 245.2 (M – I)+, calcd 372.07 (M ) C15H21N2OI). Anal. Calcd for C15H21N2OI: C, 48.4; H, 5.69; N, 7.53. Found: C, 48.35; H, 5.39; N, 7.29. Dye 14. To a 100 mL flask were added 2 (0.37 g, 1.04 mmol), diphenylformamidine (0.10 g, 0.5 mmol), sodium acetate (0.13 g, 1.6 mmol), and acetic anhydride (5 mL). The mixture was heated at 110 °C for 4.5 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar that was dissolved in concentrated hydrochloric acid (20 mL). The solution was stirred for 3 h and then was diluted by addition to water (500 mL). The mixture was loaded onto a column of RP silica gel (3 cm × 12 cm) in water. The column was washed with water (approximately 1 L) to load all of the compound onto the column and to remove the excess acid. Gradient elution from 0% to 40% (v/v) methanol in water (the methanol was increased in steps of 5%, and volumes from 300 mL to 1 L of eluent were used for each step) eluted the dye. Fractions were evaporated under vacuum to leave a red residue that was treated with the minimum 20% (v/v) water in methanol to dissolve the dye. The solution was filtered through a medium frit and then was evaporated under vacuum. The residue was dried under vacuum to afford 14 (0.080 g, 22%): Rf ) 0.56 (C18-SiO2, water/methanol, 3:1), 0.22 (C18SiO2, RPA/methanol, 3:1); 1H NMR (D2O + 2% (v/v) of 30% NaOD) δ 1.42–1.50 (m, 4H), 1.64 (quintet, J ) 7.5 Hz, 4H), 1.78 (s, 12H), 1.88 (quintet, J ) 7.5 Hz, 4H), 2.20 (t, J ) 7.4 Hz, 4H), 4.12 (t, J ) 7.3 Hz, 4H), 6.33 (d, J ) 13.7 Hz, 2H), 7.30 (dd, J ) 1.6 and 8.3 Hz, 2H), 7.72–7.78 (m, 2H), 7.81 (d, J ) 11 Hz, 2H) and 8.56 (t, J ) 13.6 Hz, 2H); 13C NMR δ 28.6, 29.0, 29.5, 30.1, 40.3, 46.9, 42.1, 105.1, 113.2 (d, J ) 14 Hz), 127.0 (d, J ) 9 Hz), 133.5 (d, J ) 10 Hz), 140.8 (d, J ) 168 Hz), 143.3 (d, J ) 13 Hz), 145.3 (d, J ) 3 Hz), 153.7, 178.0, 186.6; ESI-MS obsd (+) 717.3 (M + H)+, (–) 715.1 (M – H)-, calcd 716.26 (M ) C35H46N2O10P2). Anal. Calcd for C35H46N2O10P2 · H2O: C, 57.22; H, 6.59; N, 3.81; P, 8.43. Found: C, 57.18; H, 6.38; N, 4.08; P, 7.90. Dye 15. To a 100 mL flask were added 2 (0.37 g, 1.04 mmol), pyridine (25 mL), and triethyl orthoformate (0.28 mL). The mixture was heated at 110 °C for 1.5 h. Triethyl orthoformate (0.25 mL) was added, and heating was continued for 2.5 h. After the mixture was cooled to room temperature, the volatile components were removed under vacuum to leave a tar. The tar was worked up as described for compound 14 (concentrated hydrochloric acid (20 mL), RP column chromatography (3 cm × 12 cm), 0–40% (v/v) methanol in water) to afford 15 as a red solid (0.195 g, 24 %): Rf ) 0.56 (C18-SiO2, water/methanol,

Technical Notes

3:1), 0.55 (C18-SiO2, RPA/methanol, 3:1); 1H NMR (D2O + 2% (v/v) of 30% NaOD) δ 1.21 (t, J ) 7.2 Hz, 6H), 1.41–1.49 (m, 4H), 1.64 (quintet, J ) 7.4 Hz, 4H), 1.75 (s, 12H), 1.86 (quintet, J ) 7.3 Hz, 4H), 2.20 (t, J ) 7.5 Hz, 4H), 3.85 (quintet, J ) 7.2 Hz, 4H), 4.11 (t, J ) 7.2 Hz, 4H), 6.38 (d, J ) 13.4 Hz, 2H), 7.40 (dd, J ) 1.3 and 8.1 Hz, 2H), 7.74–7.82 (m, 4H) and 8.55 (t, J ) 13.4 Hz, 1H); 13C NMR δ 18.6, 28.5, 28.9, 29.5, 30.0, 40.2, 47.0, 52.0, 64.0, 105.9, 114.0 (d, J ) 15 Hz), 127.5 (d, J ) 11 Hz), 132.6 (d, J ) 181 Hz), 134.7 (d, J ) 12 Hz), 143.9 (d, J ) 15 Hz), 147.0 (d, J ) 3 Hz), 154.4, 178.3, 186.4; ESI-MS obsd (+) 773.4 (M + H)+, (–) 771.3 (M – H)-, calcd 772.33 (M ) C39H54N2O10P2). Dye 16. To a 50 mL flask were added 1 (0.31 g, 1.16 mmol), diphenylformamidine (0.30 g, 1.53 mmol), acetic acid (2 mL), and acetic anhydride (2 mL). The mixture was heated at 120 °C for 1.5 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar that was washed with ether (2 × 30 mL). The residue was dissolved in acetic acid (2 mL), and then pyridine (4 mL) and 10 (0.40 g, 1.14 mmol) were added. The mixture was heated at 120 °C for 0.75 h and then was cooled to room temperature. The volatile components were removed under vacuum, and the residue was dissolved in concentrated hydrochloric acid (30 mL). After being stirred for 3 h, the mixture was diluted by addition to water (200 mL) and then was basified, first by addition of 4 M sodium hydroxide solution until the pH was approximately 4 and then with saturated sodium bicarbonate solution. Initially dye was precipitated from the mixture, but then it redissolved. The aqueous solution was filtered and was loaded onto a column of RP silica gel (4 cm × 15 cm) in water. The column was washed with water (200 mL), 1 M hydrochloric acid (100 mL), and water until the pH of the effluent was neutral. Gradient elution from 0% to 50% (v/v) methanol in water (the methanol was increased in steps of 5% and volumes from 300 mL to 1 L of eluent were used for each step) eluted the symmetrical diphosphonate dye first and then the unsymmetrical dye. The fractions were evaporated under vacuum to leave a red residue that was taken up in methanol. The solution was filtered through a medium frit, and the methanol was removed under vacuum. The residue was washed with acetonitrile and was dried under vacuum to give dye 16 as a red solid (0.32 g, 51%): Rf ) 0.26 (C18-SiO2, water/methanol, 3:1), 0.17 (C18-SiO2, RPA/ methanol, 3:1); 1H NMR (CD3OD) δ 1.42 (t, J ) 7.3 Hz, 3H), 1.51–1.58 (m, 2H), 1.71 (quintet, J ) 7.3 Hz, 2H), 1.78 (s, 14H), 1.88 (quintet, J ) 7.4 Hz, 2H), 2.33 (t, J ) 7.4 Hz, 2H), 4.18–4.24 (m, 4H), 6.48–6.56 (m, 2H), 7.32–7.50 (m, 4H), 7.58 (d, J ) 7.0 Hz, 1H), 7.86–7.93 (m, 2H), and 8.57 (t, J ) 13.4 Hz, 1H). 13C NMR δ 12.5, 25.7, 27.3, 28.2, 28.3, 34.6, 40.3, 45.3, 50.4, 50.9, 103.5, 104.6, 111.5 (d, J ) 16 Hz), 112.8, 123.6, 125.9 (d, J ) 12 Hz), 127.2, 130.1, 132.8 (d, J ) 194 Hz), 133.2 (d, J ) 11 Hz), 141.8 (d, J ) 16 Hz), 142.4, 143.2, 145.0 (d, J ) 3 Hz), 152.5, 175.4, 176.8, 177.2; ESI-MS obsd (+) 551.4 (M + H)+, (–) 549.3 (M – H)-, calcd 550.26 (M ) C31H39N2O5P). Anal. Calcd for C31H39N2O5P: C, 67.62; H, 7.14; N, 5.09; P, 5.63. Found: C, 67.25; H, 6.87; N, 5.01; P, 5.6. Dye 17. To a 100 mL flask were added 1 (0.29 g, 1.08 mmol), diphenylformamidine (0.40 g, 2.0 mmol), acetic acid (5 mL), and acetic anhydride (5 mL). The mixture was heated at 125 °C for 3 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar that was washed with ether (2 × 30 mL) and ethyl acetate (2 × 50 mL). The residue was dissolved in acetic acid (5 mL), and then pyridine (8 mL) and 11 (0.370 g, 1.0 mmol) were added. The mixture was heated at 125 °C for 3 h. The volatile components were removed under vacuum to leave a tar. The tar was worked up as described for compound 16 (concentrated hydrochloric acid (30 mL), RP column chromatography (3 cm × 15 cm),


0–55% (v/v) methanol in water) to afford 17 as a red solid (0.072 g, 14%): Rf ) 0.19 (C18-SiO2, water/methanol, 3:1), 0.24 (C18SiO2, RPA/methanol, 3:1); 1H NMR (CD3OD) δ 1.40–1.46 (m, 6H), 1.78 (s, 6H), 1.79 (s, 6H), 3.73 (s, 2H), 4.18–4.30 (m, 4H), 6.48 (d, J ) 13.2 Hz, 1H), 6.58 (d, J ) 13.6 Hz, 1H), 7.38–7.44 (m, 3H), 7.53 (s, 1H), 7.85–7.91 (m, 2H), and 8.57 (t, J ) 13.4 Hz, 1H); 13C NMR δ 12.5, 12.8, 28.1, 28.3, 40.2, 40.8, 41.5, 50.2, 51.1, 103.3, 104.9, 111.6 (d, J ) 15 Hz), 112.7, 124.9, 125.9 (d, J ) 12 Hz), 129.2 (d, J ) 190 Hz), 131.3, 133.4 (d, J ) 11 Hz), 134.8, 141.7, 142.0 (d, J ) 15 Hz), 142.9, 145.9 (d, J ) 3 Hz), 152.5, 174.9, 175.1, 176.9; ESI-MS obsd (+) 523.3 (M + H)+, 545.3 (M + Na)+, calcd 522.3 (M ) C29H35N2O5P). Dye 18. To a 250 mL flask were added 12 (0.60 g, 2.04 mmol), diphenylformamidine (0.50 g, 2.6 mmol), acetic acid (21 mL), and acetic anhydride (15 mL). The mixture was heated at 115 °C for 3 h and then was cooled to room temperature. The volatile components were removed under vacuum, and the residue was washed with ethyl acetate (3 × 100 mL) to give a powder. To the powder were added 2 (0.70 g, 2.0 mmol) and acetic acid (25 mL). The mixture was warmed until the solids dissolved, and then pyridine (35 mL) was added. The mixture was heated to 115 °C for 0.5 h and then acetic anhydride (15 mL) was added. The mixture was heated for a further 2 h during which time the mixture became deep-red. The mixture was cooled to room temperature, and then the volatile components were removed under vacuum. The residue was treated with saturated aqueous sodium bicarbonate solution (250 mL) to dissolve the dye. The solution was filtered and was loaded onto a column of RP silica gel (4 cm × 20 cm) in water. The column was washed with water (approximately 200 mL) and 0.5 M hydrochloric acid (approximately 500 mL), RPA buffer (500 mL) and then was eluted with a gradient from 0 to 30% (v/v) methanol in RPA buffer (the methanol was increased in steps of 5%, and volumes from 500 mL to 1 L of eluent were used for each step). The fractions were evaporated under vacuum to leave a red residue that was dissolved in 1 M hydrochloric acid (approximately 100 mL). The solution was loaded onto a column of RP silica gel (3 cm × 10 cm) in water. The column was washed with 1.0 M hydrochloric acid (200 mL) and then with water until the pH of the effluent was neutral. The column was eluted with a sharp gradient from 0 to 50% (v/v) methanol in water. The dye-containing fractions were evaporated under vacuum to afford 18 as a red solid (0.45 g, 34%): Rf ) 0.62 (C18-SiO2, water/methanol, 3:1), 0.55 (C18-SiO2, RPA/ methanol, 3:1); 1H NMR (D2O) δ 1.38–1.47 (m, 2H), 1.59–1.72 (m, 14H), 1.78–1.94 (m, 6H), 2.20 (t, J ) 7.4 Hz, 2H), 2.95 (t, J ) 7.5 Hz, 2H), 4.03–4.09 (m, 4H), 6.27–6.32 (m, 2H), 7.24–7.38 (m, 4H), 7.50 (d, J ) 7.0 Hz, 1H), 7.75–7.80 (m, 2H), and 8.43 (t, J ) 13.5 Hz, 1H); 13C NMR δ 24.4, 28.4, 28.9, 29.4, 29.9, 30.0, 40.1, 40.2, 46.3, 46.9, 51.8, 52.0, 53.2, 105.3, 113.6 (d, J ) 15 Hz), 114.0, 125.1, 126.9 (d, J ) 11 Hz), 128.3, 131.5, 133.8 (d, J ) 11 Hz), 136.3 (d, J ) 175 Hz), 143.4 (d, J ) 14 Hz), 143.7, 144.5, 146.2, 153.8, 177.7, 177.8, 186.2; ESI-MS obsd (+) 681.3 (M + Na)+, (–) 657.2 (M – H)-, calcd 658.25 (M ) C33H43N2O8PS). Anal. Calcd for C33H43N2O8PS · H2O: C, 58.57; H, 6.70; N, 4.14; P, 4.58. Found: C, 58.78; H, 6.83; N, 4.41; P, 4.80. Dye 18 Disodium Salt. Dye 18 (0.40 g) was dissolved in a mixture of deionized (DI) water (200 mL) and methanol (50 mL). The solution was passed through a column (2.5 cm × 10 cm) of Dowex 50-8X anion exchange resin, sodium form. After the remaining dye was washed off the column with DI water, the solution was concentrated to approximately 20 mL, filtered through a medium frit, and then evaporated under vacuum to afford the disodium salt of 18 as a red solid (0.41 g, 97%). Anal. Calcd for C33H41N2O8PSNa2 · H2O: C, 54.99; H, 6.01; N,


3.89; P, 4.30; Na, 6.38. Found: C, 54.15; H, 6.03; N, 4.03; P, 3.90; Na, 6.30. Dye 19. To a 100 mL flask were added 2 (0.41 g, 1.15 mmol), malonaldehyde dianilide hydrochloride (0.142.g, 0.55 mmol), acetic anhydride (5 mL), and sodium acetate (0.13 g). The mixture was heated at 110 °C for 4.5 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar. The tar was worked up as described for compound 14 (concentrated hydrochloric acid (30 mL), RP column chromatography (4 cm × 15 cm), 0–40% (v/v) methanol in water) to afford 19 as a blue solid (0.23 g, 51%): Rf ) 0. 36 (C18-SiO2, water/methanol, 3:1), 0.17 (C18-SiO2, RPA/ methanol, 3:1); 1H NMR (D2O + 2% (v/v) of 30% NaOD) δ 1.41–1.47 (m, 4H), 1.59–1.66 (m, 16H), 1.83 (quintet, J ) 7.3 Hz, 4H), 2.20 (t, J ) 7.5 Hz, 4H), 4.07 (t, J ) 7.4 Hz, 4H), 6.28 (d, J ) 13.6 Hz, 2H), 6.57 (t, J ) 12.5 Hz, 1H), 7.26 (dd, J ) 1.9 and 8.3 Hz, 2H), 7.64–7.67 (m, 2H), 7.75 (d, J ) 11.5 Hz, 2H), and 7.99 (t, J ) 13.1 Hz, 2H); 13C NMR δ 28.6, 29.0, 29.5, 29.7, 40.2, 46.7, 51.8, 106.3, 113.2 (d, J ) 15 Hz), 126.9 (d, J ) 11 Hz), 127.6, 133.5 (d, J ) 11 Hz), 136.9 (d, J ) 175 Hz), 143.8 (d, J ) 15 Hz), 146.2 (d, J ) 3 Hz), 156.3, 176.3, 186.5; ESI-MS obsd (+) 743.3 (M + H)+, (–) 741.3 (M – H)-, calcd 742.28 (M ) C37H48N2O10P2). Anal. Calcd for C37H48N2O10P2 · (H2O)2: C, 57.06; H, 6.73; N, 3.60; P, 7.95. Found: C, 56.70; H, 6.62; N, 3.67; P, 7.4. Dye 20. To a 50 mL flask were added 1 (0.36 g, 1.34 mmol), malonaldehyde dianilide hydrochloride (0.36 g, 1.40 mmol), acetic acid (4 mL), and acetic anhydride (4 mL). The mixture was heated at 110 °C for 1.5 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar. The tar was dissolved in acetic acid (4 mL), and then pyridine (5 mL) and compound 2 (0.46 g, 1.30 mmol) were added. The mixture was heated at 110 °C for 3 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a dark-blue tar. The tar was worked up as described for 14 (concentrated hydrochloric acid (30 mL), RP column chromatography (4 cm × 15 cm), 0–45% (v/v) methanol in water) to afford 20 as a blue solid (0.205 g, 24%): Rf ) 0.46 (C18-SiO2, water/methanol, 3:1), 0.31 (C18-SiO2, RPA/methanol, 3:1); 1H NMR (CD3OD) δ 1.43 (t, J ) 7.2 Hz, 3H), 1.51–1.57 (m, 2H), 1.69–1.89 (m, 16H), 2.35 (t, J ) 7.1 Hz, 2H), 4.14–4.23 (m, 4H), 6.27–6.37 (m, 2H), 6.68 (t, J ) 12.4 Hz, 1H), 7.31–7.34 (m, 2H), 7.87–7.95 (m, 4H) and 8.24–8.36 (m, 2H); ESI-MS obsd (+) 657.3 (M + H)+, (–) 655.3 (M – H)-, calcd 656.24 (M ) C33H42N2O8P2). Dye 21. To a 100 mL flask were added 1 (1.87 g, 7.0 mmol), malonaldehyde dianilide hydrochloride (2.0 g, 7.73 mmol), acetic acid (20 mL), and acetic anhydride (20 mL). The mixture was heated at 110 °C for 2.5 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar that was washed with ether (2 × 60 mL). The tar was dissolved in acetic acid (30 mL), and then pyridine (40 mL) and 10 (2.5 g, 7.1 mmol) were added. The mixture was heated at 110 °C for 1 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar. The tar was worked up as described for compound 16 (concentrated hydrochloric acid (70 mL), RP column chromatography (4 cm × 20 cm), 0–50% (v/v) methanol in water) to afford a 21 as a blue solid (1.54 g, 38%): Rf ) 0.09 (C18-SiO2, water/methanol, 3:1), 0.10 (C18-SiO2, RPA/ methanol, 3:1); 1H NMR (CD3OD) δ 1.37 (t, J ) 7.4 Hz, 3H), 1.46–1.55 (m, 2H), 1.64–1.75 (m, 14H), 1.84 (quintet, J ) 7.4 Hz, 2H), 2.32 (t, J ) 7.3 Hz, 2H), 4.11–4.17 (m, 4H), 6.26–6.34 (m, 2H), 6.64 (t, J ) 12.4 Hz, 1H), 7.25–7.33 (m, 3H), 7.39–7.44 (m, 1H), 7.50 (d, J ) 7.2, 1H), 7.84–7.90 (m, 2H), and 8.22–8.31 (m, 2H); 13C NMR δ 12.5, 25.7, 27.4, 27.9, 28.2,

Reddington

34.7, 39.9, 44.9, 50.4, 50.7, 104.0, 104.8, 110.8 (d, J ) 15 Hz), 112.2, 123.5, 125.9 (d, J ) 10 Hz), 126.5, 126.8, 129.8, 132.8 (d, J ) 9 Hz), 135.7 (d, J ) 181 Hz), 141.9 (d, J ) 15 Hz), 142.8, 143.5, 144.2 (d, J ) 3 Hz), 155.5, 155.8, 174.2, 175.2, 177.4; ESI-MS obsd (+) 577.4 (M + H)+, (–) 575.4 (M – H)-, calcd 576.28 (M ) C33H41N2O5P). Anal. Calcd for C33H41N2O5P · (H2O)2: C, 64.69; H, 7.40; N, 4.57; P, 5.06. Found: C, 64.34; H, 7.44; N, 4.66; P, 5.0. Dye 22. To a 50 mL flask were added 11 (0.387 g, 1.04 mmol), malonaldehyde dianilide hydrochloride (0.321 g, 1.24 mmol), acetic acid (5 mL), and acetic anhydride (5 mL). The mixture was heated at 110 °C for 1 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar that was washed with ether (2 × 60 mL). The tar was dissolved in acetic acid (5 mL), and then pyridine (10 mL) and 1 (0.27 g, 1.0 mmol) were added. The mixture was heated at 110 °C for 1 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar. The tar was worked up as described for compound 16 (concentrated hydrochloric acid (30 mL), RP column chromatography (4 cm × 15 cm), 0–60% (v/v) methanol in water) to afford 22 as a blue solid (0.176 g, 31%): Rf ) 0.19 (C18-SiO2, water/methanol, 3:1), 0.20 (C18-SiO2, RPA/ methanol, 3:1); 1H NMR (CD3OD) δ 1.32–1.41 (m, 6H), 1.72 (s, 6H), 1.73 (s, 6H), 3.70 (s, 2H), 4.10–4.20 (m, 4H), 6.26 (d, J ) 13.6 Hz, 1H), 6.33 (d, J ) 14.0 Hz, 1H), 6.62 (t, J ) 12.4 Hz, 1H), 7.24–7.29 (m, 2H), 7.34–7.37 (m, 1H), 7.45 (d, J ) 1.4 Hz, 1H), 7.83–7.90 (m, 2H), and 8.21–8.30 (m, 2H); 13C NMR δ 12.5, 12.6, 27.7, 28.9, 39.9, 40.2, 41.6, 50.3, 50.8, 103.8, 104.6, 110.7 (d, J ) 14 Hz), 111.9, 124.7, 125.9 (d, J ) 10 Hz), 126.8, 131.0, 132.8 (d, J ) 10 Hz), 133.9, 134.9 (d, J ) 175 Hz), 141.8 (d, J ) 14 Hz), 141.9, 143.2, 144.3, 155.3, 155.9, 173.9, 174.9, 175.5; ESI-MS obsd (+) 549.3 (M + H)+, (–) 547.3 (M – H)-, calcd 548.24 (M ) C31H37N2O5P). Dye 23. To a 250 mL flask were added 12 (0.60 g, 2.04 mmol), malonaldehyde dianilide hydrochloride (0.60 g, 2.3 mmol), acetic acid (25 mL), and acetic anhydride (25 mL). The mixture was heated at 115 °C for 2 h and was cooled to room temperature, and then the volatile components were removed under vacuum. The residue was washed with ethyl acetate (3 × 100 mL) to give a powder. To the powder were added 2 (0.70 g, 2.0 mmol) and acetic acid (25 mL). The mixture was warmed until the solids dissolved, and then pyridine (35 mL) was added. The mixture was heated to 115 °C for 3 h during which time the mixture became deep-blue. The mixture was cooled to room temperature, and the volatile components were removed under vacuum. The residue was worked up as described for compound 18 (RP column chromatography (4 cm × 20 cm, 0–30% (v/v) methanol in RPA) to afford 23 as a blue solid (0.95 g, 69 %): Rf ) 0.46 (C18-SiO2, water/methanol, 3:1), 0.41 (C18-SiO2, RPA/methanol, 3:1); 1H NMR (D2O) δ 1.38–1.46 (m, 2H), 1.50 (s, 6H), 1.54–1.66 (m, 8H), 1.73–1.97 (m, 6H), 2.21 (t, J ) 7.4 Hz, 2H), 2.96 (t, J ) 7.4 Hz, 2H), 4.00 (t, J ) 7.4 Hz, 2H), 4.11 (t, J ) 7.4 Hz, 2H), 6.10 (d, J ) 13.6 Hz, 1H), 6.24 (d, J ) 13.8 Hz, 1H), 6.48 (t, J ) 12.5 Hz, 1H), 7.11–7.16 (m, 1H), 7.24–7.29 (m, 3H), 7.44 (d, J ) 7.5 Hz, 1H), 7.68–7.74 (m, 2H), and 7.82–7.95 (m, 2H). 13C NMR δ 24.7, 28.6, 28.8, 29.1, 29.6, 29.9, 40.2, 46.5, 46.8, 51.8, 52.2, 53.5, 106.0, 106.7, 113.4 (d, J ) 15 Hz), 114.1, 125.4, 127.1 (d, J ) 9 Hz), 127.8, 128.2, 131.5, 133.9 (d, J ) 13 Hz), 134.7 (d, J ) 179 Hz), 143.9 (d, J ) 14 Hz), 144.4, 144.9, 146.9 (d, J ) 2 Hz), 156.3, 156.9, 176.0, 176.8, 186.2; ESI-MS obsd (+) 729.3 (M – H + 2Na)+, 707.3 (M + Na)+, (–) 683.2 (M – H)-, calcd 684.26 (M ) C35H45N2O8PS). Anal. Calcd for C35H45N2O8PS: C, 61.39; H, 6.62; N, 4.09; P, 4.52; S, 4.68. Found: C, 61.03; H, 6.62; N, 3.91; P, 4.4; S, 5.64.

Technical Notes

Dye 23 Disodium Salt. Dye 23 (0.50 g, 0.73 mmol) was dissolved in a mixture of DI water (200 mL) and methanol (50 mL). The solution was passed through a column (2.5 cm × 10 cm) of Dowex 50-8X anion exchange resin, sodium form. After the remaining dye was washed off the column with DI water, the aqueous solution was concentrated to approximately 20 mL. The solution was filtered through a medium frit and was evaporated under vacuum to afford the sodium salt of dye 23 as a blue solid (0.49 g, 92%). Anal. Calcd for C35H43N2O8PSNa2 · (H2O)2: C, 54.97; H, 6.19; N, 3.66; P, 4.05; Na, 6.01. Found: C, 54.66; H, 5.97; N, 3.66; P, 3.90; Na, 6.30. Dye 24. To a 100 mL flask were added 1 (0.50 g, 1.87 mmol), malonaldehyde dianilide hydrochloride (0.50 g, 1.93mmol), acetic acid (6 mL), and acetic anhydride (6 mL). The mixture was heated at 110 °C for 2.5 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a tar that was washed with ether (2 × 40 mL). The tar was dissolved in acetic acid (4 mL), and then pyridine (5 mL) and 13 (0.75 g, 2.0 mmol) were added. The mixture was heated at 110 °C for 1 h and then was cooled to room temperature. The volatile components were removed under vacuum to leave a dark-blue tar. The tar was worked up as described for compound 16 (concentrated hydrochloric acid (30 mL), RP column chromatography (4 cm × 15 cm), 0–55% (v/ v) methanol in water) to afford 24 as a blue solid (0.47 g, 46%): Rf ) 0.26 (C18-SiO2, water/methanol, 1:1), 0.18 (C18-SiO2, RPA/methanol, 3:1); 1H NMR (CD3OD) δ 1.36–1.40 (m, 6H), 1.73 (s, 12H), 4.12–4.18 (m, 4H), 4.47 (s, 2H), 6.27–6.32 (m, 2H), 6.62 (t, J ) 12.4 Hz, 1H), 7.25–7.29 (m, 2H), 7.36–7.39 (m, 1H), 7.46 (d, J ) 1.2 Hz, 1H), 7.83–7.90 (m, 2H), 7.18 (s, 1H) and 8.22–8.30 (m, 2H); 13C NMR δ 12.5, 12.6, 27.7, 27.9, 39.9, 40.2, 42.6, 50.4, 50.7, 104.0, 104.4, 110.8 (d, J ) 14 Hz), 111.9, 123.0, 125.9 (d, J ) 10 Hz), 126.8, 129.3, 132.8 (d, J ) 12 Hz), 135.7 (d, J ) 192 Hz), 137.2, 141.9 (d, J ) 15 Hz), 142.4, 143.3, 144.2 (d, J ) 3 Hz), 155.5, 155.9, 163.8, 174.2, 174.7; ESI-MS obsd (+) 548.4 (M + H)+, (–) 546.4 (M – H)-, calcd 547.26 (M ) C31H38N3O4P). Dye 25. To a 500 mL flask were added dye 24 (0.39 g, 0.713 mmol), methanol (100 mL), and concentrated HCl (10 mL). The mixture was stirred at room temperature for 40 h. The mixture was diluted with water (200 mL) and then was basified by addition of saturated sodium bicarbonate solution. The aqueous solution was filtered and was loaded onto a column of RP silica gel (3 cm × 10 cm) in water. The column was washed with water to remove the salts and excess base, 1.0 M HCl (50 mL) and finally with water (500 mL) to remove the excess acid. Gradient elution from 0% to 80 % (v/v) methanol in water (the methanol was increased in steps of 10% and volumes from 500 mL to 1 L of eluent were used for each step) first eluted residual starting material and then 25. The fractions were evaporated under vacuum to leave a blue residue that was taken up in methanol (10 mL). The solution was filtered through a medium frit, and the methanol was removed under vacuum to afford 25 as a blue solid (0.266 g, 72%): Rf ) 0.12 (C18-SiO2, water/ methanol, 1:1), 0.12 (C18-SiO2, RPA/methanol, 3:1); 1H NMR (CD3OD) δ 1.23–1.33 (m, 6H), 1.63 (s, 12H), 4.00–4.12 (m, 6H), 6.20 (d, J ) 13.8 Hz, 1H), 6.28 (d, J ) 13.8 Hz, 1H), 6.56 (t, J ) 12.3 Hz, 1H), 7.19–7.25 (m, 2H), 7.41–7.43 (m, 1H), 7.53 (s, 1H), 7.72–7.84 (m, 2H), and 8.12–8.22 (m, 2H); 13 C NMR δ 12.5, 12.6, 27.8, 27.9, 40.0, 40.2, 42.3, 50.4, 50.7, 104.0, 104.9, 111.2 (d, J ) 14 Hz), 111.9, 124.4, 126.0 (d, J ) 11 Hz), 127.1, 130.8, 132.3, 132.8 (d, J ) 10 Hz), 136.7 (d, J ) 177 Hz), 142.1 (d, J ) 15 Hz), 143.4, 143.7, 144.0 (d, J ) 3 Hz), 155.5, 156.2, 173.9, 175.3; ESI-MS obsd (+) 520.3 (M + H)+, 542.3 (M + Na)+, calcd 519.27 (M ) C30H38N3O3P). Dye 26. To a 100 mL flask were added 2 (0.41 g, 1.15 mmol), squaric acid (0.063 g, 0.54 mmol), n-butanol (50 mL), and


toluene (30 mL). The flask was fitted with a Dean–Stark trap, and then the mixture was heated at reflux overnight. After the mixture was cooled to room temperature, the solid was filtered off, washed with DCM (3 × 30 mL), and dried. The solid was dissolved in 0.2 M potassium carbonate solution (150 mL), and the solution was stirred for 6 days. Hydrochloric acid (1.0 M) was added to lower the pH to approximately 1–2. A suspension formed that was stirred for a further 1 h. The solid was filtered off and was washed with water (2 × 3 mL). The solid was dried under vacuum to afford dye 26 as a blue solid (0.32 g, 72%): Rf ) 0.11 (C18-SiO2, water/methanol, 3:1), 0.16 (C18SiO2, RPA/methanol, 3:1); 1H NMR (DMSO-d6) δ 1.35–1.42 (4H, m), 1.56 (quintet, J ) 7.3 Hz, 4H), 1.65–1.75 (m, 16H), 2.21 (t, J ) 7.2 Hz, 4H), 4.07–4.14 (m, 4H), 5.85 (s, 2H), 7.39–7.42 (m, 2H), 7.63–7.69 (m, 2H), 7.73 (d, J ) 11.8 Hz, 2H); 13C NMR δ 24.4, 26.0, 26.5, 26.7, 33.7, 43.2, 48.8, 87.1, 110.2 (d, J ) 16 Hz), 124.4 (d, J ) 12 Hz), 129.1 (d, J ) 184 Hz), 131.3 (d, J ) 11 Hz), 141.2 (d, J ) 16 Hz), 144.5 (d, J ) 3 Hz), 169.5, 174.5, 179.9, 180.8; ESI-MS obsd (–) 783.2 (M – H)-, 805.3 (M + Na – 2H)-, calcd 784.26 (M ) C38H46N2O12P2). Anal. Calcd for C38H46N2O12P2: C, 58.16; H, 5.91; N, 3.27; P,7.89. Found: C, 57.84; H, 6.13; N, 3.60; P, 7.70. Dye 14-(NHS Ester)2. To a 25 mL flask were added 14 (44.8 mg, 0.059 mmol), water (0.3 mL), DMF (3 mL), and diisopropylethylamine (DIEA) (0.2 mL). The mixture was stirred until the solid dissolved, and then succinimidotetramethyluronium tetrafluoroborate (TSTU) (35.8 mg, 0.119 mmol) was added. Stirring was continued for 1 h, and then the volatile components were removed under high vacuum without heating. The residue was washed with ethyl acetate (2 × 20 mL), THF (2 × 20 mL), and acetonitrile (2 × 20 mL). The residue was dried under high vacuum overnight with phosphorus pentoxide to afford 14-(NHS ester)2 as a dark-red solid (48.1 mg). Dye content by RP HPLC was 71% 14-(NHS ester)2, 24% 14-NHS ester, and 5% 14. ESI-MS obsd (–) 909.2 (M – H)-, calcd 910.3 (M ) C43H52N4O14P2). Dye 16-NHS Ester. To a 25 mL flask were added 16 (89.5 mg, 0.161 mmol), water (0.7 mL), and DMF (1.4 mL). The mixture was stirred until the solid dissolved, and then DIEA (0.24 mL) and DMF (4.8 mL) were added. After the mixture was stirred for 10 min, TSTU (50.3 mg, 0.167 mmol) was added. Stirring was continued for 1 h, and then the volatile components were removed under high vacuum without heating. The residue was dissolved in DCM, and the solution was filtered through a medium frit. The DCM was evaporated under vacuum without heating, and the residue was dried under high vacuum overnight with phosphorus pentoxide to afford 16-NHS ester as a darkred solid (147 mg). Dye content by RP HPLC was 96% 16NHS ester and 4% 16. ESI-MS obsd (–) 646.2 (M – H)-, calcd 647.3 (M ) C35H42N3O7P). Dye 18-NHS Ester. To a 50 mL flask were added 18 (102.5 mg, 0.150 mmol), water (0.8 mL), and DMF (1.8 mL). The mixture was stirred until the solid dissolved, and then DIEA (0.16 mL) and DMF (3.6 mL) were added. After the mixture was stirred for 10 min, TSTU (70 mg, 0.23 mmol) was added. Stirring was continued for 1 h, and then the volatile components were removed under high vacuum without heating. The residue was washed with DCM (3 × 30 mL) to afford a solid. The solid was filtered off, washed with acetonitrile (3 × 20 mL), and dried under high vacuum overnight with phosphorus pentoxide to afford 18-NHS ester as a dark-red solid (109 mg). Dye content by RP HPLC was 92% 18-NHS ester and 8% 18. ESI-MS obsd (–) 754.1 (M – H), calcd 755.3 (M ) C37H46N3O10PS). Dye 20-NHS Ester. To a 25 mL flask were added 20 (42 mg, 0.064 mmol), water (0.3 mL), DMF (3 mL), and DIEA


(0.2 mL). The mixture was stirred until the solid dissolved, and then TSTU (19 mg, 0.119 mmol) was added. Stirring was continued for 1 h, and then the volatile components were removed under high vacuum without heating. The residue was washed with ethyl acetate (2 × 20 mL), THF (2 × 20 mL), and acetonitrile (2 × 20 mL). The residue was dried under high vacuum overnight with phosphorus pentoxide to afford 20-NHS ester as a dark-blue solid (37.2 mg). Dye content by RP HPLC was 82% 20-NHS ester and 18% 20. ESI-MS obsd (–) 752.1 (M – H)-, calcd 753.3 (M ) C37H45N3O10P2). Dye 21-NHS Ester. Procedure as for compound 16-NHS ester with 21 (93 mg, 0.163 mmol), water (0.7 mL), DMF (1.4 mL), DIEA (0.20 mL), DMF (4.9 mL), and TSTU (50 mg, 0.166 mmol) gave 21-NHS ester as a blue solid (137 mg). Dye content by RP HPLC was 90% 21-NHS ester and 10% 21. ESI-MS obsd (–) 672.2, calcd 673.3 (M ) C37H44N3O7P). Dye 23-NHS Ester. Procedure as for compound 18-NHS ester with 23 (95 mg, 0.161 mmol), water (0.7 mL), DMF (1.4 mL), DIEA (0.14 mL), DMF (2.8 mL), and TSTU (60 mg, 0.20 mmol) gave 23-NHS ester as a dark-blue solid (101 mg). Dye content by RP HPLC was 94% 23-NHS ester and 6% 23. ESIMS obsd (–) 780.1 (M – H), calcd 781.28 (M ) C39H48N3O10PS) Dye 26-(NHS ester)2. To a 50 mL flask were added 26 (49.7 mg, 0.061 mmol), water (0.3 mL), and DMF (3.0 mL). The mixture was stirred until the solid dissolved, and then DIEA (0.3 mL) was added. After the mixture was stirred for 20 min, TSTU (36.7 mg, 0.121 mmol) was added. Stirring was continued for 1 h, and then the mixture was filtered. The volatile components were removed under high vacuum without heating, and the residue was washed with ethyl acetate (3 × 30 mL), THF (30 mL), and acetonitrile (2 × 30 mL). The solid was dried under high vacuum overnight with phosphorus pentoxide to afford 26-(NHS ester)2 as a dark-blue solid (109 mg). Dye content by RP HPLC was 83% 26-(NHS ester)2 and 17% 26NHS ester. ESI-MS obsd (–) 977.2 (M – H), calcd 978.3 (M ) C46H52N4O16P2). Protein Labeling with Dye Succinimidyl Esters. A stock solution of the sheep γ globulin (S-IgG) (Sigma-G9887) was prepared at a concentration of 2 mg/mL (approximately 1.33 × 10-5 M) in carbonate–bicarbonate buffer, pH 9.3. One milliliter aliquots of the stock were dispensed into vials. Stock solutions of the active esters of dyes 16, 18, 21, 23, and 26 were prepared at a concentration of 0.01 M in DMSO. Saturated solutions of the active esters of dyes 14 and 20 were prepared by adding sufficient material to produce a 0.01 M solution to the appropriate volume of DMSO and shaking the mixture for 2 h at room temperature. Aliquots of the active ester stock solutions were added to the protein solutions to provide a range of starting dye to protein ratios from 0.7:1 to 50:1. The solutions were mixed well and left to stand at room temperature overnight. The samples were divided into two or three equal portions and were purified by size exclusion chromatography on Sephadex G50 (0.7 cm × 12 cm) using either carbonate–bicarbonate buffer, pH 9.3, BBS, pH 8.1, or PBS, pH 7.3, as effluent. The labeled proteins, which eluted from the column prior to the hydrolyzed active esters, were stored at 4 °C. Solutions were warmed to room temperature and were filtered before absorption and fluorescence spectra were recorded. Conjugates of Cy3 and of Cy5 were prepared by following the methods supplied with the products. Extinction Coefficients and Fluorescence Quantum Yields. Stock solutions of dyes were prepared from weighed samples of dried material and then were serially diluted to provide five solutions with absorbance values in the range 0.2–1. Extinction coefficients (ε) were determined from plots of the absorbance values vs concentration according to Beer’s law. Measurements were repeated with fresh solutions, and average

Reddington

values were calculated. Solutions used for absorbance measurements were diluted 20× prior to use in fluorescence measurements. Quantum yields (QYs) were determined relative to standards (rhodamine 6G (QY ) 0.86 in ethanol (14)) for dyes with absorption maxima in the range 540–560 nm; Cy5.18 (QY ) 0.27 in PBS pH 7.3 (4)) for dyes with absorption maxima in the range 630–680 nm) and are intended to indicate trends rather than absolute values. For dyes used in labeling experiments, QYs are an average of at least three measurements. Variation between measurements used to calculate extinction coefficients and quantum yields fell within 15% of each other. Values for quantum yields were rounded to two decimal places. Determination of Dye to Protein Ratios and Conjugate Brightness. Dye to protein (D/P) ratios were determined as outlined in ref 5. In cases where absorbance spectra recorded in aqueous solution indicated significant dye aggregation, the solutions were diluted (1:2) with formamide and absorbance spectra recorded again. For each conjugate solution the concentration of dye was estimated using the absorbance of the dye at the peak maximum and the extinction coefficient of free dye according to Beer’s law. The absorbance of the conjugated dye at 280 nm was calculated using the ratio of the extinction coefficients of the free dye at 280 nm to that at the peak maximum. (For squarane dye 26, ε280nm/εpeak max ) 0.041. For trimethine cyanine dyes 14, 16, and 18, ε280nm/εpeak max ) 0.083. For pentamethine cyanine dyes 20, 21, and 23, ε280nm/εpeak max ) 0.026.) The concentration of the protein (MW ) 155 000, ε280nm ) 165 000 L mol-1 cm-1) was determined from the absorbance value of the solution at 280 nm after correction for the absorbance of the conjugated dye at 280 nm. Conjugate brightness is defined as the product of the extinction coefficient, the quantum yield, and the number of dyes per protein molecule ((ε)(QY)(D/P)). Quantum yields of conjugates were determined in wholly aqueous solutions. Oligonucleotide Labeling with Dye Succinimidyl Esters. A T10 DNA oligonucleotide was prepared using standard automated synthesis regimes on a Biosearch 8700 oligonucleotide synthesizer. For Quasar 570 and Quasar 670, amidites of the dyes were coupled to the DNA fragment on the controlled pore size glass (CPG) solid support. For the other dyes, a monomethoxytrityl (MMT) protected amino C6 functionalized amidite (Biosearch Technologies, Inc.) was manually coupled to the DNA fragment and then the protecting group was removed to afford the amino-modified oligonucleotides still attached to CPG. For cyanine monophosphonates 16, 18, 21, and 23, the CPG (10 mg) was shaken overnight in a DMSO solution (0.30 mL) of the active ester (3 mg) and N-methylmorpholine (0.020 mL). For Cy3 and Cy5, the CPG (10 mg), DMSO (0.3 mL), and N-methylmorpholine (0.020 mL) were added to the vial of reactive dye supplied by the manufacturer and the mixtures were shaken overnight. The DNA was cleaved from the CPG using concentrated aqueous ammonia at room temperature for 1 h. The solutions were frozen, and the volatile components were removed using a speed vac. The crude oligonucleotides were purified by a combination of AX and RP HPLC to afford the labeled oligonucleotides that were characterized by electrospray MS. T10-16: obsd (–) 3692, calcd 3691. T10-18: obsd (–) 3800, calcd 3801. T1021: obsd (–) 3718, calcd 3717. T10-23: obsd (–) 3825, calcd. 3827. T10-Q570: obsd (–) 3600, calcd 3601. T10-Q670: obsd (–) 3626, calcd 3627. T10-Cy3.29: obsd (–) 3772, calcd 3771. T10-Cy5.29: obsd (–) 3798 (M – H)-, calcd 3797.

RESULTS AND DISCUSSION 1. Synthesis of Dyes. Key intermediates for the syntheses of the cyanine and squarane dyes are the indolium phosphonates 1 and 2 (Scheme 1). p-Bromoaniline was converted into the hydrazine hydrochloride 3, which was then subjected to Fischer indole condensation with methylbutanone to afford bromoindole

Technical Notes


Scheme 1. Synthetic Route To Indolium Phosphonates

4 (15). Preliminary investigation of the nickel-catalyzed substitution reaction of the bromine for phosphonate (16) demonstrated that bromoindole 4 could be phosphonated in high yield, but partial ethylation of the indole nitrogen also occurred resulting in a mixture of products. It should be noted that this reaction is extremely vigorous once it begins because of the generation of ethyl bromide in the reaction mixture at temperatures of 160–180 °C. Further study indicated that bromoindo-

lium salts 5 and 6 could be phosphonated with this method. To avoid the possibility of partial ethylation of the carboxylic acid of 6 and a mixture of reaction products, 6 was converted to the Chart 1

ethyl ester 7. The phosphonation reaction of salts 5 and 7 generated the charge neutral compounds 8 and 9, which proved to be difficult to isolate in analytically pure form. Hydrolysis of 8 and 9 using refluxing in 6 M hydrochloric acid gave indolium phosphonates 1 and 2 and also small quantities of byproducts in which the phosphonic acid residue was removed from the indolium residue. Compounds 8 and 9 turned pinkred upon exposure to air in a matter of hours, whereas 1 and 2 became colored over a few days. All of these compounds can be stored indefinitely in a desiccator under argon. Indolium salts 10–12 were prepared by literature methods (17). Formyl protected aminomethyl indolium salt 13 was prepared from the corresponding aminomethylindole (18) by sequential treatment with methyl formate and with ethyl iodide. Symmetrical and unsymmetrical cyanine dyes and squarane dyes 14–25 (Chart 1) were prepared from salts 1 and 2, and the nonphosphonate indolium salts 10–13 were prepared using literature methods with some minor changes as noted below. Attempts to prepare the symmetrical trimethine cyanine dye 14 with the commonly used reaction mixture of triethyl orthoformate in pyridine generated dye 15 in which monophosphonate ethyl esters were formed. Treatment of 15 with refluxing in 6 M hydrochloric acid to hydrolyze these esters resulted in stepwise cleavage of the phosphonate groups from the dye in


addition to phosphonate ester cleavage. Symmetrical trimethine and pentamethine cyanine dyes were prepared in acetic anhydride with sodium acetate as base catalyst using either diphenylformamidine or malonaldehyde dianilide, respectively. Unsymmetrical trimethine and pentamethine cyanine dyes were prepared by first reacting one salt with either diphenylformamidine or malonaldehyde dianilide, respectively, in a mixture of acetic anhydride and pyridine and then treating the resulting intermediate with the second salt in a mixture of pyridine and acetic acid. Analysis of the crude reaction mixtures by RP HPLC indicated that in addition to the desired dyes, significant quantities of byproducts with absorption spectra resembling those of cyanine dye dimers were present. Treatment of the crude reaction mixtures with 1.0 M hydrochloric acid or with 1.0 M sodium carbonate solution for 16 h altered the HPLC trace profile by reducing the products with dimerlike absorption spectra and enhancing those with monomer like spectra. Treatment with concentrated hydrochloric acid for 3 h eliminated these byproducts completely. It seems likely that under the conditions of the condensation reactions dimeric and oligomeric species in which the dyes are connected through the phosphonate groups are formed. These byproducts were more prevalent the longer the phosphonic acid containing salts were heated with acetic anhydride. In the case of some of the unsymmetrical dyes the byproducts were avoided by using the indolium phosphonate in the second step of synthesis rather than the first. In the case of the formyl protected dye 24 partial cleavage of the formyl group was observed during the acid treatment. The formyl group was removed from purified dye 24 using methanolic hydrochloric acid. All of the dyes were isolated by RP column chromatography using gradient elution of methanol in either water or RPA buffer. Elemental analysis indicated that cyanine monophosphonates and diphosphonates were isolated as the zwitterionic forms. Squaraine diphosphonate 26 was prepared by condensation of indolium salt 2 with squaric acid in a boiling mixture of butanol and toluene (19, 20), followed by cleavage of any butyl esters that formed with aqueous potassium carbonate, and was isolated in 72% yield as the zwitterion without the need for chromatography.

Reddington Table 1. Photophysical Data for Cyanine Phosphonates, Squaraine Phosphonates, and Related Dyes

dye 14 15 16 17 18 19

20

21

22

23

25

26

Cy5.18 (4) Cy5.29 (4) Cy3.18 (4) Sq635-b (20)

pHa 9.3 7.3 7.3 9.3 7.3 7.3 9.3 7.3 9.3 7.3 4.0 1.0 9.3 7.3 4.0 1.0 9.3 7.3 4.0 1.0 9.3 7.3 4.0 1.0 9.3 7.3 4.0 1.0 9.3 7.3 4.0 1.0 9.3 7.3 4.0 1.0 7.3 7.3 7.3

extinction coefficient, L mol-1 cm-1

absorption max, nm

emission max, nm

556 555 552 549 548 552 551 550 654 652 651 649 653 651 649 647 647 646 645 643 650 649 647 645 648 647 646 644 650 646 645 644 637 635 634 insoluble 649

571 569 564 564 563 567 565 564 672 668 666 663 669 668 666 663 665 663 662 662 668 667 665 664 666 665 663 663 668 663 662 661 647 644 643

550

665

250 000 250 000 150 000

635

648

140 000

666

b

133 000 133 000 133 000

240 000 240 000

210 000

220 000

260 000

QYb 0.07 0.07 0.08 0.04 0.04 0.03 0.05 0.05 0.17 0.17 0.20 0.18 0.14 0.15 0.19 0.19 0.14 0.15 0.13 0.05 0.12 0.11 0.11 0.06 0.14 0.16 0.15 0.08 0.12 0.13 0.15 0.16 0.04 0.04 0.04 0.27 0.20 0.04 0.07 0.15

a

pH 1, 1.0 M hydrochloric acid; pH 4, 1.0 M NaH2PO4; pH 7.3, PBS; pH 9.3, carbonate–bicarbonate buffer. b Errors are estimated to be (10%.

2. Properties of Dyes. All of the dyes are soluble in water to a greater or lesser degree. The cyanine diphosphonates 14, 19, and 20 are readily soluble in aqueous media over a wide range of pH (2–12). In contrast, the zwitterionic cyanine monophosphonates such as 16, 17, 21, and 22 have limited solubility in water and in buffers below pH 7. Sodium salts of cyanine monophosphonates 16 and 21 are highly soluble in water. Cyanine monophosphonates 18, 23, and 25, which have additional water solubilizing groups, maintained good solubility at lower pH (2–7). Cyanine monophosphonates and diphosphonates and squarane diphosphonate 26 may be precipitated from aqueous solution by lowering the pH to 1–2 with hydrochloric acid. After the precipitates were washed with water, the dyes were isolated in their zwitterionic forms. The cyanine and squaraine phosphonates have similar absorption maxima, emission maxima, and extinction coefficients as their sulfonated analogues (Table 1). In the case of the pentamethine cyanine dyes 19–25, the pH sensitivity of absorp-

tion maxima, emission maxima, and fluorescence quantum yields (QYs) were investigated to determine the effect of the protonation state of the phosphonic acid group on photophysical properties of the dyes. On the basis of the pK values for phenylphosphonic acid, 1.83 and 7.07 (21), it was assumed that at pH 9.3 the phosphonate group will carry two negative charges; at pH 4 it will have one charge, and at pH 1 it would be neutral. For the pentamethine dyes 19–25 the absorption and emission maxima shift 4–10 nm to the blue when the pH is lowered from 9.3 to 1. The QYs of cyanine diphosphonates 19 and 20 increase slightly as the pH is lowered, whereas those of the cyanine monophosphonates 21–23 decrease dramatically when the pH is lowered from 4 to 1. Comparison of the absorption spectra of 21 at the different pH values shows that at pH 1 the shorterwavelength shoulder (approximately 605 nm) is significantly higher with respect to the peak maximum than at pH 4. This suggests that aggregation, which usually causes fluorescence quenching, is occurring (Figure 1 in Supporting Information). No such absorption spectrum changes were seen with cyanine diphosphonate 19. Cyanine monophosphonate 23, which has the pendent sulfonate group, shows intermediate behavior. The QY of amine containing monophosphonate 25 increases as the pH is lowered presumably because the protonated amine assists

Technical Notes

in preventing aggregation. For biological applications, measurements are typically made in the pH range 6–8 within which likely falls the second pK of the phosphonic acid group of the dyes. Consequently, the phosphonic acid group should not be considered completely in either the deprotonated or the monoprotonated forms within this pH range. The cyanine diphosphonates 19 and 20 have lower QYs than do their respective sulfonate analogues Cy5.18 and Cy5.29. Cyanine diphosphonate 14 was found to have similar QY as Cy3.18, although the current study was unable to reproduce the reported QY of 0.04 for Cy3.18 (4). Squarane diphosphonate 26 has a higher extinction coefficient but lower QY than its sulfonated analogue Sq635-b (20). 3. Synthesis of Succinimidyl Esters. Succinimidyl esters are one of the most commonly used reactive forms of dyes for labeling of amine containing substrates such as proteins and were thought to be a suitable form for the testing of the new dyes. Treatment of the cyanine monophosphonates, such as 16 and 21, with commonly used activating reagents such as disuccinimidyl carbonate/pyridine, dicyclohexylcarbodiimide/ N-hydroxysuccinimide, or succinimidotetramethyluronium tetrafluoroborate (TSTU)/diispropylethylamine (DIEA) under anhydrous conditions resulted in mixtures of products some of which exhibited cyanine dimerlike absorption maxima. LC–MS confirmed the presence of dye dimers. Treatment of these mixtures with carbonate-bicarbonate buffer, pH 9.3, for several days did not convert the mixtures back to the starting material, but rather the dimer impurities persisted indicating that the dyes had been linked to each other through the phosphonate groups. The reactivity of phosphonate groups to these activating agents under anhydrous conditions is serious enough to prevent selective activation of carboxylic acid of the dye. Treatment of the cyanine monophosphonates with TSTU/DIEA under partially aqueous (10–15% water) conditions suppressed the formation of the dimer byproducts. It is important to avoid excess TSTU and to ensure that the dye is completely dissolved before the TSTU is added. Active esters of cyanine monophosphonates 16, 18, 21, and 23 were prepared with approximately 90% yield, but separation of the nondye byproducts of the reaction proved to be difficult, and therefore, the crude mixtures were used in labeling experiments without purification. To permit accurate estimation of stoichiometry for labeling experiments, effective molecular weights were calculated on the basis of the mass of starting dye, the mass of final product, and the amount of the dye in the active ester form as determined by RP HPLC at the absorption maximum of the dye. When the disodium salts of dyes 18 and 23 were used, then the active esters were easily separated from the nondye byproducts of the activation reaction. HPLC analysis of the cyanine monophosphonate active esters indicated little decomposition or hydrolysis during storage in a freezer at -8 °C at for at least 3 months. Application of the partially aqueous activating conditions to the squaraine diphosphonate 26 generated the disuccinimidyl ester in good yield, but activation of cyanine diphosphonates 14, 19, and 20 under similar conditions was problematic. After removal of the volatile components from the reaction mixtures and washing, the residues exhibited poor solubility in solvents such as DMSO and DMF. The active esters of 14 and 20 are highly soluble in aqueous media, and solid samples that were added to carbonate–bicarbonate buffer dissolved immediately. RP HPLC analysis of these solutions indicated that the active esters were hydrolyzed back to the starting acids in a few hours. Saturated solutions of the active esters of 14 and 20 in DMSO were used for labeling studies. 4. Protein Labeling Studies. Sheep IgG was labeled in carbonate-bicarbonate buffer at pH 9.3 using the active esters of diphosphonate dyes 14, 20, and 26 and of monophosphonate


Figure 1. Absorption spectra of dyes (solid lines) and conjugates (dashed lines): 14 (solid squares) and conjugate IgG–(14)6 (transparent squares) in BBS, pH 8.1; 20 (solid diamonds) and conjugate IgG–(20)5 (transparent diamonds) in BBS, pH 8.1; 26 (solid circles) and conjugate IgG–(26)3 (transparent circles) in PBS, pH 7.3.

dyes 16, 18, 21, and 23 to produce conjugates with a range of dye to protein (D/P) ratios. Samples were divided and were purified by G50 size exclusion chromatography using PBS, pH 7.3, BBS, pH 8.1, or carbonate-bicarbonate buffer, pH 9.3. All conjugates remained soluble during labeling at pH 9.3 and during purification in the three buffer systems, but solutions of conjugates of cyanine monophosphonates 16 and 21 with high (>5) D/P ratios developed precipitates when stored for several days at 4 °C in PBS at pH 7.3. Some of the precipitates dissolved when the solutions were warmed to room temperature. Conjugates of the other dyes did not form precipitates in any of the buffer media. Sheep IgG that was mixed with the parent dyes under conditions identical to those used for labeling separated completely from the dyes when subjected to size exclusion chromatography, indicating that nonspecific binding of the dyes to the protein does not occur. Sheep IgG was also labeled with Cy3.29 and Cy5.29 (known commercially as Cy3 monofunctional and Cy5 monofunctional, respectively) using methods supplied by the manufacturer. Previous studies have determined that labeling of proteins with disuccinimidyl active esters of cyanine and squaraine dyes under conditions similar to those used here does not cause a noticeable amount of protein cross-linking (17, 20). Conjugation of the cyanine dyes and the squaraine dye to the protein resulted in a red shift to the absorption maximum of the dyes by 2–3 and 6 nm, respectively. Typical absorption spectra of the dyes 14, 20, and 26 and of their conjugates are shown in Figure 1. Most noticeable is that the absorption spectra of conjugates are almost identical to those of the parent dyes despite the relatively high labeling densities. Usually, in aqueous media, organic dyes held in proximity to each other on the surface of a substrate clump together to form dimers and higher aggregates. Electronic ground-state coupling between the dyes in these aggregates results in different absorption and emission properties than those of the free dye. While some exceptions exist (5), it is most common for aggregates of cyanine dyes to show peak maxima that are blueshifted relative to the free dye. The absorption spectrum of the labeled substrate is the sum of the spectra of the aggregates and of the dye that is still independent. Consequently, aggregation manifests itself in the absorption spectrum as a shoulder on the shorter-wavelength side of the peak maximum of the independent dye, and in extreme cases this shoulder becomes the peak maximum. Thus, the relative height of the shoulder to the peak maximum is an indication of whether aggregation is occurring. Additionally, such aggregates have significantly lower


Figure 2. Plots of conjugate brightness (circles), quantum yields (QYs) (squares), and shoulder to peak maximum height (sh/pk) ratio (diamonds) as a function of dye to protein ratio for IgG–26 conjugates at pH 9.3 (solid symbols) and pH 7.3 (transparent symbols).

Figure 3. Plots of conjugate brightness (circles), quantum yields (QY) (squares), and shoulder to peak maximum height (sh/pk) ratio (diamonds) as a function of dye to protein ratio for IgG–20 conjugates at pH 9.3 (solid symbols), pH 8.1 (empty symbols), and pH 7.3 (transparent symbols).

QYs of fluorescence than the free fluorophore and so reduce the QY and the brightness of the labeled substrate (5, 17, 22). At pH 7.3, absorption spectra of the IgG–26 conjugates show a slight increase in the ratio of the short wavelength shoulder to the peak maximum height (sh/pk ratio) as the D/P ratio increases, whereas in pH 9.3 buffer there is little change (Figure 2, diamonds). The QY of squaraine 26 increases 4-fold when attached to the protein but then decreases as the dye D/P ratio increases (Figure 2, squares). The QY is slightly less sensitive to the D/P ratio at pH 9.3 than at pH 7.3. These differences in the observed absorption and fluorescence properties at the two pH values most likely result from increased ionization of the phosphonic acid residues at the higher pH, with concomitant increases in repulsive dye–dye interactions. The brightness of the conjugates ((ε)(QY)(D/P)) reaches a maximum with a D/P ratio of 3 at pH 7.3 and with a D/P ratio of 4 at pH 9.3 (Figure 2, circles). For comparison, the sulfonate analogue of dye 26, Sq635-b, was reported to show an increase in QY from 0.15 to 0.68 upon conjugation to bovine serum albumin, but no D/P ratio for such conjugates was presented (20). The absorption and fluorescence properties of conjugates of cyanine diphosphonate 20 exhibit similar trends as those of squarane diphosphonate 26 (Figure 3). In all three buffers there is a slight increase in the sh/pk ratio and a decrease in QY as the D/P ratio increases, but at lower pH the changes are more pronounced. The optimal labeling density is pH-dependent with values of 3.5D/P at pH 7.3, 5D/P at pH 8.1, and 6D/P at pH 9.3. For comparison, optimal labeling of IgG with Cy5.29 is

Reddington

Figure 4. Plots of conjugate brightness (circles), quantum yields (QY) (squares), and shoulder to peak maximum height (sh/pk) ratio (diamonds) as a function of dye to protein ratio for IgG–14 conjugates at pH 9.3 (solid symbols), pH 8.1 (empty symbols), and pH 7.3 (transparent symbols).

achieved with only 2–3D/P at which the QY is 0.2 and the conjugate brightness is approximately 1 × 105 (derived from data in ref 22). Thus, at pH 7.3 the cyanine diphosphonate 20 generates a brighter conjugate than Cy5. If experimental conditions permit the use of media of higher pH, then considerably greater advantage may be gained by use of dye 20. Absorption spectra of conjugates of cyanine diphosphonate 14 are relatively insensitive to the D/P ratio and to pH (Figure 4). The sh/pk ratio remained constant throughout the D/P range investigated. QY of the dye doubles upon attachment to the protein and then decreases as the D/P ratio increases. The optimal labeling density with dye 14 is greater than 6D/P compared 5–6D/P for Cy3, with the latter having a conjugate brightness of approximately 0.9 × 105 (derived from data in ref 22). Cyanine diphosphonate 14 offers at least equivalent performance as Cy3. Cyanine monophosphonates 16, 18, 21, and 23 are more prone to aggregate when attached to sheep IgG than are the cyanine diphosphonates 14 and 20, as evidenced by the increase of the sh/pk ratio (Figure 2 of Supporting Information). Absorption spectra of dyes 18 and 21, Cy3 and Cy5, and their conjugates are shown in Figure 5. It is common practice to estimate the concentration of dye in solutions of labeled conjugates through application of Beer’s law using the absorbance value at the peak maximum and the extinction coefficient of the free dye. This approach underestimates the D/P ratio when dye–dye aggregation is significant. Conjugates of the cyanine monophosphonates dyes were diluted with formamide (1:2) to break up the aggregates and permit more accurate estimations of the D/P ratio. Following the manufacturer’s recommended procedures for labeling with Cy3 and Cy5 generated conjugates with 5D/P and 7D/P, respectively. In the latter case this D/P ratio is far from the optimal of 2–3D/P (22). The absorption and fluorescence properties of conjugates of dyes 16, 18, 21, and 23 followed the same trends as those observed for the conjugates of the cyanine diphosphonates 14 and 20 except that the QY of dye 23 did not increase when the dye was attached to the protein but instead decreased (Figure 6). Optimal labeling densities for the conjugates were determined to be 4D/P for IgG–16, 5D/P for IgG–18, 2–3D/P for IgG–21, and 4D/P for IgG–23 (Figure 6). These are lower than for the cyanine diphosphonates 14 and 20 because of increased aggregation and fluorescence quenching of less highly charged dyes. Surprisingly, at optimal labeling densities, dye 21 gives significantly brighter conjugates with 2D/P than does the sulfonate containing dye 23 with 4D/P. In all cases, at higher labeling densities the conjugates are brighter at pH 9.3 than at

Technical Notes

Figure 5. Absorption spectra of dyes (solid lines) and conjugates (dashed lines) in PBS, pH 7.3: (A) 18 (transparent circles), IgG–(18)4 (solid circles), Cy3 (transparent squares), IgG–(Cy3) (solid squares); (B) 21 (transparent circles), IgG–(21)2 (solid circles), Cy5 (transparent squares), IgG–Cy5 (solid squares).

pH 7.3 whereas at lower labeling densities little difference is seen, again indicating that increased ionization of the phosphonic acid reduces quenching interactions. The absorption spectra and fluorescence properties of IgG conjugates with cyanine dyes such as Cy3 and Cy5 have been studied at length (4–6, 8, 17, 22). The initial increase in QY that is seen on conjugation to IgG of the trimethine cyanine dyes such as Cy3, 14, 16, and 18 is attributed to a rigidifying effect on the dye caused by association of the dye with the surface of the protein (4, 22). In contrast, QYs of pentamethine cyanine dyes, such as Cy5, are less dependent on the environment of the dye, and QYs of conjugates of Cy5 with D/P ratios less than 1 are similar to those of the free dye (22). Labeling of IgG with Cy5 was proposed to proceed in a nonrandom manner with binding of one Cy5 to the protein predisposing the region around the dye to further labeling (22), thereby accentuating the likelihood of dye dimer formation on the surface and leading to greater fluorescence quenching than would be seen with random labeling. If this is indeed the case, then labeling with the phosphonated cyanines at pH 9.3, where the phosphonate groups are expected to carry two negative charges, should result in more random labeling and in conjugates that are not so quenched. For cyanine diphosphonate 20, which is structurally similar to Cy5, the differences between the sh/pk ratios and the large differences in brightness of identical conjugates at pH 9.3 compared to those at pH 7.3 strongly suggest that electronic repulsion between the attached dyes is greater at pH 9.3. Reasons for the differences between the labeling properties of dyes 21 and 23 are not so obvious. The additional sulfonate group of dye 23 has the expected effect of increasing the optimal D/P ratio, presumably through assisting in solubilizing the dye and offering some inhibition to aggregation. The decrease in QY


Figure 6. Plots of conjugate brightness (solid lines) and quantum yields (dashed lines) at pH 9.3 (solid symbols) and at pH 7.3 (transparent symbols): (A) IgG–16 (circles) and IgG–18 (squares); (B) IgG–21 (circles) and IgG–23 (squares).

of dye 23 that is seen when the first dye is attached to the protein is in contrast to all of the other cyanine phosphonates studied here but is commonly seen with cyanine dyes that do not have charges appended to the aromatic residues (17). Whatever the mechanisms of labeling and quenching may be, it is clear that the cyanine diphosphonates can produce conjugates that are as bright or brighter than those of commercial Cy3 and Cy5. Conjugates of the monophosphonate cyanines dyes are not as bright as those of Cy3 and Cy5 because of the inherently lower QYs of the parent dyes and because they have a greater tendency to aggregate when attached to the protein. 5. Oligonucleotide Labeling Studies. 5′-Labeled oligodeoxyribonucleotides (ODNs) were prepared by conjugation of the active esters of dyes 16, 18, 21, 23, Cy3.29, and Cy5.29 to an amino functionalized T10-ODN or by coupling of the amidites of Quasar 570 (Q570) and Quasar 670 (Q670) to a T10-ODN. The pentamethine cyanine dyes 21, 23, Q670, and Cy5 in particular decomposed somewhat during the ammonia workup process. On conjugation to the ODNs, the absorbance maxima of the cyanine monophosphonates are shifted 4 nm to the red and their QYs increase (Table 2), which is consistent with previous observations for cyanine dyes with and without anionic substituents (9). ODNs T10-16 and T10-18 have similar QYs as ODN T10-Cy3.29, which is lower than that of ODN T10-Q570. ODNs T10-21 and T10-23 have lower quantum yields than ODNs T10Cy5.29 and T10-Q670, which have similar values. The cyanine monophosphonates, and other cyanines that carry anionic charges, such as Cy3 and Cy5, offer no advantage in QY over cyanines that do not carry anionic charges as 5′-fluorescent labels for ODNs, although they may be advantageous when multiple labeling of ODNs or DNA is required. In conclusion, synthetic methods for the preparation of phosphonic acid containing cyanine and squarane dyes were


Reddington

Table 2. Photophysical Data for T10-Dye Oligonucleotide Conjugates in PCR Buffer 5′-labeled oligonucleotide

absorption max, nm

emission max, nm

QY

T10-16 T10-18 T10-Cy3.29 T10-Q570 T10-21 T10-23 T10-Cy5.29 T10-Q670

552 553 551 549 650 651 649 647

568 567 563 564 668 669 666 666

0.10 0.11 0.11 0.14 0.21 0.23 0.32 0.31

developed. The new dyes have similar spectral properties to existing sulfonic acid containing analogues and can be used to generate brightly labeled dye–protein conjugates. As protein labels, trimethine cyanine diphosphonates, such as dye 14, are more or less equivalent to the commercial sulfonate analogue Cy3, whereas pentamethine cyanine diphosphonates, such as dye 20, are superior to the commercial sulfonate analogue Cy5, particularly when used in higher pH environments.

ACKNOWLEDGMENT The author thanks Alex Herrault for assistance in acquiring LC–MS spectra. Supporting Information Available: Absorption spectra for dyes 19, 21, and 23 and plots of shoulder to peak maximum vs dye to protein ratios for dyes 16, 18, 21, and 23. This material is available free of charge via the Internet at http://pubs.acs.org.

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(8) Reddington, M. V. (1998) New glycoconjugated cyanine dyes as fluorescent labeling reagents. J. Chem. Soc., Perkin Trans. 1, 143–147. (9) Gousou, C., Vasseur, J.-J., Bazin, H., Trinquet, E., Maurin, F., and Morvan, F. (2005) Optimized synthesis of functionalized fluorescent oligodeoxynucleotides for protein labeling. Bioconjugate Chem. 16, 465–470. (10) Devlin, R., Studholme, R. M., Dandliker, W. B., Fahy, E., Blumeyer, K., and Ghosh, S. S. (1993) Homogeneous detection of nucleic acids by transient-state polarized fluorescence. Clin. Chem. 39, 1939–1943. (11) Wolfbeis, O. S. (2001) Methods for Solubilizing Optical Markers. WO 01/36,973. (12) Borbas, K. E., Mroz, P., Hamblin, M., and Lindsey, J. S. (2006) Bioconjugatable porphyrins bearing a compact swallowtail motif for water solubility. Bioconjugate Chem. 17, 638–653. (13) Oswald, B., Gruber, M., Bohmer, M., Lehman, F., Probst, M., and Wolfbeis, O. S. (2001) Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem. Photobiol. 74, 237–245. (14) Olmstead, J., III (1979) Calorimetric determinations of absolute fluorescence quantum yields. J. Phys. Chem. 83, 2581– 2584. (15) Kawakami, M., and Kitaguchi, H. (2005) Cyanine Dyes. U.S. Patent 6,939,975. (16) Tavs, P. (1970) Reaction of aryl halides with trialkyl phosphites and dialkyl benzenephosphonites to aromatic phosphonates and phosphinates by nickel salt catalysed arylation. Chem. Ber. 103, 2428–2436. (17) Southwick, P. L., Earnst, L. A., Tauriello, E. W., Parker, S. R., Mujumbdar, R. B., Mujumbdar, S. R., Lewis, H. A., and Waggoner, A. S. (1990) Cyanine dye labeling reagents: carboxymethylindocyanine succinimidyl esters. Cytometry 11, 418– 430. (18) Gale, D. J., Lin, J., and Wilshire, J. F. K. (1970) The amidomethylation of Fischer’s base. The preparation of some new polymethine dyes. Aust. J. Chem. 30, 689–694. (19) Yee, K.-Y., Bailey, F. C., and Bluett, L. J. (1986) Squaraine chemistry. On the anomalous mass spectra of bis(4-dimethylaminophenyl)squaraine and its derivatives. Can. J. Chem. 64, 1607–1619. (20) Oswald, B., Patsenker, L., Duschl, J., Szmacinski, H., Wolfbeis, O., and Terpetschnig, E. (1999) Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels. Bioconjugate Chem. 10, 925–931. (21) Albert, A., and Sergent, E. P. (1962) Ionization Constants of Acids and Bases Wiley, New York. (22) Gruber, H. J., Hahn, C. D., Kada, G., Reiner, C. K., Harms, G. S., Ahrer, W., Dax, T. G., and Knaus, H.-G. (2000) Anomalous fluorescence enhancement of Cy3 and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin. Bioconjugate Chem. 11, 696–704. BC070090Y

Synthesis and Properties of Phosphonic Acid Containing Cyanine and

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