Synthesis of a Toolbox of Clickable Rhodamine B Derivatives

Abstract

An efficient method for the large-scale preparation of rhodamine B clickable derivatives has been developed. Starting from inexpensive rhodamine B as the starting material it was possible to functionalize the carboxylic functionality of rhodamine B with an azide, a strained-alkyne, a substituted triphenylphosphine, a thiol, and a maleimide. Through the synthetic strategy it was possible to obtain stable and pure clickable rhodamine compounds that can be readily used not only for chemoselectively probing biomolecules, but also for materials science.

Key words

click chemistry - fluorescence spectroscopy - chromophore synthesis - Diels–Alder reaction - amide coupling

• References and Notes

• 1a Karstens T, Kobs K. J. Phys. Chem. 1980; 84: 1871
• 1b Beija M, Afonso CA. M, Martinho JM. G. Chem. Soc. Rev. 2009; 38: 2410
• 2a Sharifzadeh Z, Abedi S, Morsali A. J. Mater. Chem. A 2014; 2: 4803
• 2b Voigt N, Henrich-Noack P, Kockentiedt S, Hintz W, Tomas J, Sabel BA. J. Nanopart. Res. 2014; 16: 2379
• 2c Menon JU, Ravikumar P, Pise A, Gyawali D, Hsia CC. W, Nguyen KT. Acta Biomater. 2014; 10: 2643
• 3a Xu L, Wang S, Lv YN, Son YA, Cao DR. Spectrochim. Acta A 2014; 128: 567
• 3b Pu SZ, Ding HC, Liu G, Zheng CH, Xu HY. J. Phys. Chem. C 2014; 118: 7010
• 3c Li K, Xiang Y, Wang XY, Li J, Hu RR, Tong AJ, Tang BZ. J. Am. Chem. Soc. 2014; 136: 1643
• 4a Shiraishi Y, Miyamoto R, Zhang X, Hirai T. Org. Lett. 2007; 9: 3921
• 4b Shiraishi Y, Miyamoto R, Hirai T. J. Photochem. Photobiol. A 2008; 200: 432
• 4c Rauch S, Eichhorn KJ, Oertel U, Stamm M, Kuckling D, Uhlmann P. Soft Matter 2012; 8: 10260
• 5a Zhang JG, Zhang L, Wei YL, Ma J, Shuang SM, Cai ZW, Dong C. Spectrochim. Acta A 2014; 122: 731
• 5b Hazra S, Balaji S, Banerjee M, Ganguly A, Ghosh NN, Chatterjee A. Anal. Methods 2014; 6: 3784
• 5c Culzoni MJ, de la Pena AM, Machuca A, Goicoechea HC, Babiano R. Anal. Methods 2013; 5: 30
• 6a Yuan L, Lin WY, Zheng KB, Zhu SS. Acc. Chem. Res. 2013; 46: 1462
• 6b Yapici NB, Mandalapu SR, Chew TL, Khuon S, Bi L. Bioorg. Med. Chem. Lett. 2012; 22: 2440
• 6c Mitronova GY, Belov VN, Bossi ML, Wurm CA, Meyer L, Medda R, Moneron G, Bretschneider S, Eggeling C, Jakobs S, Hell SW. Chem. Eur. J. 2010; 16: 4477
• 6d Zhou LY, Zhang XB, Wang QQ, Lv YF, Mao GJ, Luo AL, Wu YX, Wu Y, Zhang J, Tan WH. J. Am. Chem. Soc. 2014; 136: 9838
• 6e Nakajima T, Sano K, Sato K, Watanabe R, Harada T, Hanaoka H, Choyke PL, Kobayashi H. Cancer Sci. 2014; 105: 308
• 6f Kuerschner L, Thiele C. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2014; 1841: 1031
• 7a Lang K, Chin JW. Chem. Rev. 2014; 114: 4764
• 7b King M, Wagner A. Bioconjugate Chem. 2014; 25: 825
• 8 Chen X, Wu QZ, Henschke L, Weber G, Weil T. Dyes Pigments 2012; 94: 296
• 9 Nguyen T, Francis MB. Org. Lett. 2003; 5: 3245
• 10 MacKenzie DA, Sherratt AR, Chigrinova M, Cheung LL, Pezacki JP. Curr. Opin. Chem. 2014; 21: 81
• 11 Patterson DM, Nazarova LA, Prescher JA. Chem. Biol. 2014; 9: 592
• 12a Synthesis of Rhodamine B–Piperazine 1 To a solution of rhodamine B (1.0 g, 2.1 mmol) and DIPEA (0.42 mL, 2.4 mmol) in MeCN (150 mL) and at 0 °C was added a solution of HBTU (0.91 g, 2.4 mmol) in MeCN (50 mL). After stirring the mixture for 30 min at 0 °C, an ice-cold solution of mono-Boc-piperazine (0.45 g, 2.4 mmol) in MeCN (20 mL) was added. The ice-bath was removed, and the reaction was left at r.t. for overnight. The solvent was removed by rotary evaporation, and the crude was directly purified by column chromatography on silica gel using CH₂Cl₂–EtOH 95% (15:1) as the eluent. To deprotect the piperazine moiety, the product was redissolved in 10% TFA (20 mL) in CH₂Cl₂, and the reaction mixture was stirred vigorously for 2 h at r.t. in an uncovered flask. The crude was concentrated by rotary evaporation, and TFA was removed by repeatedly redissolving the residue in acetone and reapplying the vacuum. The crude product was then purified by column chromatography using CH₂Cl₂–EtOH 95% (3:1) as the eluent and switching to CH₂Cl₂–EtOH 95% (3:2) as the eluent to collect pure rhodamine B–piperazine 1 as a shiny purple solid in 95% yield (1.25 g, 2.0 mmol). R_f  = 0.3 (CH₂Cl₂–95% EtOH, 3:1). ¹H NMR (400 MHz, CDCl₃): δ = 7.69–7.66 (m, 2 H), 7.59–7.57 (m, 1 H), 7.34–7.32 (m, 1 H), 7.16 (d, J = 12 Hz, 2 H), 6.92 (dd, J ₁ = 12 Hz, J ₂ = 4 Hz, 2 H), 6.73 (d, J = 4 Hz, 2 H), 3.75–3.51 (m, 12 H), 3.13 (br s, 2 H), 3.06 (br s, 2 H), 1.31 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CDCl₃): δ = 167.2, 157.7, 155.67, 155.51, 134.5, 130.84, 130.23, 130.20, 130.11, 127.7, 114.3, 113.6, 96.3, 90.5, 53.4, 46.0, 12.4. ¹⁹F NMR (376 MHz, CD₃OD): δ = –76.89. ESI-HRMS: m/z calcd for C₃₂H₃₉N₄O₂ ⁺ [M]: 511.3068; found: 511.3074. IR (KBr): 3271,2983, 1683, 1647, 1590, 1528, 1468, 1415, 1339, 1276, 1248, 1203, 1179, 1131, 1075, 1010, 975, 922, 833, 799, 722, 682 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 91,573 M^–1 cm^–1.
• 12b Synthesis of Rhodamine B–Azide 2 To a solution of 1 (0.243 g, 0.389 mmol) and DIPEA (140 μL, 0.804 mmol) in MeCN (3 mL) was added dropwise and at 0 °C a solution of 1-azido-3-iodopropane (2, 0.115 g, 0.545 mmol) in MeCN (1 mL). After stirring the mixture at r.t. overnight, the solvent was removed by rotary evaporation, and the resulting crude residue was purified by flash column chromatography (CH₂Cl₂–95% EtOH, 15:2) to yield 2 as a purple solid in 51% yield (0.140 g, 0.198 mmol). ¹H NMR (400 MHz, CDCl₃): δ = 7.71–7.69 (m, 2 H), 7.57–7.55 (m, 1 H), 7.38–7.36 (m, 1 H), 7.30 (d, J = 8 Hz, 2 H), 7.06 (d, J = 8 Hz, 2 H), 6.79 (d, J = 4 Hz, 2 H), 3.72–3.65 (m, 8 H), 3.42 (br s, 4 H), 3.35 (t, J = 8 Hz, 2 H), 2.38 (t, J = 8 Hz, 2 H), 2.35 (br s, 2 H), 2.28 (br s, 2 H), 1.74 (quin, J = 8 Hz, 2 H), 1.35 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CD₃OD): δ = 169.3, 159.3, 157.19, 157.00, 136.8, 133.35, 132.02, 131.61, 131.31, 131.11, 128.9, 115.5, 114.9, 97.4, 56.2, 54.1, 53.5, 50.5, 47.0, 42.7, 26.9, 12.9. ¹⁹F NMR (376 MHz, CDCl₃): δ = –74.62. ESI-HRMS: m/z calcd for C₃₅H₄₄N₇O₂ ⁺ [M]: 594.3551; found: 594.3549. IR (KBr): 3262, 2924, 2029, 1630, 1588, 1413, 1339, 1273, 1179, 1131, 1073, 1007, 920,823, 680 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 91,102 M^–1 cm^–1.
• 12c Synthesis of Rhodamine B–Trityl-Protected Thiol 5 To a solution of 1 (0.416g, 0.660 mmol) in acetone (2 mL) was added DIPEA (0.5 mL, 2.9 mmol). To this mixture was added a solution of (3-bromopropyl)(trityl)sulfane (4, 0.573 mg, 1.44 mmol) in acetone (12 mL). The mixture was stirred for 6 d at r.t. under Ar (g), after which the solvent was removed by rotary evaporation. The remaining crude residue was purified by flash column chromatography (CH₂Cl₂–95% EtOH, 7:2) to yield compound 5 as a shiny purple solid in 20% yield (120 mg, 0.128 mmol). ¹H NMR (400 MHz, CD₃OD): δ = 7.77–7.75 (m, 2 H), 7.64–7.63 (m, 1 H), 7.52–7.51 (m, 1 H), 7.37 (d, J = 8 Hz, 6 H), 7.28–7.18 (m, 11 H), 7.06 (dd, J ₁ = 8 Hz, J ₂ = 4 Hz, 2 H), 6.98 (d, J = 4 Hz, 2 H), 3.67 (q, J = 8 Hz, 8 H), 3.31 (br s, 4 H), 2.17–2.12 (m, 4 H), 2.03 (br s, 4 H), 1.41 (quin, J = 8 Hz, 2 H), 1.29 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CD₃OD): δ = 169.5, 159.4, 157.4, 156.7, 135.7, 133.5, 132.54, 132.04, 131.81, 131.59, 131.15, 130.6, 128.54, 127.6, 115.3, 114.7, 97.2, 67.7, 57.9, 53.8, 46.8, 42.5, 30.6, 26.4, 15.8, 12.8. ESI-HRMS: m/z calcd for C₅₄H₅₉N₄O₂S⁺ [M]: 827.4353; found: 827.4375.
• 12d Synthesis of Rhodamine B–Furan-Protected Maleimide 8 To a solution of compound 1 (0.273 g, 0.437 mmol) in MeCN (1 mL) was added DIPEA (0.2 mL, 1.1 mmol). To this mixture was added a solution of compound 7 (0.242, 0.85mmol) MeCN (1 mL). The mixture was stirred for 3 d at r.t., after which the solvent was removed by rotary evaporation. The remaining crude residue was purified by flash column chromatography (CHCl₃–95% EtOH, 3:2) to yield compound 8 as a shiny purple solid in 34% yield (105 mg, 0.127mmol). ¹H NMR (400 MHz, CDCl₃): δ = 7.66–7.63 (m, 2 H), 7.51–7.49 (m, 1 H), 7.33–7.31 (m, 1 H), 7.20 (d, J = 8 Hz, 2 H), 6.92 (dd, J ₁ = 8 Hz, J ₂ = 4 Hz, 2 H), 6.77 (d, J = 4 Hz, 2 H), 6.49 (s, 2 H), 5.19 (s, 2 H), 3.63 (q, J = 8 Hz, 8 H), 3.45 (t, J = 8 Hz, 2 H), 3.37 (br s, 2 H), 3.29 (br s, 2 H), 2.83 (s, 2 H), 2.29–2.16 (m, 6 H), 1.62 (quin, J = 8 Hz, 2 H), 1.31 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CDCl₃): δ = 176.0, 166.9, 157.5, 155.61, 155.34, 136.2, 135.2, 131.8, 130.3, 129.95, 129.78, 129.60, 127.3, 113.85, 113.45, 96.1, 80.7, 54.7, 52.69, 52.07, 47.28, 47.13, 45.90, 41.5, 36.6, 24.2, 12.4. ESI-HRMS: m/z calcd for C₄₃H₅₀N₅O₅ ⁺ [M]: 716.3806; found: 716.3817. IR (KBr): 3264, 2971, 1767, 1695, 1630, 1588, 1468, 1411, 1340, 1275, 1180, 1131, 1074, 1010, 920, 877, 822, 679 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 91,221 M^–1 cm^–1.
• 12e Synthesis of Rhodamine B–Thiol 6 Compound 1 (0.135 g, 0.143 mmol) was dissolved in a solution of CH₂Cl₂–20% TFA (5 mL). To this solution was added i-Pr₃SiH (60 μL, 0.29 mmol). The mixture was stirred for 2 d at r.t., after which the solvent was removed by rotary evaporation. The remaining crude residue was purified by flash column chromatography using CH₂Cl₂–95% EtOH (9:2) as the eluent to yield product 6 as a shiny purple solid in 50% yield (50 mg, 0.072 mmol). This compound must be handled and stored under inert atmosphere because it easily oxidizes to the corresponding disulphide. ¹H NMR (400 MHz, CDCl₃): δ = 7.66–7.59 (m, 2 H), 7.55–7.50 (m, 1 H), 7.32–7.25 (m, 1 H), 7.20 (d, J = 8 Hz, 2 H), 6.92 (dd, J ₁ = 8 Hz, J ₂ = 4 Hz, 2 H), 6.74 (d, J = 4 Hz, 2 H), 3.60 (q, J = 8 Hz, 8 H), 3.38 (br s, 2 H), 3.32 (br s, 2 H), 2.61 (t, J = 4 Hz, 2 H), 2.50–2.21 (m, 6 H), 1.82–1.62 (m, 3 H), 1.29 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CDCl₃): δ = 169.5, 159.4, 157.4, 156.7, 135.7, 133.5, 132.54, 132.04, 131.81, 131.59, 128.94, 128.14, 115.5, 114.9, 97.5, 56.9, 53.8, 47.68, 47.02, 39.5, 29.0, 22.0, 13.0. ¹⁹F NMR (376 MHz, CDCl₃): δ = –74.62. ESI-HRMS: m/z calcd for C₃5H₄₅N₄O₂S⁺ [M]: 585.3258; found: 585.3263. IR (KBr): 3362, 3064, 2969, 2927, 2806, 1689, 1635, 1589, 1469, 1415, 1340, 1275, 1250, 1180, 1228, 1074, 1008, 972, 921, 822, 792, 721, 680 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 89,842 M^–1 cm^–1.
• 12f Synthesis of Rhodamine B–Maleimide 9 Compound 8 (0.050g, 0.080 mmol) was dissolved in DMF (0.5 mL) and heated to 110 °C for 5 h. The DMF was removed by rotary evaporation by dilution with toluene. The resulting residue was purified by flash column chromatography (CH₂Cl₂–95% EtOH, 7:1) to yield product 9 as a shiny purple solid in 66% yield (0.040 g, 0.053 mmol). ¹H NMR (400 MHz, CDCl₃): δ = 7.67–7.65 (m, 2 H), 7.51–7.49 (m, 1 H), 7.34–7.32 (m, 1 H), 7.22 (d, J = 8 Hz, 2 H), 6.95 (dd, J ₁ = 8 Hz, J ₂ = 4 Hz, 2 H), 6.79 (d, J = 4 Hz, 2 H), 6.67 (s, 2 H), 3.64 (q, J = 8 Hz, 8 H), 3.52 (t, J = 8 Hz, 2 H), 3.39 (br s, 2 H), 3.33 (br s, 2 H), 2.33–2.20 (m, 6 H), 1.70 (quin, J = 8 Hz, 2 H), 1.31 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CD₃OD): δ = 172.6, 159.4, 157.03, 157.07, 135.6, 133.5, 131.71, 131.43, 131.20, 129.0, 115.54, 115.00, 97.5, 95.81, 56.8, 54.2, 53.7, 47.1, 42.9, 40.6, 30.8, 26.3, 13.02. ¹⁹F NMR (376 MHz, CD₃OD): δ = –50.00. ESI-HRMS): m/z calcd for C₃₉H₄₆N₅O₄ ⁺ [M]: 648.3544; found: 648.3543. IR (KBr): 3256, 2973, 2930, 2872, 1705, 1631, 1590, 1556, 1528, 1508, 1469, 1414, 1341, 1276, 1249, 1181, 1133, 1075, 1011, 976, 922, 827, 761, 683 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 90,419 M^–1 cm^–1.
• 12g Synthesis of Rhodamine B–BCN 11 To a solution of 1 (0.27 g, 0.43 mmol) and DIPEA (150 μL, 0.861 mmol) in MeCN was added dropwise and at 0 °C a solution of compound 10 (0.080 g, 0.25 mmol) in MeCN (1 mL). After the reaction mixture was stirred at r.t. for 20 h, the solvent was removed by rotary evaporation, and the resulting crude residue was purified by flash column chromatography (CH₂Cl₂–i-PrOH, 7:3) to yield product 11 as a shiny purple solid in 88% yield (175 mg, 0.219 mmol). ¹H NMR (400 MHz, CD₃OD): δ = 7.78–7.76 (m, 2 H), 7.70–7.68 (m, 1 H), 7.53–7.50 (m, 1 H), 7.28 (d, J = 8 Hz, 2 H), 7.07 (dd, J ₁ = 8 Hz, J ₂ = 4 Hz, 2 H), 7.67 (d, J = 4 Hz, 2 H), 3.98 (d, J = 8 Hz, 2 H), 3.69 (q, J = 8 Hz, 8 H), 3.39 (br s, 4 H), 3.31 (br s, 4 H), 2.37–2.29 (m, 2 H), 2.29–2.06 (m, 4 H), 1.42–1.29 (m, 14 H), 0.75–0.64 (m, 3 H). ¹³C NMR (150 MHz, CD₃CN): δ = 168.5, 162.52, 162.27, 159.0, 157.2, 156.93, 156.54, 136.8, 133.2, 132.04, 131.39, 131.11, 130.9, 128.8, 116.7, 115.3, 114.8, 99.8, 97.2, 71.0, 46.9, 34.3, 31.1, 24.7, 23.9, 22.0, 14.4, 13.0. ¹⁹F NMR (376 MHz, CD₃CN): δ = –76.26. ESI-HRMS: m/z calcd for C₄₃H₅₁N₄O₄ ⁺ [M]: 687.391; found: 687.393. IR (KBr): 3255, 2977,2923, 1675,1589, 1419, 1340, 1254, 1183, 1132, 1077, 1006, 922, 831, 721, 679 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 91,299 M^–1 cm^–1.
• 12h Synthesis of Rhodamine B–Triphenylphosphine 13 To a flask containing compound 1 (50 mg, 0.080 mmol), HBTU (520 mg, 0.137 mmol), and 3-(diphenylphosphino)-4-(methoxycarbonyl)benzoic acid (12, 390 mg, 0.108 mmol) were added MeCN (2 mL) and DIPEA (0.040 mL, 0.23 mmol). The reaction mixture was stirred for 18 h. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography using i-PrOH–CH₂Cl₂ (1:10) as the eluent to give the product 13 as a purple solid in 51% yield (40 mg, 0.041 mmol). ¹H NMR (400 MHz, CDCl₃): δ = 8.08 (br s, 1 H), 7.72–7.62 (m, 3 H), 7.56–7.39 (m, 4 H), 7.37–7.18 (m, 11 H), 6.94–6.70 (m, 4 H), 3.71 (s, 3 H), 3.64–3.51 (m, 10 H), 3.37 (br s, 4 H), 3.22 (br s, 2 H), 1.29 (t, J = 8 Hz, 12 H). ¹³C NMR (150 MHz, CDCl₃): δ = 169.3, 167.6, 166.5, 157.7, 155.6, 141.1, 140.8, 137.63, 137.31, 134.6, 134.0, 133.8, 132.4, 131.9, 131.3, 130.44, 130.30, 128.81, 128.66, 128.58, 128.41, 127.75, 127.48, 114.1, 113.7, 96.3, 64.3, 52.2, 46.0, 41.7, 30.9, 25.3,12.5. ³¹P NMR (162 MHz, CDCl₃): δ = –3.80 (s). ESI-HRMS: m/z calcd for C₅₃H₅₄N₄O₅P⁺ [M]: 857.3826; found: 857.3822. IR (KBr): 3254, 3054, 2973, 2929, 2869, 1718, 1635, 1589, 1527, 1466, 1414, 1338, 1274, 1247, 1180, 1159, 1132, 1073, 1004, 975, 921, 840, 789, 748, 698, 682, 664 cm^–1. UV-vis (DMSO, λ_max, ε): 571 nm, 88,970 M^–1 cm^–1.
• 13 Fery-Forgues S, Lavabre D. J. Chem. Educ. 1999; 76: 1260