A cationic water-soluble cyclophane (1a) having a rhodamine moiety as a red-fluorescence fluorophore was prepared by reaction of a monoamine derivative of tetraaza[6.1.6.1]paracyclophane having three N-t-butoxycarbonyl- β -alanine residues with rhodamine B isothiocyanate, followed by removal of the protecting groups. The guest-binding behavior of 1a toward anionic guests such as dabsyl derivative and 4-(1-pyrene)butanoate was investigated by fluorescence spectroscopy. The results suggested the formation of host-guest complexes with a stoichiometric ratio of 1:1 and the binding constants (K) of the host-guest complexes were evaluated.
In recent years, much attention has been focused on development of fluorescent host sensor systems, which are able to detect small organic compounds [
On the other hand, many types of fluorescent dyes such as fluorescein isothiocyanate [
HEPES (N-(2-hydroxyethyl) piperazine-N’-2-ethanesulfonic acid) buffer (0.01 M, pH 7.4, with 0.15 M NaCl) was purchased from GE Healthcare. A cyclophane derivative bearing N-protected amines (2) was prepared after a method reported previously [
were recorded on JASCO FP-750, Perkin-Elmer spectrum one, and JMS-T100 CS spectrometers, respectively.
Piperidine (1.0 mL) was added to a solution of cyclophane derivative bearing N-protected amines (2) (179 mg, 0.14 mmol) in dry dichloromethane (DCM, 5 mL), and the mixture was stirred for 5 h at room temperature. Then the solvent was evaporated off under reduced pressure to give a pale yellow solid (monoamine of cyclophane). The monoamine of cyclophane was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant. The precursor fraction was evaporated to dryness under reduced pressure to give a pale yellow solid (cyclophane monoamine, 152 mg). Triethylamine was added to a solution of the monoamine of cyclophane (140 mg, 0.13 mmol) in dry DCM (8 mL) at room temperature, and the mixture was allowed to stand at same temperature. The mixture was added to a solution of rhodamine B isothiocyanate (91 mg, 0.17 mmol) in dry DCM (2 mL), and the resulting mixture was stirred for 1 day at the same temperature. After being dried (Na2SO4), the solution was evaporated to dryness under reduced pressure to give a dark purple solid. The crude product was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant. Evaporation of the product fraction under reduced pressure gave a dark purple solid (151 mg, 73%): mp 144˚C - 145˚C. 1H NMR (400 MHz, CDCl3, 293 K) δ 1.3 (m, 12H), 1.4 (m, 35H), 2.1 (m, 8H), 3.3 (m, 8H), 3.5 (m, 8H), 3.6 (m, 8H), 3.9 (m, 4H), 5.3 (m, 5H), 6.6 (m, 4H), 7.0 (m, 10H), 7.1 (m, 10H) and 7.5 (m, 1H). 13C NMR (100 MHz, CDCl3, 293 K) δ 12.8, 25.1, 28.6, 35.0, 36.5, 40.3 - 41.5, 45.8, 48.9, 79.1, 96.2 - 96.9, 112 - 113, 128 - 129, 130 - 131, 132, 140 - 141, 155, 156, 157, 171, 172 and 181. IR 1646 cm−1 (C=O). Found: C, 59.72; H, 7.47; N, 8.74. Calcd for C90H113ClN11NaO13 S· 9H2O: C, 59.74; H, 7.30; N, 8.51. ESI-TOF MS (positive mode): m/z 1589 [M + H]+, 1611 [M + Na]+, where M denotes zwitterionic form of cyclophane (M, C90H113N11 O13S).
Trifluoroacetic acid (1.0 mL) was added to a solution of CP-Boc3RhB (153 mg, 0.096 mmol) in dry DCM (6 mL), and the mixture was stirred for 1 h at room temperature. Evaporation of the solvent under reduced pressure gave a dark purple solid. The crude product was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant. Evaporation of the product fraction under reduced pressure gave a dark purple solid (139 mg, 89%): mp 182˚C - 190˚C (decomp.). 1H NMR (400 MHz, CD3OD, 293 K) δ 1.1 - 1.2 (m, 12H), 1.3 - 1.5 (m, 8H), 2.2 - 2.5 (m, 8H), 2.9 - 3.1 (m, 8H), 3.5 - 3.8 (m, 16H), 3.9 - 4.1 (m, 4H), 6.7 - 7.0 (m, 8H), 7.1 - 7.4 (m, 16H) and 8.1 (m, 1H). 13C NMR (100 MHz, CD3OD, 293K) δ 11.8, 23.6 - 25.0, 31.4, 35.8, 40.6, 45.5, 48.6, 95.8, 113 - 114, 116 - 121, 128 - 129, 130 - 131, 132, 139 - 141, 142, 155, 158, 161 - 162, 170 and 181. IR 1645 cm−1 (C=O). Found: C, 58.52; H, 6.00; N, 9.28. Calcd for C81H92F9N11O13S·2H2O: C, 58.37; H, 5.81; N, 9.24. ESI-TOF MS (positive mode): m/z 1289 [M + H]+, 1311 [M + Na]+, where M denotes triamine derivative of cyclophane as a free base (M, C75H89N11O7S).
Succinic anhydride (59 mg, 0.59 mmol) was added to a solution of cyclophane 1a (106 mg, 0.06 mmol) and triethylamine (0.5 mL) in dry DCM (4 mL) at room temperature, and the mixture was stirred for 1 day. Ethylenediamine (0.1 mL, 1.5 mmol) was added to the mixture to quench the reaction. After being dried (Na2SO4), the solution was evaporated to dryness under reduced pressure to give a dark purple solid. The crude product was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant. Evaporation of the product fraction under reduced pressure gave a dark purple solid. Then added 0.1 M NaOH aq. (2 ml) and stirred 20 min at room temperature. After dialysis (1.0 kDa cut-off) for 4 h, the solvent was freeze-dried to gave a dark purple solid (88 mg, 82 %): mp 170˚C - 172˚C (decomp.). 1H NMR (400 MHz, CD3OD, 293 K) δ 1.0 - 1.5 (m, 20H) 2.2 (m, 6H), 2.4 (m, 14H), 3.3 (m, 8H), 3.4 - 3.8 (m, 16H), 3.9 - 4.0 (m, 4H), 6.8 (m, 4H), 6.9 - 7.1 (m, 10H), 7.2 - 7.4 (m, 8H), 7.7 - 7.9 (m, 2H) and 8.1 (m, 1H). 13C NMR (100 MHz, CD3OD, 293K) δ 11.7, 23.1, 23.9, 32.8, 33.5, 34.1, 35.4, 40.6, 45.6, 52.0, 96.0, 113, 114, 128 - 129, 130 - 131, 132, 140, 141 - 142, 155, 158, 171 - 172, 174 ,179, 180 and 181. IR 1736, 1635 cm−1 (C=O). Found: C, 63.37; H, 6.35; N, 9.56. Calcd for C87H101N11O16S·3H2O: C, 63.60; H, 6.56; N, 9.38. ESI-TOF MS (negative mode): m/z 1589 [M − H]−, 1610 [M − 2H + Na]−, 1632 [M − 3H + 2Na]−, where M denotes carboxylic acid of cyclophane (M, C87H101N11O16S).
Triethylamine (0.5 mL) was added to a solution of β-alanine t-butyl ester hydrochloride (95 mg, 0.52 mmol) in dry DCM (10 ml) at room temperature. The mixture was added to a solution of 4-dimethyl-aminoazobenzene- 4-sulfonyl chloride (DabsylCl, 149 mg, 0.46 mmol) in dry DCM (5 ml), and the resulting mixture was stirred for day at room temperature. The residue was chromatographed on a column of silica gel (SiO2) with chloroform-methanol (95:5 v/v) as eluant. Evaporation of the product fraction under reduced pressure gave a orange-red solid (158 mg, 79%): mp 170˚C - 171˚C. 1H NMR (400 MHz, CDCl3, 293 K) δ 1.4 (s, 9H), 2.4 (m, 2H), 3.2 (m, 6H), 3.5 (m, 2H), 6.8 (m, 2H) and 7.9 - 8.0 (m, 6H). 13C NMR (100 MHz, CDCl3, 293K) δ 28.3, 35.1, 39.2, 40.5, 81.8, 112, 122, 126, 140, 144, 153, 156 and 172. IR 1708 cm−1 (C=O). Found: C, 57.11; H, 6.38; N, 12.63. Calcd for C21H28N4O4S∙0.5 H2O: C, 57.12; H, 6.62; N, 12.69. ESI-TOF MS (positive mode): m/z 433 [M + H]+, 455 [M + Na ]+.
Trifluoroacetic acid (1.0 ml) was added to a solution of 4 (75 mg, 0.17 mmol) in dry DCM (5 ml), and the mixture was stirred for 4 h at room temperature. The residue was chromatographed on a column of silica gel (SiO2) with chloroform-methanol (9:1 v/v) as eluant. The product fraction was evaporated to dryness under reduced pressure to give a orange-red solid (53 mg, 82%): mp 164˚C - 165˚C. 1H NMR (400 MHz, CD3OD, 293 K) δ 2.5 (m, 2H), 3.1 (m, 6H), 3.2 (m, 2H), 6.9 (m, 2H) and 7.9 - 8.0 (m, 6H). 13C NMR (100 MHz, CD3OD, 293K) δ 28.3, 35.1, 39.2, 40.5, 81.8, 112, 122, 126, 140, 144, 153, 156 and 172. IR 1709 cm−1 (C=O). Found: C, 54.24; H, 5.36; N, 14.88. Calcd for C17H20N4O4S: C, 54.00; H, 5.40; N, 15.11. ESI-TOF MS (positive mode): m/z 377 [M + H]+, 399 [M + Na ]+.
The calculations were carried out on a Pentium 4 3.2 GHz × 2 machine using Macro Model 9.1 molecular modeling software on a Red Hat Enterprise Linux WS 4.3 operating system. The geometry of 1a and 1b was optimized using molecular mechanics employing the OPLS_2005 force field for the simulation of the hosts. The geometry was optimized without any constraints allowing all atoms, bonds, and dihedral angles to change simultaneously.
To each solution of fluorescent cyclophane (0.5 μM) in HEPES buffer were added increasing amounts of 5 and 6, and the fluorescence intensity was monitored after each addition by excitation at 558 nm. Aqueous stock solution of 5 was prepared after addition of NaOH. The binding constants were calculated on the basis of the Benesi-Hildebrand method for titration data.
From a viewpoint of development of cyclophanes emitting in the red region of visible spectrum, we have designed water-soluble cyclophanes having a rhodamine moiety. Actually, we have adopted a simple strategy to prepare rhodamine-appended cyclophanes by introducing a rhodamine moiety into tetraaza[6.1.6.1]paracyclo- phane [
As mentioned above, rhodamine derivatives have an intense visible absorption. Actually, rhodamine-appended water-soluble cyclophanes 1a and 1b had high absorption coefficients and absorption in the visible region owing to the rhodamine moieties. In addition, they showed fluorescence emission spectra originated rhodamine moieties with a fluorescence maximum at 579 nm in aqueous media in aqueous HEPES (2-[4-(2-hydroxy- ethyl)-1-piperazinyl]ethanesulfonic acid) buffer (0.01 M, pH 7.4, 0.15 M with NaCl) at 298 K (
host-guest complexation. A similar fluorescence feature was observed when 4-(1-pyrene)butanoate (6) was employed as an anionic florescence guest. That is, upon addition of 6 to an aqueous solution containing 1a, fluorescence intensity originated from 1a decreased, as shown in
Rhodamine-appended cyclophanes bearing three cationic polar side chains 1a were successfully prepared by
reaction of RITC with a monoamine derivative of cyclophane, followed by removal of the protecting groups in a fairly good yield. 1a showed fluorescence bands with a fluorescence maximum at 579 nm in an aqueous HEPES buffer. Formation of the host-guest complexes of the present cyclophane with anionic guests was demonstrated by fluorescence quenching experiments. The fluorescence intensity originating from 1a was subjected to decrease, upon complexation with anionic guests such as 5 and 6.
The present work is partially supported by Grant-in-Aid (No. 24550166) from the Ministry of Education, Culture, Science, Sports and Technology of Japan.