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Photochemistry and Photobiology Vol. 56, No. 3, pp. 31 1-317, 1992 Printed in Great Britain. All rights reserved
PHOTOPHYSICAL PROPERTY OF SANGUINARINE IN THE EXCITED SINGLET STATE A. DAS, R. NANDIand M. MAITI* Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology, Calcutta 700 032, India
(Received 30 August 1991; accepted 30 January 1992) Abstract-The photophysical property of the alkanolamine form of sanguinarine has been studied in aqueous and organic medium under various environmental conditions from the measurement of absorption, fluorescence and NMR spectroscopy. Alkanolamine fluorescence shows an excitation time dependent fluorescence quenching and the rate of quenching increases significantly with increasing pH and concentration of the species, while it decreases with increasing temperature. This phenomenon is explained by excited state intramolecular proton transfer from a 6-OH group to the lone pair of nitrogen through the formation of zwitterion.
INTRODUCTION
Sanguinarine, a benzophenanthridine alkaloid, has displayed antimicrobial and antitumour activity (Stermitz et al., 1973; Preininger, 1975; Cordell and Farnsworth, 1977; Nandi et al., 1983; Kornman, 1986). Its salt has been used in toothpastes and dental rinses (Boulware ef al., 1985). It forms a molecular complex with DNA and preferentially binds to G-C rich DNA by intercalation (Maiti et al., 1982; 1984; Smekal et al., 1984; Faddejeva et al., 1984; Nagao et al., 1985; Nandi et al., 1985; Nandi and Maiti, 1985; Maiti and Nandi, 1987). It has also been reported to be a phototoxic H202 producing alkaloid (Tuveson ef al., 1989). Sanguinarine has pH dependent structural equilibrium between structure I (iminium form) and structure I1 (alkanolamine form) with pK value of 7.4 as revealed by spectrophotometric and spectrofluorometric measurements (Maiti et al., 1983). The stability of two structural forms have further been confirmed by Jones et al. (1986) from the analysis of absorption spectrophotometry, HPLC and NMR spectroscopy and they have suggested that conversion from the iminium ion to the alkanolamine form may enhance antimicrobial activity by increasing cellular availability of the alkaloid due to its (alkanolamine form) greater lipophilicity. During our recent comparative studies on the interaction of iminium and alkanolamine forms of sanguinarine with various DNA (Maiti and Nandi, 1987; Chakraborty et al., 1991), we have found an excitation time dependent fluorescence quenching (ETDFQ)? of the alkanolamine form, while steady state fluorescence is observed in iminium form. The present study reports on the equilibrium and sta'To whom correspondence should be addressed. tAbbreviations: DMSO, dimethyl sulfoxide; ESIPT, excited state intramolecular proton transfer; ETDFQ, excitation time dependent fluorescence;EtOH, ethanol; MeOH, methanol.
31 I
bility of the iminium and neutral form of sanguinarine in various solvent systems from the standpoint of spectrophotometric and spectrofluorometric studies and the effect of solvent, pH, temperature and concentration of the species on ETDFQ rate value in the excited singlet state. MATERIALS AND METHODS
Sanguinarine chloride was purchased from Aldrich Chemical Co., Milwaukee. WI and was used after checking its purity by thin layer chromatography and melting point and characterizing by mass and NMR spectrum. The alkaloid solution was freshly prepared by dissolving appropriate amounts in water and the concentration was obtained spectrophotometricallyusing a molar extinction coefficient (E) of 30700 M - ' cm-' at 327 nm in 0.1 N HCI (Jones er al., 1986). Ethanol (EtOH), methanol (MeOH), dimethylsulfoxide (DMSO), deuterium oxide (D20), and glycerol were obtained from Sigma Chemical Co., St. Louis, MO. Ethyl-d, alcohol-d (C2D50D), methyl-d, alcohol-d (CD,OD) and DMSO-d, [(CD,),SO] were obtained from Aldrich Chemical Co. All other reagents used were of analytical grade. Deionized double distilled water was used throughout. The alkaloid obeyed Beer's law in the concentration range used. Experiments were carried out in (i) 0.1 N HCI, (ii) 0.1 N NaOH, (iii) aqueous solution of various pH values prepared by the addition of appropriate amounts of concentrated HCI or NaOH. (iv) MeOH. (v) MeOH-NaOH of various pH, (vi) EtOH, (vii) Tris-HCI buffer, pH 8.5, (viii) DMSO. (ix) glycerol and (x) MeOH, EtOH and DMSO with 0.1 N HCI of various proportions. The solution pH was measured in a pH M84 Research pH meter (Radiometer Copenhagen, Denmark) with an accuracy of +0.001. Absorption spectroscopy. Ultraviolet visible absorption spectra of the alkaloid were recorded at 25°C in quartz cell of 1 cm path length on a Shimadzu model UV 260 automatic recording spectrophotometer equipped with a temperature programmer (KPC 5 ) and a temperature controller (SPR 5 ) (Shimadzu Corporation, Japan). N M R spectroscopy. 'H NMR spectra were recorded on a JEOL FT-100 NMR spectrometer operating at 99.6 MHz. All 'H shifts are relative to Me,% Fluorescence spectroscopy. Fluorescence spectra were recorded in quartz cell of 0.5 cm path length on Hitachi model F-4010(Hitachi Ltd., Tokyo, Japan) equipped with a thermoelectric temperature controller model EYELA
312
A.
0.0
1
250
3 50
450
DASel al.
550
WAVELENGTH [nm)
Figure 1. Representative absorption spectra of sanguinarine (structure I) of 3.94 pM in H,O-HCI and H,O-NaOH solution of different pH values. (1) pH 1.0, (2) pH 6.7, (3) pH 7.0, (4) pH 7.6, (5) pH 8.0, (6) pH 13.0 and (7) pH 13.0 converted to pH 3.0 with the addition of concentrated HCI. unit cool UC-55 (Tokyo Rikakikai Co. Ltd., Japan). Change of fluorescence intensity at a particular wavelength with time were also recorded. Computer programs supplied by Hitachi was employed in the processing of the fluorescence data. Uncorrected fluorescence spectrum was reported in all cases. Reversibility. The reversibility of spectral changes was done using 0.1 N HCI or 0.1 N NaOH following the method described earlier (Maiti et a / . , 1983; Chakraborty et al., 1990): RESULTS
Absorption spectra
0.0-
I L I '. , '..Ji, 250 350 450 550 250 350 450 550
WAVELENGTH (nm)
Figure 2. Absorption spectra of sanguinarine (structure I) of 11.88 pM in (A) Tris-NaOH buffer, pH 11.0, (B) EtOH. (C) MeOH and (D) DMSO represented by solid line. Dotted lines represent the spectra at pH 3.0 after addition of concentrated HCI to the respective solvent for each case. pattern absorption spectrum of sanguinarine is observed. It is worth noting that the spectral changes are completely reversible when sanguinarine solution, at one end of pH, is titrated with HCI or NaOH to the other end of pH.
N M R spectra
'H NMR spectra of sanguinarine (iminium form) It has been reported earlier (Maiti et al., 1983) that sanguinarine has pH dependent structural in D 2 0 , C2Ds0D, C D 3 0 D and DMSO-d6 were changes as followed from UV-absorption spectra measured and chemical shifts were found to be (240-350 nm range). Here Fig. 1 illustrates the UV- identical in all the cases. The results showed that visible absorption spectra (220-550 nm) of sanguin- NMR spectroscopy could not detect the formation arine at various pH values. The spectra are more of alkoxide or dimethyl sulfoxide at the Ch position or less superimposable in pH range 1.0-6.0 with in the high concentration of sanguinarine peaks at 273, 327 and 468 nm. The peak intensities (>1200 pM) of the sample preparation where the at 327 and 468 nm diminish with increasing pH from addition product formation was only about 1%. 6.1 to upwards. It is interesting to note that with These observations are rationalized from the the gradual abolition of the 468 nm peak, the absorption spectra of various concentrations appearance of a new peak around 235 nm is (ranging from 10 pM to 500 p M ) of sanguinarine observed. Ultimately the long wavelength band at (iminium form) in EtOH, MeOH or DMSO. It 468 nm is completely abolished at pH 8.6 and has been observed that an increase in the initial above. The short wavelength peak at 273nm is concentration of sanguinarine causes decrease in gradually shifted to a higher wavelength peak at the conversion of sanguinarine (iminium form) into 283 nm with increasing pH. Moreover, the series of ethoxide, methoxide or dimethyl sulfoxide formaspectra at various pH give three isosbestic points tion, while a dilute ethanolic, methanolic or dimearound 239, 283 and 303 nm which indicate clearly thy1 sulfoxide solution of sanguinarine (
Photochemistry and Photobiology Vol. 56, No. 3, pp. 31 1-317, 1992 Printed in Great Britain. All rights reserved
PHOTOPHYSICAL PROPERTY OF SANGUINARINE IN THE EXCITED SINGLET STATE A. DAS, R. NANDIand M. MAITI* Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology, Calcutta 700 032, India
(Received 30 August 1991; accepted 30 January 1992) Abstract-The photophysical property of the alkanolamine form of sanguinarine has been studied in aqueous and organic medium under various environmental conditions from the measurement of absorption, fluorescence and NMR spectroscopy. Alkanolamine fluorescence shows an excitation time dependent fluorescence quenching and the rate of quenching increases significantly with increasing pH and concentration of the species, while it decreases with increasing temperature. This phenomenon is explained by excited state intramolecular proton transfer from a 6-OH group to the lone pair of nitrogen through the formation of zwitterion.
INTRODUCTION
Sanguinarine, a benzophenanthridine alkaloid, has displayed antimicrobial and antitumour activity (Stermitz et al., 1973; Preininger, 1975; Cordell and Farnsworth, 1977; Nandi et al., 1983; Kornman, 1986). Its salt has been used in toothpastes and dental rinses (Boulware ef al., 1985). It forms a molecular complex with DNA and preferentially binds to G-C rich DNA by intercalation (Maiti et al., 1982; 1984; Smekal et al., 1984; Faddejeva et al., 1984; Nagao et al., 1985; Nandi et al., 1985; Nandi and Maiti, 1985; Maiti and Nandi, 1987). It has also been reported to be a phototoxic H202 producing alkaloid (Tuveson ef al., 1989). Sanguinarine has pH dependent structural equilibrium between structure I (iminium form) and structure I1 (alkanolamine form) with pK value of 7.4 as revealed by spectrophotometric and spectrofluorometric measurements (Maiti et al., 1983). The stability of two structural forms have further been confirmed by Jones et al. (1986) from the analysis of absorption spectrophotometry, HPLC and NMR spectroscopy and they have suggested that conversion from the iminium ion to the alkanolamine form may enhance antimicrobial activity by increasing cellular availability of the alkaloid due to its (alkanolamine form) greater lipophilicity. During our recent comparative studies on the interaction of iminium and alkanolamine forms of sanguinarine with various DNA (Maiti and Nandi, 1987; Chakraborty et al., 1991), we have found an excitation time dependent fluorescence quenching (ETDFQ)? of the alkanolamine form, while steady state fluorescence is observed in iminium form. The present study reports on the equilibrium and sta'To whom correspondence should be addressed. tAbbreviations: DMSO, dimethyl sulfoxide; ESIPT, excited state intramolecular proton transfer; ETDFQ, excitation time dependent fluorescence;EtOH, ethanol; MeOH, methanol.
31 I
bility of the iminium and neutral form of sanguinarine in various solvent systems from the standpoint of spectrophotometric and spectrofluorometric studies and the effect of solvent, pH, temperature and concentration of the species on ETDFQ rate value in the excited singlet state. MATERIALS AND METHODS
Sanguinarine chloride was purchased from Aldrich Chemical Co., Milwaukee. WI and was used after checking its purity by thin layer chromatography and melting point and characterizing by mass and NMR spectrum. The alkaloid solution was freshly prepared by dissolving appropriate amounts in water and the concentration was obtained spectrophotometricallyusing a molar extinction coefficient (E) of 30700 M - ' cm-' at 327 nm in 0.1 N HCI (Jones er al., 1986). Ethanol (EtOH), methanol (MeOH), dimethylsulfoxide (DMSO), deuterium oxide (D20), and glycerol were obtained from Sigma Chemical Co., St. Louis, MO. Ethyl-d, alcohol-d (C2D50D), methyl-d, alcohol-d (CD,OD) and DMSO-d, [(CD,),SO] were obtained from Aldrich Chemical Co. All other reagents used were of analytical grade. Deionized double distilled water was used throughout. The alkaloid obeyed Beer's law in the concentration range used. Experiments were carried out in (i) 0.1 N HCI, (ii) 0.1 N NaOH, (iii) aqueous solution of various pH values prepared by the addition of appropriate amounts of concentrated HCI or NaOH. (iv) MeOH. (v) MeOH-NaOH of various pH, (vi) EtOH, (vii) Tris-HCI buffer, pH 8.5, (viii) DMSO. (ix) glycerol and (x) MeOH, EtOH and DMSO with 0.1 N HCI of various proportions. The solution pH was measured in a pH M84 Research pH meter (Radiometer Copenhagen, Denmark) with an accuracy of +0.001. Absorption spectroscopy. Ultraviolet visible absorption spectra of the alkaloid were recorded at 25°C in quartz cell of 1 cm path length on a Shimadzu model UV 260 automatic recording spectrophotometer equipped with a temperature programmer (KPC 5 ) and a temperature controller (SPR 5 ) (Shimadzu Corporation, Japan). N M R spectroscopy. 'H NMR spectra were recorded on a JEOL FT-100 NMR spectrometer operating at 99.6 MHz. All 'H shifts are relative to Me,% Fluorescence spectroscopy. Fluorescence spectra were recorded in quartz cell of 0.5 cm path length on Hitachi model F-4010(Hitachi Ltd., Tokyo, Japan) equipped with a thermoelectric temperature controller model EYELA
312
A.
0.0
1
250
3 50
450
DASel al.
550
WAVELENGTH [nm)
Figure 1. Representative absorption spectra of sanguinarine (structure I) of 3.94 pM in H,O-HCI and H,O-NaOH solution of different pH values. (1) pH 1.0, (2) pH 6.7, (3) pH 7.0, (4) pH 7.6, (5) pH 8.0, (6) pH 13.0 and (7) pH 13.0 converted to pH 3.0 with the addition of concentrated HCI. unit cool UC-55 (Tokyo Rikakikai Co. Ltd., Japan). Change of fluorescence intensity at a particular wavelength with time were also recorded. Computer programs supplied by Hitachi was employed in the processing of the fluorescence data. Uncorrected fluorescence spectrum was reported in all cases. Reversibility. The reversibility of spectral changes was done using 0.1 N HCI or 0.1 N NaOH following the method described earlier (Maiti et a / . , 1983; Chakraborty et al., 1990): RESULTS
Absorption spectra
0.0-
I L I '. , '..Ji, 250 350 450 550 250 350 450 550
WAVELENGTH (nm)
Figure 2. Absorption spectra of sanguinarine (structure I) of 11.88 pM in (A) Tris-NaOH buffer, pH 11.0, (B) EtOH. (C) MeOH and (D) DMSO represented by solid line. Dotted lines represent the spectra at pH 3.0 after addition of concentrated HCI to the respective solvent for each case. pattern absorption spectrum of sanguinarine is observed. It is worth noting that the spectral changes are completely reversible when sanguinarine solution, at one end of pH, is titrated with HCI or NaOH to the other end of pH.
N M R spectra
'H NMR spectra of sanguinarine (iminium form) It has been reported earlier (Maiti et al., 1983) that sanguinarine has pH dependent structural in D 2 0 , C2Ds0D, C D 3 0 D and DMSO-d6 were changes as followed from UV-absorption spectra measured and chemical shifts were found to be (240-350 nm range). Here Fig. 1 illustrates the UV- identical in all the cases. The results showed that visible absorption spectra (220-550 nm) of sanguin- NMR spectroscopy could not detect the formation arine at various pH values. The spectra are more of alkoxide or dimethyl sulfoxide at the Ch position or less superimposable in pH range 1.0-6.0 with in the high concentration of sanguinarine peaks at 273, 327 and 468 nm. The peak intensities (>1200 pM) of the sample preparation where the at 327 and 468 nm diminish with increasing pH from addition product formation was only about 1%. 6.1 to upwards. It is interesting to note that with These observations are rationalized from the the gradual abolition of the 468 nm peak, the absorption spectra of various concentrations appearance of a new peak around 235 nm is (ranging from 10 pM to 500 p M ) of sanguinarine observed. Ultimately the long wavelength band at (iminium form) in EtOH, MeOH or DMSO. It 468 nm is completely abolished at pH 8.6 and has been observed that an increase in the initial above. The short wavelength peak at 273nm is concentration of sanguinarine causes decrease in gradually shifted to a higher wavelength peak at the conversion of sanguinarine (iminium form) into 283 nm with increasing pH. Moreover, the series of ethoxide, methoxide or dimethyl sulfoxide formaspectra at various pH give three isosbestic points tion, while a dilute ethanolic, methanolic or dimearound 239, 283 and 303 nm which indicate clearly thy1 sulfoxide solution of sanguinarine (
