Journal of Photochemistry
and Photobiology,
B: Biology, 5 (1990)
85 - 93
85
MERBROMIN (MERCUROCHROME) A PHOTOSENSITIZER FOR SINGLET OXYGEN REACTIONS KLAUS GOLLNICKT and STEPHAN HELD Znstitut fiir Organ&he (F.R.G.) (Received June 12,1989;
Chemie der Universitct Miinchen, Karl&r. 23, D-8000 Miinchen 2 accepted August 22, 1989)
Keywords. Merbromin, mercurochrome, photosensitizer, photodynamic effects.
singlet oxygen,
singlet oxygen
Summary Merbromin, produced in many countries and used world wide as an antiseptic under the trademark “mercurochrome”, is shown to be an efficient sensitizer for type II (singlet oxygen) photo-oxygenations by using 2-methyl-2-butene, (+)-limonene, (+)-a-pinene, a,a’-dimethylstilbenes and (-)-L-methionine as oxygen acceptors. Type I photo-oxygenations are negligible. An estimate of the quantum yield of singlet oxygen formation by merbromin in methanol gives a value of about 0.1.
1. Introduction Merbromin (MR), prepared by White [l] in 1925 by reaction of 2’,7’dibromofluorescein with mercuric acetate, is manufactured in many countries such as F.R.G. (MercurochromTM), France (CurichromeTM, MercurochromeTM), Italy (MercurocromoTM ) and U.S.A. (MercurochromeTM) and is used world wide as an efficient, inexpensive antiseptic.
MR: R, = H; R2 = HgOH; EO:R,=R,=Br,
TAuthor to whom correspondence loll-1344/90/$3.50
should be addressed. 0 Elsevier Sequoia/Printed
in The Netherlands
86
Of the many naturally occurring and artificial dyes, fluorescein derivatives, such as rose bengal (RB) and eosin (EO), are the longest-known artificial sensitizers of photodynamic effects and diseases caused by light in the presence of molecular oxygen [2]. These dyes are efficient photosensitizers for the generation of singlet oxygen ( ‘02) in so-called “type II photo-oxygenation reactions” [ 31 (f or a recent review of RB, see Neckers [ 41). It is mainly this activated oxygen species which, in photodynamic processes, damages proteins, cell membranes and nucleic acids by oxidizing certain amino acids (tryptophan, histidine, methionine, tyrosine, cysteine) in proteins, unsaturated fatty acids and cholesterol in cell membranes and guanine in nucleic acids. However, “type I photo-oxygenation reactions” [ 31 also seem to be responsible in some of the destruction processes (for reviews, see, for example, Spikes and MacKnight [ 51, Knowles [6], Gollnick [ 71, Foote [ 81 and Jori and Spikes [ 91). Because MR is closely related to EO, we may expect it to be a photosensitizer for ‘0, generation and, therefore, to be able to act as a photodynamic sensitizer. However, in spite of the widespread use of MR as an antiseptic, nothing with regard to its photosensitizing properties or its possible photodynamic side effects seems to be known. Such side effects could occur, for example, when wounds treated with MR are exposed to light and air. To the best of our knowledge (and to our surprise), there is only one report in the literature that MR may act as a photodynamic substance in trypsin inactivation [lo]. Therefore it seemed desirable to study the sensitizing properties of MR. In this paper, we report that MR sensitizes the photo-oxygenation of olefins (used as model compounds for lipids) and the photodynamically sensitive cx-amino-acid methionine in a type II process, i.e. via singlet oxygen. Type I processes, induced by hydrogen atom abstraction or electron transfer, are apparently negligible.
2. Materials and methods 2.1. Materials 2,5-Dimethylfuran (1) (Janssen), a-terpinene (4) (Aldrich), 2-methyl-2butene (6) (Aldrich), (+)-limonene (9) (EGA) and (+)-a-pinene (16) (Janssen) were distilled in a spinning band column in a nitrogen atmosphere before use. (-)-L-Methionine (23) (Merck) was recrystallized from ethanol, and cisand trans-a&-dimethylstilbenes (18a and 18b respectively) were synthesized by reductive coupling of acetophenone according to the method described by Lenoir [ll]. Fractional crystallization of the cis-trans mixture from ethanol yielded cis-a,a’-dimethylstilbene (18a, m.p. 64 “C) and tram-cy’dimethylstilbene (lab, m.p. 102 “C) [12]. RB (EGA), EO (EGA) and MR (Fluka) were purified by extraction with methanol. MercurochromTM, a 2% aqueous solution of merbromin, was purchased from Krewel. Acetonitrile (MeCN) (Merck) was distilled over P,O, and K&O, in sequence; methanol
87 (MeOH) (Fluka) and acetone (Fluka) were used w i t h o u t further purification. Distilled water was used to prepare the MeCN-H20 (9:1, v/v) and a c e t o n e H20 (9:1, v/v) mixtures.
2.2. Instrumentation UV spectra were obtained on a UV spectrometer (Zeiss RPQ 20c). 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker WP-80-CW spectrometer. Vapour phase chromatography analyses were performed on a Varian 1445 gas chromatograph equipped with a flame ionization detector. All products were identified by their IH NMR spectra. Products 3, 5, 17, 19 and 24 were isolated; their properties are in accord with those reported in the literature, e.g. 3: m.p. 79 - 81 °C; 81 °C [13]; 5: m.p. 3.5 °C; 4.2 °C [14]; 17: b.p. 59 - 60 °C (2 Torr); 208 - 209 °C (760 Torr) [15]; 19: m.p. 33 - 34 °C; 33 - 34 °C [12]; 24: m.p. 2 2 8 - 229 °C; 228 - 229 °C [16]. After reduction of the hydroperoxide mixture with sodium sulphite in m e t h a n o l - w a t e r , alcohols 10 - 15 were analysed on a polypropylene glass capillary column (50 m) and compared with authentic samples [17]. 2.3. Irradiation procedures Irradiations were carried o u t in an irradiation unit (25 ml) with automatic recording of oxygen consumption [18]. The irradiation unit, the oxygen burette and the tubing connecting the unit with the burette were kept at 13 + 0.1 °C by cooling with water (thermostat JULABO-P). A high pressure mercury lamp (Philips HP 125 W) was used as light source. A Schott glass filter (cut-off at 400 nm) was applied. The sensitizer concentrations were 3 X 10 -4 M in all solvents and solvent mixtures. The photo-oxygenation of methionine was carried out in acetone-water (1:1, v/v). For the MRsensitized oxygenation reactions in MeCN-water and acetone-water mixtures, MR and Mercurochrom TM solution, appropriately diluted with the organic solvents, were used. The initial concentrations of the substrates were 2.5 X 10 -2 M in all solvents. Various amounts of 1,4-diazabicyclo[2.2.2.]octane (DABCO) Cup to 2 X 10 -2 M) were added for quenching MR-sensitized photo-oxygenations of methionine.
3. Results and discussion The UV spectra of MR and EO in methanol (shown in Fig. 1) have similar band shapes and absorption maxima (MR: kmax = 512 nm, log e = 4.79; EO: kmax = 523 nm, log e = 4.90), indicating the close relationship between the two dyes. Figure 1 also contains the UV spectrum of RB (Xmax = 557 nm, log e = 4.97) for comparison. When oxygen-saturated methanolic solutions of 2,5-dimethylfuran (1) or a-terpinene (4) are irradiated in the presence of MR, EO or RB, one moleequivalent of oxygen is consumed in each case and cis-2,5-dimethyl-2hydroperoxy-5-methoxy-2,5-dihydrofuran (3) [19] and ascaridole (5) [20]
88
5
logr I L
3
2
1 10
I
I
I
I
I
I
15
20
25
30
3s
10
wave
45
runber~[~cm-g
Fig. 1. UV absorption spectra of merbromin, eosin and rose bengal in methanol: -, MR; ---, EO; . . . . , RB.
respectively are formed. Both reactions proceed as [ 4 + 21 cycloadditions of ‘0,. With furan 1, ozonide 2 is the primary product; in an immediate secondary reaction, 2 adds one molecule of methanol to yield cis-methoxyhydroperoxide 3 [ 131.
89
By using a cut-off filter at 400 nm and by applying MR and EO at concentrations of 3 X 10V4 M, an approximately equal number of photons should be absorbed by these dye solutions. Using this assumption and the fact that an initial concentration of 2.5 X 10v2 M of 1 causes the quantitative trapping of ‘0, in methanol until more than 95% of 1 is consumed [6,13], an estimate of the quantum yield of ‘02 production by MR ($(102)MR) in methanol can be made. Thus a ratio of ~zo/~Ma = 3 (where uzo and UMRare the rates of oxygen consumption in the presence of EO and MR respectively) indicates that $( ‘0,) MR should be approximately one-third of the singlet oxygen quantum yield obtained in the presence of EO. With a value of about 0.4 for the latter [21], $( ‘0,) MRshould be about 0.1. (Quantum yield determinations of triplet MR formation and of MR-sensitized singlet oxygen production are in progress; the results will be published later.) Because [4 + 21 cycloaddition of oxygen to cis-1,3-diene systems may also result from interactions of radical cations of 1,3-dienes with superoxide anions 02’ [22 - 251, the appearance of endoperoxides is not unambiguous proof of type II (singlet oxygen) oxygenation reactions. However, monoolefins that react with singlet oxygen to give products different from those obtained in type I (hydrogen-atom-abstraction induced or electron-transfer induced) processes may be used as reliable probes to distinguish between these two types of photo-oxygenation. Olefins such as 2-methyl-2-butene (6), (+)-limonene (9), (+)-a-pinene (16) and a$!-dimethylstilbenes (18a, 18b) can be used for this purpose.
CH3 (1)
H3C
A/
\
m3
mwz
_.J/H2
+
(2) reduction OH
9 a) sens/hv/O~
10
;
11 b) reduction
/. 6 OH
(1) MWW02 (2) reduction 16
b
17
JyH3
HO
90
Ph
Ph
CH3
NH2C
IPh
Ph
n
kH3
H3d
n
OOH 19
I
\_
cis:
18a
truns:
lgb
Ph H3C
cH3
H3C
22
21
20
--,-
TABLE 1 Photo-oxygenation
of (+)-limonene (9) in methanol at 13 “C
Sensitizer
(+)-IO
(+)-II
(+)-12
(+) -13
(-) -14
(-)-15
MR EO RB None ( ‘02 b, TPPC Noned
3Ba 38 34 41 39 17
9 9 10 9 12 18
18 18 20 18 20 0
21 21 21 22 20 0
9 9 10 7 7 34e
5 5 5 3 2 31e
aProduct distribution in per cent. b102 produced from 302 by microwave discharge [ 26 ] CTetraphenylporphin in CC14 [ 121. dAutoxidation [ 27 1. eRacemates of 14 and 15 are formed.
Irradiation of these olefins in the presence of oxygen and MR produces the ene products 7 and 8 (about l:l), 10 - 15 (Table 1) and 17 (more than 95%) from 6, 9 and 16 respectively, after mild reduction of the original hydroperoxides or hydroperoxide mixtures. Autoxidation of (+)-limonene (9) produces the tertiary alcohols 10 and 11 in a ratio of 1 :l, the racemic carveols 14 and 15 in a ratio of 1: 1 and no secondary alcohols with exocyclic double bonds (12 and 13), which can be clearly distinguished from the product distribution obtained with singlet oxygen generated by microwave discharge [26] or by sensitization with EO, RB or tetraphenylporphin (TPP) (Table 1). Singlet oxygen reacts with a-pinene (16) in an ene reaction to give truns-pinocarveol (17) [ 15, 281, whereas autoxidation, i.e. hydrogenabstraction-induced oxygenation of 16, leads to only about 15% of 17; 85% of other oxidation products are formed [ 211. Similarly, 9,10-dicyanoanthracene (DCA) photosensitizes the oxygenation of 16 to 17 to only about 20%; 80% of other oxidation products are obtained, which are assumed to be formed by an electron-transfer-induced process [ 291.
91
DCA is an efficient photosensitizer of singlet oxygen oxygenations in non-polar solvents and in polar solvents such as MeCN. However, in polar solvents it also acts as a photosensitizer of electron-transfer-induced oxygenation reactions [30 - 321. With cis-a+‘-dimethylstilbene (18a) and trunsar,a’-dimethylstilbene (18b) as substrates and DCA as sensitizer in MeCN, competition between the two modes of photo-oxygenation may be studied, yielding hydroperoxide 19 via the singlet oxygen pathway and products 20 22 via electron-transfer-induced oxygenation [ 121. However, photosensitization by MR leads to ene product 19 exclusively, as does photosensitization by EO or RB [12,18], indicating that only ‘0, is involved in the photooxygenation sensitized by these xanthene dyes.
Finally, the photodynamically sensitive a-amino-acid (-)L-methionine (23) was used as a substrate to distinguish between type I and type II photooxygenation processes. According to Sysak et al. [16], 23 reacts with IO, to give sulphoxide 24 in aqueous solution at a pH of less than 6, whereas loss of the carboxyl group and formation of methional occurs during type I photo-oxygenation [ 51. Irradiation of 23 in acetone-water (1: 1, v/v; pH 5.5) in the presence of oxygen and MR or EO produces sulphoxide 24 (yields of isolated 24, better than 90%). This again proves that MR is an efficient sensitizer for the generation of singlet oxygen, but a rather inefficient sensitizer for type I photo-oxygenations. In accord with this result, we found a decrease in the rate of oxygen uptake by 23 with increasing amounts of DABCO, a well-known singlet oxygen quencher [ 331. At the largest DABCO concentration used (2 X lop2 M), the rate of oxygen consumption was reduced to about one-fifth of that obtained in the absence of DABCO. However, it should be kept in mind that a decrease in oxygen consumption rate or rate of oxygenation product formation in the presence of DABCO is not proof of singlet oxygen quenching per se, because other mechanisms such as electron transfer between DABCO and electronically excited sensitizers may occur (for a discussion of the difficulties encountered in distinguishing between singlet oxygen and other oxygenation reactions, see Davidson et al. [ 341).
4. Conclusions The mercury-containing xanthene dye merbromin (MR) has been shown to be a photosensitizer for oxygenation reactions that occur via the
92
singlet oxygen pathway. Its photosensitizing efficiency is about one-third of that exhibited by the structurally closely related eosin (EO). With olefinic substrates and sulphides such as methionine, the efficiency of MR as a photosensitizer for type I oxygenation reactions (whether induced by hydrogen atom abstraction or by electron transfer) appears to be negligible. The results obtained so far lead us to expect that MR (and thus the commercially available antiseptic mercurochromeTM) will be a rather efficient photodynamic substance. References 1 E. C. White, Herstellung eines mercurierten Dibromfluoresceins, Chem. Zentralbl. ZZ,I (1925) 614. 2 H. F. Blum, Photodynamic Action and Diseases Caused by Light, Rhinehold, New York, 1941. 3 K. Gollnick, Type II photooxygenation reactions in solution, Adu. Photochem., 6 (1968) 1 - 122. 4 D. C. Neckers, Rose bengal, J. Photochem. PhotobioE., A, 47 (1989) 1 - 29. 5 J. D. Spikes and M. L. MacKnight, Photodynamic effects on molecules of biological importance: amino acids, peptides and proteins, in U. Gallo and L. Santamaria (eds.), Research Progress in Organic, Biological and Medicinal Chemistry, Vol. 3, NorthHolland, Amsterdam, 1972, Part I, pp. 124 - 136. 6 A. Knowles, The dye-sensitized degradation of nucleotides, in U. Gallo and L. Santamaria (eds.), Research Progress in Organic, Biological and Medicinal Chemistry, Vol. 3, North-Holland, Amsterdam, 1972, Part I, pp. 183 - 213. 7 K. Gollnick, Chemical aspects of photodynamic action in the presence of molecular oxygen, in 0. F. Nygaard, H. I. Adler and W. K. Sinclair (eds.), Radiation Research Biomedical, Chemical and Physical Properties, Academic Press, New York, 1975, pp. 590 - 611. 8 C. S. Foote, Photosensitized oxidation and singlet oxygen: consequences in biological systems, in W. A. Pryor (ed.), Free Radicals in Biology, Vol. III, Academic Press, New York, 1976, pp. 85 - 133. 9 G. Jori and J. D. Spikes, Photosensitized oxidations in complex biological structures, in M. A. J. Rodgers and E. L. Powers (eds.), Oxygen and Oxy-Radicals in Chemistry and Biology, Academic Press, New York, 1981, pp. 441 - 457. 10 J. D. Spikes, personal communication to L. Santamaria in L. Santamaria and G. Prino, List of the photodynamic substances, in U. Gallo and L. Santamaria (eds.), Research Progress in Organic, Biological and Medicinal Chemistry, Vol. 3, North-Holland, Amsterdam, 1972, Part I, pp. XI - XXXV. 11 D. Lenoir, Synthese tetrasubstituierter Ethylene durch reduktive Kupplung von Ketonen mittels Titan(II)-salzen. Zur Anwendung der Methode, Synthesis, (1977) 553 - 554. 12 K. Gollnick and A. Schnatterer, 9,10-Dicyanoanthracene-sensitized photooxygenation of o&r’-dimethylstilbenes. Mechanism and kinetics of the competing singlet oxygen and electron transfer photooxygenation reactions, Photochem. Photobiol., 43 (1986) 365 - 378. 13 K. Gollnick and A. Griesbeck, Singlet oxygen photooxygenation of furans: isolation and reactions of [ 4 + Blcycloaddition products (unsaturated sec.-ozonides), Tetrahedron, 41 (1985) 2057 - 2068. 14 G. 0. Schenck, K. G. Kinkel and H. J. Mertens, Uber die Photosynthese des Askaridols und verwandter Endoperoxyde, Liebigs Ann. Chem., 584 (1953) 125 155.
93
15 G. 0. Schenck, H. Eggert and W. Denk, Uber die Bildung von Hydroperoxyden bei photosensibilisierten Reaktionen von 0s mit geeigneten Akzeptoren, insbesondere mit 01-und fl-Pinen, Liebigs Ann. Chem., 584 (1953) 177 - 198. 16 P. K. Sysak, C. S. Foote and T. Y. Ching, Photooxygenation of methionine, Photothem. Photobiol., 26 (1977) 19 - 27. 17 G. 0. Schenck, K. Gollnick, G. Buchwald, S. Schroeter and G. Ohloff, Zur chemischen und sterischen Selektivitlt der photosensibilisierten Os-Ubertragung auf (+)-Limonen und (+)-Carvomenthen, Liebigs Ann. Chem., 674 (1964) 93 - 117. 18 H. R. Paur, Zur Photooxygenierung kernsubstituierter o,o’-Dimethylstilbene. Elektronische Effekte bei der En-Reaktion mit Singulett-Sauerstoff, Dissertation, University of Miinchen, 1982. 19 C. S. Foote, M. T. Wuesthoff, S. Wexler, I. G. Burstain, R. Denny, G. 0. Schenck and K. H. Schulte-Elte, Photosensitized oxygenation of alkyl-substituted furans, Z’etrahedron, 23 (1967) 2583 - 2599. 20 G. 0. Schenck and K. Ziegler, Die Synthese des Askaridols, Naturwissenschoften, 32 (1945) 157. 21 K. Gollnick and G. 0. Schenck, Mechanism and stereoselectivity of photosensitized oxygen transfer reactions, Pure Appl. Chem., 9 (1964) 507 - 525. 22 D. H. R. Barton, G. Leclerc, P. D. Magnus and I. D. Menzies, An unusual synthesis of ergosterol acetate peroxide, J. Chem. Sot., Chem. Commun., (1972) 447 - 449. 23 J. Eriksen, C. S. Foote and T. L. Parker, Photosensitized oxygenation of alkenes, J. Am. Chem. Sot., 99 (1977) 6455 - 6456. 24 R. K. Haynes, Lewis-acid-catalysed oxygenation of l,l-bicyclohexenyl and o-terpinene. Reactions in dichloromethane and liquid sulfur dioxide, Aust. J. Chem., 31 (1978) 131 - 138. 25 R. Tang, H. J. Yue, J. F. Wolf and F. Mares, Oxygenation of cyclic dienes to endoperoxides, J. Am. Chem. Sot., 100 (1978) 5248 - 5249. 26 K. Gollnick and G. Schade, Mikrowellenentladung von CO*: eine neue, ergiebige Quelle fur Singulett-Sauerstoff, 0s (lag), Tetrahedron Lett., (1973) 857 - 860. 27 G. 0. Schenck, 0. A. Neumiiller, S. Schroeter and G. Ohloff, Zur Autoxydation des (+)-Limonens, Liebigs Ann. Chem., 687 (1965) 26 - 39. 28 C. W. Jefford, A. F. Boschung, R. M. Moriarty, C. G. Rimbault and M. H. Laffer, The reaction of singlet oxygen with (Y- and /3-pinenes, Helv. Chim. Acta, 56 (1973) 2649 2659. 29 B.-W. Zhang, Y.-F. Ming and Y. Cao, Sensitized photooxidation of pinenes by 9,10dicyanoanthracene, Photochem. Photobiol., 40 (1984) 581 - 587. 30 D. S. Steichen and C. S. Foote, Indirect sensitized photooxygenation of aryl olefins, J. Am. Chem. Sot., 103 (1981) 1855 - 1857. 31 J. Santamaria, Photo-oxygenation de composes aromatiques sensibilisees par des accepteurs d’electron, Tetrahedron Lett., 22 (1981) 4511 - 4515. 32 A. Schnatterer, Dimethylstilbene als Sonde fur Singulett- Sauerstoff- und ElektronenTransfer-Photooxygenierungen, Diplomarbeit, University of Miinchen, 1982. 33 C. Ouannes and T. Wilson, Quenching of singlet oxygen by tertiary aliphatic amines. Effect of DABCO, J. Am. Chem. Sot., 90 (1968) 6527 - 6528. 34 R. S. Davidson, D. Goodwin and J. Pratt, Problems associated with distinguishing between singlet oxygen and electron transfer photooxygenation reactions, Free Rad. Res. Commun., 2 (1987) 313 - 320.
and Photobiology,
B: Biology, 5 (1990)
85 - 93
85
MERBROMIN (MERCUROCHROME) A PHOTOSENSITIZER FOR SINGLET OXYGEN REACTIONS KLAUS GOLLNICKT and STEPHAN HELD Znstitut fiir Organ&he (F.R.G.) (Received June 12,1989;
Chemie der Universitct Miinchen, Karl&r. 23, D-8000 Miinchen 2 accepted August 22, 1989)
Keywords. Merbromin, mercurochrome, photosensitizer, photodynamic effects.
singlet oxygen,
singlet oxygen
Summary Merbromin, produced in many countries and used world wide as an antiseptic under the trademark “mercurochrome”, is shown to be an efficient sensitizer for type II (singlet oxygen) photo-oxygenations by using 2-methyl-2-butene, (+)-limonene, (+)-a-pinene, a,a’-dimethylstilbenes and (-)-L-methionine as oxygen acceptors. Type I photo-oxygenations are negligible. An estimate of the quantum yield of singlet oxygen formation by merbromin in methanol gives a value of about 0.1.
1. Introduction Merbromin (MR), prepared by White [l] in 1925 by reaction of 2’,7’dibromofluorescein with mercuric acetate, is manufactured in many countries such as F.R.G. (MercurochromTM), France (CurichromeTM, MercurochromeTM), Italy (MercurocromoTM ) and U.S.A. (MercurochromeTM) and is used world wide as an efficient, inexpensive antiseptic.
MR: R, = H; R2 = HgOH; EO:R,=R,=Br,
TAuthor to whom correspondence loll-1344/90/$3.50
should be addressed. 0 Elsevier Sequoia/Printed
in The Netherlands
86
Of the many naturally occurring and artificial dyes, fluorescein derivatives, such as rose bengal (RB) and eosin (EO), are the longest-known artificial sensitizers of photodynamic effects and diseases caused by light in the presence of molecular oxygen [2]. These dyes are efficient photosensitizers for the generation of singlet oxygen ( ‘02) in so-called “type II photo-oxygenation reactions” [ 31 (f or a recent review of RB, see Neckers [ 41). It is mainly this activated oxygen species which, in photodynamic processes, damages proteins, cell membranes and nucleic acids by oxidizing certain amino acids (tryptophan, histidine, methionine, tyrosine, cysteine) in proteins, unsaturated fatty acids and cholesterol in cell membranes and guanine in nucleic acids. However, “type I photo-oxygenation reactions” [ 31 also seem to be responsible in some of the destruction processes (for reviews, see, for example, Spikes and MacKnight [ 51, Knowles [6], Gollnick [ 71, Foote [ 81 and Jori and Spikes [ 91). Because MR is closely related to EO, we may expect it to be a photosensitizer for ‘0, generation and, therefore, to be able to act as a photodynamic sensitizer. However, in spite of the widespread use of MR as an antiseptic, nothing with regard to its photosensitizing properties or its possible photodynamic side effects seems to be known. Such side effects could occur, for example, when wounds treated with MR are exposed to light and air. To the best of our knowledge (and to our surprise), there is only one report in the literature that MR may act as a photodynamic substance in trypsin inactivation [lo]. Therefore it seemed desirable to study the sensitizing properties of MR. In this paper, we report that MR sensitizes the photo-oxygenation of olefins (used as model compounds for lipids) and the photodynamically sensitive cx-amino-acid methionine in a type II process, i.e. via singlet oxygen. Type I processes, induced by hydrogen atom abstraction or electron transfer, are apparently negligible.
2. Materials and methods 2.1. Materials 2,5-Dimethylfuran (1) (Janssen), a-terpinene (4) (Aldrich), 2-methyl-2butene (6) (Aldrich), (+)-limonene (9) (EGA) and (+)-a-pinene (16) (Janssen) were distilled in a spinning band column in a nitrogen atmosphere before use. (-)-L-Methionine (23) (Merck) was recrystallized from ethanol, and cisand trans-a&-dimethylstilbenes (18a and 18b respectively) were synthesized by reductive coupling of acetophenone according to the method described by Lenoir [ll]. Fractional crystallization of the cis-trans mixture from ethanol yielded cis-a,a’-dimethylstilbene (18a, m.p. 64 “C) and tram-cy’dimethylstilbene (lab, m.p. 102 “C) [12]. RB (EGA), EO (EGA) and MR (Fluka) were purified by extraction with methanol. MercurochromTM, a 2% aqueous solution of merbromin, was purchased from Krewel. Acetonitrile (MeCN) (Merck) was distilled over P,O, and K&O, in sequence; methanol
87 (MeOH) (Fluka) and acetone (Fluka) were used w i t h o u t further purification. Distilled water was used to prepare the MeCN-H20 (9:1, v/v) and a c e t o n e H20 (9:1, v/v) mixtures.
2.2. Instrumentation UV spectra were obtained on a UV spectrometer (Zeiss RPQ 20c). 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker WP-80-CW spectrometer. Vapour phase chromatography analyses were performed on a Varian 1445 gas chromatograph equipped with a flame ionization detector. All products were identified by their IH NMR spectra. Products 3, 5, 17, 19 and 24 were isolated; their properties are in accord with those reported in the literature, e.g. 3: m.p. 79 - 81 °C; 81 °C [13]; 5: m.p. 3.5 °C; 4.2 °C [14]; 17: b.p. 59 - 60 °C (2 Torr); 208 - 209 °C (760 Torr) [15]; 19: m.p. 33 - 34 °C; 33 - 34 °C [12]; 24: m.p. 2 2 8 - 229 °C; 228 - 229 °C [16]. After reduction of the hydroperoxide mixture with sodium sulphite in m e t h a n o l - w a t e r , alcohols 10 - 15 were analysed on a polypropylene glass capillary column (50 m) and compared with authentic samples [17]. 2.3. Irradiation procedures Irradiations were carried o u t in an irradiation unit (25 ml) with automatic recording of oxygen consumption [18]. The irradiation unit, the oxygen burette and the tubing connecting the unit with the burette were kept at 13 + 0.1 °C by cooling with water (thermostat JULABO-P). A high pressure mercury lamp (Philips HP 125 W) was used as light source. A Schott glass filter (cut-off at 400 nm) was applied. The sensitizer concentrations were 3 X 10 -4 M in all solvents and solvent mixtures. The photo-oxygenation of methionine was carried out in acetone-water (1:1, v/v). For the MRsensitized oxygenation reactions in MeCN-water and acetone-water mixtures, MR and Mercurochrom TM solution, appropriately diluted with the organic solvents, were used. The initial concentrations of the substrates were 2.5 X 10 -2 M in all solvents. Various amounts of 1,4-diazabicyclo[2.2.2.]octane (DABCO) Cup to 2 X 10 -2 M) were added for quenching MR-sensitized photo-oxygenations of methionine.
3. Results and discussion The UV spectra of MR and EO in methanol (shown in Fig. 1) have similar band shapes and absorption maxima (MR: kmax = 512 nm, log e = 4.79; EO: kmax = 523 nm, log e = 4.90), indicating the close relationship between the two dyes. Figure 1 also contains the UV spectrum of RB (Xmax = 557 nm, log e = 4.97) for comparison. When oxygen-saturated methanolic solutions of 2,5-dimethylfuran (1) or a-terpinene (4) are irradiated in the presence of MR, EO or RB, one moleequivalent of oxygen is consumed in each case and cis-2,5-dimethyl-2hydroperoxy-5-methoxy-2,5-dihydrofuran (3) [19] and ascaridole (5) [20]
88
5
logr I L
3
2
1 10
I
I
I
I
I
I
15
20
25
30
3s
10
wave
45
runber~[~cm-g
Fig. 1. UV absorption spectra of merbromin, eosin and rose bengal in methanol: -, MR; ---, EO; . . . . , RB.
respectively are formed. Both reactions proceed as [ 4 + 21 cycloadditions of ‘0,. With furan 1, ozonide 2 is the primary product; in an immediate secondary reaction, 2 adds one molecule of methanol to yield cis-methoxyhydroperoxide 3 [ 131.
89
By using a cut-off filter at 400 nm and by applying MR and EO at concentrations of 3 X 10V4 M, an approximately equal number of photons should be absorbed by these dye solutions. Using this assumption and the fact that an initial concentration of 2.5 X 10v2 M of 1 causes the quantitative trapping of ‘0, in methanol until more than 95% of 1 is consumed [6,13], an estimate of the quantum yield of ‘02 production by MR ($(102)MR) in methanol can be made. Thus a ratio of ~zo/~Ma = 3 (where uzo and UMRare the rates of oxygen consumption in the presence of EO and MR respectively) indicates that $( ‘0,) MR should be approximately one-third of the singlet oxygen quantum yield obtained in the presence of EO. With a value of about 0.4 for the latter [21], $( ‘0,) MRshould be about 0.1. (Quantum yield determinations of triplet MR formation and of MR-sensitized singlet oxygen production are in progress; the results will be published later.) Because [4 + 21 cycloaddition of oxygen to cis-1,3-diene systems may also result from interactions of radical cations of 1,3-dienes with superoxide anions 02’ [22 - 251, the appearance of endoperoxides is not unambiguous proof of type II (singlet oxygen) oxygenation reactions. However, monoolefins that react with singlet oxygen to give products different from those obtained in type I (hydrogen-atom-abstraction induced or electron-transfer induced) processes may be used as reliable probes to distinguish between these two types of photo-oxygenation. Olefins such as 2-methyl-2-butene (6), (+)-limonene (9), (+)-a-pinene (16) and a$!-dimethylstilbenes (18a, 18b) can be used for this purpose.
CH3 (1)
H3C
A/
\
m3
mwz
_.J/H2
+
(2) reduction OH
9 a) sens/hv/O~
10
;
11 b) reduction
/. 6 OH
(1) MWW02 (2) reduction 16
b
17
JyH3
HO
90
Ph
Ph
CH3
NH2C
IPh
Ph
n
kH3
H3d
n
OOH 19
I
\_
cis:
18a
truns:
lgb
Ph H3C
cH3
H3C
22
21
20
--,-
TABLE 1 Photo-oxygenation
of (+)-limonene (9) in methanol at 13 “C
Sensitizer
(+)-IO
(+)-II
(+)-12
(+) -13
(-) -14
(-)-15
MR EO RB None ( ‘02 b, TPPC Noned
3Ba 38 34 41 39 17
9 9 10 9 12 18
18 18 20 18 20 0
21 21 21 22 20 0
9 9 10 7 7 34e
5 5 5 3 2 31e
aProduct distribution in per cent. b102 produced from 302 by microwave discharge [ 26 ] CTetraphenylporphin in CC14 [ 121. dAutoxidation [ 27 1. eRacemates of 14 and 15 are formed.
Irradiation of these olefins in the presence of oxygen and MR produces the ene products 7 and 8 (about l:l), 10 - 15 (Table 1) and 17 (more than 95%) from 6, 9 and 16 respectively, after mild reduction of the original hydroperoxides or hydroperoxide mixtures. Autoxidation of (+)-limonene (9) produces the tertiary alcohols 10 and 11 in a ratio of 1 :l, the racemic carveols 14 and 15 in a ratio of 1: 1 and no secondary alcohols with exocyclic double bonds (12 and 13), which can be clearly distinguished from the product distribution obtained with singlet oxygen generated by microwave discharge [26] or by sensitization with EO, RB or tetraphenylporphin (TPP) (Table 1). Singlet oxygen reacts with a-pinene (16) in an ene reaction to give truns-pinocarveol (17) [ 15, 281, whereas autoxidation, i.e. hydrogenabstraction-induced oxygenation of 16, leads to only about 15% of 17; 85% of other oxidation products are formed [ 211. Similarly, 9,10-dicyanoanthracene (DCA) photosensitizes the oxygenation of 16 to 17 to only about 20%; 80% of other oxidation products are obtained, which are assumed to be formed by an electron-transfer-induced process [ 291.
91
DCA is an efficient photosensitizer of singlet oxygen oxygenations in non-polar solvents and in polar solvents such as MeCN. However, in polar solvents it also acts as a photosensitizer of electron-transfer-induced oxygenation reactions [30 - 321. With cis-a+‘-dimethylstilbene (18a) and trunsar,a’-dimethylstilbene (18b) as substrates and DCA as sensitizer in MeCN, competition between the two modes of photo-oxygenation may be studied, yielding hydroperoxide 19 via the singlet oxygen pathway and products 20 22 via electron-transfer-induced oxygenation [ 121. However, photosensitization by MR leads to ene product 19 exclusively, as does photosensitization by EO or RB [12,18], indicating that only ‘0, is involved in the photooxygenation sensitized by these xanthene dyes.
Finally, the photodynamically sensitive a-amino-acid (-)L-methionine (23) was used as a substrate to distinguish between type I and type II photooxygenation processes. According to Sysak et al. [16], 23 reacts with IO, to give sulphoxide 24 in aqueous solution at a pH of less than 6, whereas loss of the carboxyl group and formation of methional occurs during type I photo-oxygenation [ 51. Irradiation of 23 in acetone-water (1: 1, v/v; pH 5.5) in the presence of oxygen and MR or EO produces sulphoxide 24 (yields of isolated 24, better than 90%). This again proves that MR is an efficient sensitizer for the generation of singlet oxygen, but a rather inefficient sensitizer for type I photo-oxygenations. In accord with this result, we found a decrease in the rate of oxygen uptake by 23 with increasing amounts of DABCO, a well-known singlet oxygen quencher [ 331. At the largest DABCO concentration used (2 X lop2 M), the rate of oxygen consumption was reduced to about one-fifth of that obtained in the absence of DABCO. However, it should be kept in mind that a decrease in oxygen consumption rate or rate of oxygenation product formation in the presence of DABCO is not proof of singlet oxygen quenching per se, because other mechanisms such as electron transfer between DABCO and electronically excited sensitizers may occur (for a discussion of the difficulties encountered in distinguishing between singlet oxygen and other oxygenation reactions, see Davidson et al. [ 341).
4. Conclusions The mercury-containing xanthene dye merbromin (MR) has been shown to be a photosensitizer for oxygenation reactions that occur via the
92
singlet oxygen pathway. Its photosensitizing efficiency is about one-third of that exhibited by the structurally closely related eosin (EO). With olefinic substrates and sulphides such as methionine, the efficiency of MR as a photosensitizer for type I oxygenation reactions (whether induced by hydrogen atom abstraction or by electron transfer) appears to be negligible. The results obtained so far lead us to expect that MR (and thus the commercially available antiseptic mercurochromeTM) will be a rather efficient photodynamic substance. References 1 E. C. White, Herstellung eines mercurierten Dibromfluoresceins, Chem. Zentralbl. ZZ,I (1925) 614. 2 H. F. Blum, Photodynamic Action and Diseases Caused by Light, Rhinehold, New York, 1941. 3 K. Gollnick, Type II photooxygenation reactions in solution, Adu. Photochem., 6 (1968) 1 - 122. 4 D. C. Neckers, Rose bengal, J. Photochem. PhotobioE., A, 47 (1989) 1 - 29. 5 J. D. Spikes and M. L. MacKnight, Photodynamic effects on molecules of biological importance: amino acids, peptides and proteins, in U. Gallo and L. Santamaria (eds.), Research Progress in Organic, Biological and Medicinal Chemistry, Vol. 3, NorthHolland, Amsterdam, 1972, Part I, pp. 124 - 136. 6 A. Knowles, The dye-sensitized degradation of nucleotides, in U. Gallo and L. Santamaria (eds.), Research Progress in Organic, Biological and Medicinal Chemistry, Vol. 3, North-Holland, Amsterdam, 1972, Part I, pp. 183 - 213. 7 K. Gollnick, Chemical aspects of photodynamic action in the presence of molecular oxygen, in 0. F. Nygaard, H. I. Adler and W. K. Sinclair (eds.), Radiation Research Biomedical, Chemical and Physical Properties, Academic Press, New York, 1975, pp. 590 - 611. 8 C. S. Foote, Photosensitized oxidation and singlet oxygen: consequences in biological systems, in W. A. Pryor (ed.), Free Radicals in Biology, Vol. III, Academic Press, New York, 1976, pp. 85 - 133. 9 G. Jori and J. D. Spikes, Photosensitized oxidations in complex biological structures, in M. A. J. Rodgers and E. L. Powers (eds.), Oxygen and Oxy-Radicals in Chemistry and Biology, Academic Press, New York, 1981, pp. 441 - 457. 10 J. D. Spikes, personal communication to L. Santamaria in L. Santamaria and G. Prino, List of the photodynamic substances, in U. Gallo and L. Santamaria (eds.), Research Progress in Organic, Biological and Medicinal Chemistry, Vol. 3, North-Holland, Amsterdam, 1972, Part I, pp. XI - XXXV. 11 D. Lenoir, Synthese tetrasubstituierter Ethylene durch reduktive Kupplung von Ketonen mittels Titan(II)-salzen. Zur Anwendung der Methode, Synthesis, (1977) 553 - 554. 12 K. Gollnick and A. Schnatterer, 9,10-Dicyanoanthracene-sensitized photooxygenation of o&r’-dimethylstilbenes. Mechanism and kinetics of the competing singlet oxygen and electron transfer photooxygenation reactions, Photochem. Photobiol., 43 (1986) 365 - 378. 13 K. Gollnick and A. Griesbeck, Singlet oxygen photooxygenation of furans: isolation and reactions of [ 4 + Blcycloaddition products (unsaturated sec.-ozonides), Tetrahedron, 41 (1985) 2057 - 2068. 14 G. 0. Schenck, K. G. Kinkel and H. J. Mertens, Uber die Photosynthese des Askaridols und verwandter Endoperoxyde, Liebigs Ann. Chem., 584 (1953) 125 155.
93
15 G. 0. Schenck, H. Eggert and W. Denk, Uber die Bildung von Hydroperoxyden bei photosensibilisierten Reaktionen von 0s mit geeigneten Akzeptoren, insbesondere mit 01-und fl-Pinen, Liebigs Ann. Chem., 584 (1953) 177 - 198. 16 P. K. Sysak, C. S. Foote and T. Y. Ching, Photooxygenation of methionine, Photothem. Photobiol., 26 (1977) 19 - 27. 17 G. 0. Schenck, K. Gollnick, G. Buchwald, S. Schroeter and G. Ohloff, Zur chemischen und sterischen Selektivitlt der photosensibilisierten Os-Ubertragung auf (+)-Limonen und (+)-Carvomenthen, Liebigs Ann. Chem., 674 (1964) 93 - 117. 18 H. R. Paur, Zur Photooxygenierung kernsubstituierter o,o’-Dimethylstilbene. Elektronische Effekte bei der En-Reaktion mit Singulett-Sauerstoff, Dissertation, University of Miinchen, 1982. 19 C. S. Foote, M. T. Wuesthoff, S. Wexler, I. G. Burstain, R. Denny, G. 0. Schenck and K. H. Schulte-Elte, Photosensitized oxygenation of alkyl-substituted furans, Z’etrahedron, 23 (1967) 2583 - 2599. 20 G. 0. Schenck and K. Ziegler, Die Synthese des Askaridols, Naturwissenschoften, 32 (1945) 157. 21 K. Gollnick and G. 0. Schenck, Mechanism and stereoselectivity of photosensitized oxygen transfer reactions, Pure Appl. Chem., 9 (1964) 507 - 525. 22 D. H. R. Barton, G. Leclerc, P. D. Magnus and I. D. Menzies, An unusual synthesis of ergosterol acetate peroxide, J. Chem. Sot., Chem. Commun., (1972) 447 - 449. 23 J. Eriksen, C. S. Foote and T. L. Parker, Photosensitized oxygenation of alkenes, J. Am. Chem. Sot., 99 (1977) 6455 - 6456. 24 R. K. Haynes, Lewis-acid-catalysed oxygenation of l,l-bicyclohexenyl and o-terpinene. Reactions in dichloromethane and liquid sulfur dioxide, Aust. J. Chem., 31 (1978) 131 - 138. 25 R. Tang, H. J. Yue, J. F. Wolf and F. Mares, Oxygenation of cyclic dienes to endoperoxides, J. Am. Chem. Sot., 100 (1978) 5248 - 5249. 26 K. Gollnick and G. Schade, Mikrowellenentladung von CO*: eine neue, ergiebige Quelle fur Singulett-Sauerstoff, 0s (lag), Tetrahedron Lett., (1973) 857 - 860. 27 G. 0. Schenck, 0. A. Neumiiller, S. Schroeter and G. Ohloff, Zur Autoxydation des (+)-Limonens, Liebigs Ann. Chem., 687 (1965) 26 - 39. 28 C. W. Jefford, A. F. Boschung, R. M. Moriarty, C. G. Rimbault and M. H. Laffer, The reaction of singlet oxygen with (Y- and /3-pinenes, Helv. Chim. Acta, 56 (1973) 2649 2659. 29 B.-W. Zhang, Y.-F. Ming and Y. Cao, Sensitized photooxidation of pinenes by 9,10dicyanoanthracene, Photochem. Photobiol., 40 (1984) 581 - 587. 30 D. S. Steichen and C. S. Foote, Indirect sensitized photooxygenation of aryl olefins, J. Am. Chem. Sot., 103 (1981) 1855 - 1857. 31 J. Santamaria, Photo-oxygenation de composes aromatiques sensibilisees par des accepteurs d’electron, Tetrahedron Lett., 22 (1981) 4511 - 4515. 32 A. Schnatterer, Dimethylstilbene als Sonde fur Singulett- Sauerstoff- und ElektronenTransfer-Photooxygenierungen, Diplomarbeit, University of Miinchen, 1982. 33 C. Ouannes and T. Wilson, Quenching of singlet oxygen by tertiary aliphatic amines. Effect of DABCO, J. Am. Chem. Sot., 90 (1968) 6527 - 6528. 34 R. S. Davidson, D. Goodwin and J. Pratt, Problems associated with distinguishing between singlet oxygen and electron transfer photooxygenation reactions, Free Rad. Res. Commun., 2 (1987) 313 - 320.
