Phorochrmisrrj and Pholohrolug?.. 1976, Vol. 24. pp. 29-40,

Pergamon Press. Printed in Great Britain

PHOTOOXIDATION OF XANTHOBILIRUBIC ACID IN AQUEOUS SOLUTION: PRODUCT AND MECHANISM STUDIES J. 0. GRUNEWALD,J. C. WALKERand E. R. STROPE Department of Biochemistry, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, KS 66103, U.S.A. (Receioed 17 July 1975;accepted 27 February 1976)

Abstract-Xanthobilirubic acid, an oxodipyrrylmethene with a chromophore very similar to that of bilirubin, was aerobically irradiated as its sodium salt in borate buffer at pH 8. Two photooxidation acid, were isolated. products, methylethylmaleimideand 5-formyl-2,4-dimethy1-1H-pyrrole-3-propanoic These products can be rationalized on the basis of either a Type I or a Type I1 (singlet oxygen) photooxygenation mechanism. The reaction is inhibited by azide, a singlet oxygen quencher, and sensit-

ized by methylene blue. However, both the self-sensitized and the methylene blue-sensitized reactions are enhanced, rather than inhibited, by high concentrations of l,Cdiazabicyclo[2.2.2]octane, another known singlet oxygen quencher. It is therefore proposed that the diazabicyclooctane can also act as an electron donor. The self-sensitized photooxidation of xanthobilirubic acid is an autocatalytic reaction. This is supported by the fact that addition of a previously irradiated solution to a freshly-prepared solution significantly increases the rate of photodegradation. A similar catalyst is formed, but much more slowly, from xanthobilirubic acid in the dark in the presence of oxygen.

McDonagh (1971) predicted that the dipyrroledicarboxaldehyde isolated by Bonnett and Stewart (1975) should be a photooxidation product of bilirubin, based on results of mechanistic studies in chloroform which implicated singlet oxygen. Analogous aldehydes have been proposed as intermediates in the formation of products isolated from either direct or dye-sensitized photooxidations of dipyrrylmethenes in methanol (Lightner and Quistad, 1972a; Lightner and Crandall, 1973). However, until our preliminary report (Grunewald and Strope, 1973a), no such compound had been isolated and characterized. The currently favored mechanism for bilirubin photooxidation in organic solvents is that in which bilirubin sensitizes the production of singlet oxygen, and this reactive oxygen then adds to sites of unsaturation in the tetrapyrrole. Results in support of this mechanism are: (1) the observed accelerated rate in perdeuterated methanol or in the presence of singlet oxygen sensitizers, methylene blue or Rose Bengal; (2) partial inhibition of the reaction by singlet oxygen quenchers, DABCO and all-trans-b-carotene; (3)competitive inhibition in the presence of singlet oxygen traps, 2,3-dimethyl-2-butene and 2,5-dimethylfuran; (4) lack of inhibition by a radical inhibitor, 26-di-t-butylphenol (McDonagh, 1971;Bonnett and Stewart, 1972% 1975) and ( 5 ) the types of products isolated (Bonnett and Stewart, 1972b, 1975; Lightner and Quistad, 1972% 1972b, 1972~).Bonnett and Stewart (1972b, 1975) have stated that the course of the reaction is highly dependent on solvent, however, and that in purified chloroform, a Type I process leading to biliverdins may be important.

INTRODUCTION

In view of the recent report (Bonnett and Stewart, 1975) of the isolation of a dipyrroldicarboxaldehyde as a minor photooxidation product of bilirubin (2) in aqueous solution, we wish' to report in detail our findings (Grunewald and Strope, 1973a) of a pyrrolecarboxaldehyde as an aqueous photooxidation product of xanthobilirubic acid (I), and results of related mechanistic studies. This compound (5-[(1,5-dihydro3-ethyl-l-methyl-5-oxo-2H-pyrrole-2-ylidene)me~yl] 2,4-dimethyl-lH-pyrrole-3-propanoic acid) has essentially the same chromophore as bilirubin, but is much more stable in the dark in aqueous buffered solution near physiological pH (McDonagh and Assisi, 1972; J. 0.Grunewald and A. E. Munhambo, unpublished). Its carboxylic acid sidecchain provides the necessary solubility to allow studies in weakly alkaline medium, in contrast to other dipyrrylmethenes which have been used as models for bilirubin in organic solvents (Lightner and Quistad, 1972a; Lightner and Crandall, 1973).

(1)

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30

J. 0. GRUNEWALD, J. C. WALKERand E. R. Smope

The two photooxidation products of xanthobilirubic acid, methylethylmaleimide (3) and 5-formy172,4dimethyl- 1H-pyrrole-3-propanoic acid (4), could also be rationalized from mechanistic reasoning involving photosensitized singlet oxygen production by XBA. However, the results of mechanistic studies presented here and in part elsewhere (Walker a n d Grunewald 1975) indicate that the photooxidation of xanthobilirubic acid in aqueous buffered solution does not proceed exclusively by a singlet oxygen (Type 11) mechanism (Gollnick, 1968). MATERIALS AND METHODS

UV-visible spectra were run on a Cary Model 14 recording spectrophotometer. IR spectra on a Beckman Model IR-8 grating spectrophotometer, nuclear magnetic resonance (NMR) spectra on a Varian Model T-60 specrrometer with tetramethylsilane as internal standard and mass spectra on a Varian Atlas CH-5 spectrometer. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Materials. Xanthobilirubic acid was synthesized by the method of Grunewald et al. (1975) and was pure according to NMR, IR and UV spectral criteria and thin layer chromatography (Silica gel F, Woelm, 0.25mm. 10% acetic acid/chloroform). The LX (borate buffer, pH 8) was 410 nm, E 33,000. 1,4-Diazabicyclo[2.2.2]octane (DABCO, triethylenediamine, Eastman) was purified by sublimation in uacuo. Methylene blue was recrystallized from absolute ethanol. Sodium azide (Sigma) was used as received. Borate buffer was prepared as directed in Dawson et al. 41969). All other reagents and solvents were analytical reagent grade and used as received unless otherwise specified. All aqueous solutions were prepared using boiled, deionized water. Silica gel G (Merck, with 2% green fluorescent indicator, Woelm) was used for preparative thin-layer chromatography (TLC) [l mm thickness, prepared from 50 g silica gel in 100 mP water, dried at 100°C for 1.5 h and stored in a desiccator over Drierite]. Irradiations. The irradiation apparatus used in all mechanistic experiments except those employing filter solutions or methylene blue consisted of an Ace Glass photochemical reaction vessel (Cat. No. 6515-03 modified to contain 250mt) and a water-jacketed Pyrex immersion well (Ace Glass Cat. No. 6517-05), with a 450W Hanovia medium pressure quartz-mercury vapor arc as the light source. For preparative-scale irradiations, a 400 md reaction vessel was used. A Pyrex filter sleeve was inserted between the lamp and immersion well. Calculations based on the manufacturers’ specifications of spectral energy distribution for the light source and the transmission characteristics of the Pyrex filter show that 99.9% of the energy incident on the solution being irradiated is due to wavelengths greater than 300nm. Ice water was circulated through the jacket of the immersion well to cool the lamp and maintain the temperature of the solution being irradiated at 10-14”C Compressed air (passed through Ascarite to remove carbon dioxide) or nitrogen was bubbled through the solution throughout the irradiation period. The pH of all solutions was 8.0 f 0.1, measured before lamp ignition on a Leeds and Northrup pH meter. During irradiation, samples were withdrawn at intervals and, after appropriate dilution, their UV-visible spectra were recorded. All xanthobilirubic acid solutions were prepared and handled in subdued light. Preparative irradiations. For preparative-scale irradiations of freshly-prepared solutions, 50 mg of xanthobilirubic acid was dissolved in 19.6 n-d of 0.1 N sodium hydroxide and 200n-d of a solution 0.1 M in both boric acid and potassium chloride was immediately added and the

mixture diluted to 400 r d with water. The solutions were aerobically irradiated (2.5 h) immediately after preparation. One preparative-scale irradiation was done using filter solution I to absorb light below 330nm, as described below. ‘Aged‘ solutions were made up similarly, using 110mg of 1/400&, and stored in the dark at 5°C for two days before aerobic irradiation (1.25 h) and photoproduct isolation. Mechanistic studies. Solutions in borate buffer were prepared by dissolving the appropriate amount of xanthobilirubic acid in 12.25 m t of 0.1 N sodium hydroxide, 125 mt of a solution 0.1 M with respect to both boric acid and potassium chloride was added and then the solution brought to 250 mt with water. Unbuffered solutions were prepared by dissolving xanthobilirubic acid in 1 mt of 0.1 N sodium hydroxide, diluting to approximately 200 mP with 0.055 M potassium chloride, acidifying to pH 8 with approximately 21-d of 1% hydrochloric acid and then diluting to 250 mt with potassium chloride solution. This procedure required 20 min. Borate solutions of xanthobilirubic acid designated as ‘fresh@-prepared‘ were allowed to stand in the reaction vessel for 30 min prior to lamp ignition, with air bubbling through the solution for the last 10 min before irradiation. Unbuffered ‘freshly-prepared‘ solutions were purged with air for 10 min before irradiation, making a total of 30 min from dissolution to irradiation. Solutions designated as ‘aged‘ were kept in the dark at 5°C for the times specified in the text. ‘Irradiated freshly-prepared’ solutions were made up and immediately irradiated until the absorbance at 410 nm did not decrease significantly on further irradiation, and either used immediately or stored in the dark at 5°C. ‘Irradiated aged’ solutions were kept in the dark at 5°C for the specified times before irradiation to minimum absorbance at 410nm. Chemical light filters were prepared as follows: filter solution I was made by dissolving 722.2 g sodium bromide and 4.5 g lead nitrate in 1.5 / of water (Schonberg, 1968); filter solution I1 was prepared using 20mg of potassium chromate in 250 mt of 0.05 N potassium hydroxide (Davies and Prue, 1955). In experiments employing solution I only, the filter was circulated through the immersion well jacket (20.65 cm pathlength). Under these conditions, no light was transmitted at wavelengths < 330 nm (calculated on the basis of the absorbance of the filter solution measured on the Cary 14 Spectrophotometer in a 0.1 cm cell). The temperature of the solution being irradiated was maintained at 5-10°C by cooling the beaker containing the filter solution in ice and by adding dry ice to the filter solution itself. The transmission characteristics of the filter solution were not changed by dry ice addition or irradiation. When both filter solutions I and I1 were used, filter I was circulated a8 described above, and filter I1 was placed in the reaction vessel (- 12 mm pathlength). Based on the absorbance of the two solutions in series (10 mm path each) on the Cary 14 Spectrophotometer, no light below 395nm was transmitted. The xanthobilirubic acid solution being irradiated (20 d, 0.24 M) was contained in a flat-sided culture tube (Pyrex 9200, 152mm long) which was taped to were the outer flask containing filter 11. Samples (1.5 d) withdrawn every 15 min and analyzed as described above. Xanthobilirubic acid solutions containing DABCO or sodium azide were prepared using the standard procedure, but with addition of a weighed amount of DABCO or sodium azide or the appropriate volume of a stock solution (0.01 M) of DABCO in boiled deionized water before dilution to 250 mt. For the methylene blue photosensitized oxidations, solutions were prepared by diluting 20 d of a nitrogen-saturated stock solution of xanthobilirubic acid in borate buffer (0.15 mM, prepared as described above) to give l 0 0 d with a final concentration of 30phf. To this was added 40 p! of an aqueous solution which was 40 pM in methylene blue, and, in the DABCO experiments,

Photooxidation of xanthobilirubic acid

31

the appropriate weighed amount of DABCO. Solutions 31 3 nm (c 20,000), sh 275 nm; IR (KBr, cm- ') 2200-3550 were irradiated at 18-20°C in 25 x 300mm test tubes (broad, COOH), 3250 (NH), 1698 (acid C==O),1598 (aldewhich were taped to the outside of the 250 m! photochemi- hyde G O ) , 1265 (C-0); NMR (DMSO-d6) 6 11.20 (br, cal reaction vessel. Mixing was accomplished by bubbling 2H, exchangeable with D 2 0 , N H and COOB), 9.25 (s, air or nitrogen through the solutions. In all of the methyl- 1 H, not exchangeable with D,O, &-C==O), 2.30-2.67 (m, ene blue experiments, a nitrogen-saturated solution of 1 4H, CB,C&COOH) and 2.26, 2.19 (2s, 6H, two (0.84 mM) was used in the large reaction vessel as a light ring-CH,); Molecular weight (mass spectrum) 195.08835 filter (- 12 mm pathlength), so that in the reaction solution, (Calc. for CloH,3 N 0 3 195.08946). This photoproduct was only methylene blue was absorbing light. Samples (4 d) characterized as 5-formyl-2,4-dimethy1-1H-pyrrole-3-prowere withdrawn every 15 min during irradiation and their panoic acid (4) on the basis of the identity of its IR, NMR, UV-visible spectra determined. UV and mass spectra and its TLC behavior with those Products of the methylene blue photosensitized oxi- of an authentic synthetic sample, prepared by hydrogenodation of xanthobilirubic acid were isolated following the lysis and decarboxylation of benzyl4-(2-methoxy-carbonylgeneral procedure described below from two experiments ethyl)-3,5-dimethyl-lH-pyrrole-2-carboxylate(Hayes et al., using 100 m4 of a 0.5 mM solution of 1 in borate buffer, 1958) and formylation according to the method of Chong with methylene blue at 2C-30 nM. et al. (1969), followed by basic hydrolysis of the methyl Product isolation from irradiation of 'aged' xanthobilirubic propanoate. acid solutions. A solution of 110 mg xanthobilirubic acid A sample (40 mg) of the crude photoaldehyde from prein 4 0 0 d borate buffer was stored in the dark at 5°C parative TLC was esterified by refluxing for 10 min with for two days and then irradiated for 1.25 h. It was extracted 5 m ! of 14% boron trifluoride in methanol. Chloroform with eight 100 m! portions of chloroform, the organic layer (20 rm") and water (80 d ) were added and the layers separfiltered through phase-separating paper (Whatman No. ated. The aqueous phase was extracted with three 20 mt IPS)and concentrated in uucuo to yield 31 mg of residue. portions of chloroform, and the combined chloroform This was combined with 36mg of similar residue from a extracts concentrated in uacuo to give 27 mg of a redsecond irradiation and extracted with 6 0 0 d of boiling brown residue, which was extracted with 75 m4 hexane in hexane in 25 m! portions, and the hexane-soluble material 25 mt portions. The hexane-soluble material (9.6 mg, dmax (44 mg) subjected to preparative TLC (10% acetic acid- (MeOH) 312 nm, sh 275 nm) was further purified by prechloroform). The band at R , 0.67 (visualized using a parative TLC (silica gel F, 1 mm, benzenejabsolute ethanol, Mineralight lamp) was eluted from the silica gel with 1OO:lO) and the material at R, 0.3-0.5 eluted with methchloroform (1-8.6mg, mp 63-66°C). This was combined anol and crystallized from hexane to give a solid with mp with identical material from several irradiations and sub- 108-115°C and UV, IR and NMR spectra identical to limed (5S°C/17 mm) to give white crystals, mp 66.S-67.8"C. those of authenticmethyl 5-formyl-2,4-dimethyI-1H-pyrroleRecrystallization from distilled petroleum ether (bp 3-propanoate, synthesized as described above. Further re4 W C ) gave a colorless crystalline solid, mp 67.5-68.5"C, crystallization from methanol gave crystals, mp 1 2 6 which was identical in all respects (mp, spectral properties, 129.5"C, UV (MeOH) 312 nm (c 20,700), which had identiTLC behavior) to authentic methylethylmaleimide(3),pre- cal TLC behavior (R, 0.38, silica gel F, benzene/ethanol, pared from methylethylmaleic anhydride (Schreiber and 100:lO) to that of the authentic methyl ester. Vermuth, 1965) by the method of Muir and Neuberger Products from irradiation of freshly-prepared solutions (1949). Spectral data: UV (MeOH) 221 nm (G 18,100) and of xanthobilirubic acid were isolated as described above, 226 nm (E 17,300); IR (CHC13,cm-I) 3455 (N-H), 3020 in similar yields. (C-H), 1772 ( w ) and 1725 (s) (imide -0); WMR (CCl,) 6 8.57 (br, s, 1 H, NH), 2.38 (4,J = 7.5 Hz, 2 H, Cl€,CH,), RESULTS 1.94 (s, 3 H, ring-C&), 1.13 (t, J = 7.5 Hz, 3 H, CH,C&); Mass spectrum (70eV, 1 9 T ) m/e 139 (M'., loOo/, 124 Photoproduct isolation. Typical UV-visible spectra (46%), 67 (62%). The basic aqueous solution from which the maleimide obtained during a preparative scale (50 mg/400mt) had been extracted was acidified (10% HC1) to pH 3 and aerobic irradiation of a freshly-prepared solution of the water removed in uucuo at 25-30°C. The residual solid was washed with eight 50 mP portions of chloroform; the xanthobilirubic acid in borate buffer (pH 8, A = 0.055) mixture was suction-filteredbetween washings. The chloro- are shown in Fig. 1. Photodecay was complete in 2.5 h; form solution was filtered through phase-separating paper under the same conditions in the dark, there were (Whatman No, 1PS) and concentrated in uacuo to yield n o observable spectral changes over a t least 6.5h. 70 mg of residue. This was combined with a similar residue During irradiation, as the absorbance of the starting (72 mg) from a second irradiation and the mixture treated with methanol. A bright yellow methanol-insoluble solid material (410 nm) decreased, there were increases in (5.5 mg) was identified as mesobilirubin XIII-cr by its visible absorbance a t 303-315 nm and 21&230 nm. The peak and mass spectral properties and TLC comparison with a t 315 nm decreased in intensity during the last part an authentic sample synthesized by the method of Fischer of the irradiation, and this decrease was not prevented and Adler (1931). The methanol-soluble material (136 mg) was chromatographed on four 20 x 20cm preparative by filtering out light below 330nm using a chemical TLC plates and the major bands at R , 0.45 and 0.31 eluted filter solution. When an 'aged' solution (2 days, 5"C, from the silica gel with chloroform and methanol. dark) of xanthobilirubic acid in borate buffer was The photoproduct with R, 0.45 (~, (MeOH) 313nm, irradiated, spectral changes were qualitatively similar sh 275 nm) from four irradiations (60 me) was further puri- to those shown in Fig. 1, but the rate of photodecay fied by partition chromatography on a microcrystalline ceflulose (Biorad CeIlex MX)'column (13 x 470 mm). The was much enhanced. Photoproducts were isolated from both freshlycolumn was eluted with 100mt hexane, 100 mP 5% chlorolorm/hexane, 100 mt 10% chloroform/hexane, 1850 m& prepared and aged solutions in similar yields. From 25% chloroform/hexane, 850 mG 50% chloroform/hexane, a typical irradiation of an aged (2 days) solution 1500 mP 75% chloroform/hexane, 250 mP chloroform and 500 d methanol. Material which eluted with the last three (110 mg 1/400 mt borate buffer) there was obtained solvents was combined (41 mg) and crystallized from water a 20% yield (based on molar ratios) of methylethylto give light tan crystals, mp 148.5-151.0"C. UV (MeOH) maleimide (3), identified on the basis of its solubility

32

200

500

400

300

WAVELENGTH,

NM

Figure 1. Absorption spectra during aerobic preparative-scale irradiation of a freshly-prepared xanthobilirubic acid (1)solution in borate buffer. Numbers on curves are irradiation times in minutes. Initial [l] = 50 mg/400 d ; pH = 8, A = 0.055. All samples diluted 1:25.

properties, UV, IR, NMR and mass spectral characteristics and comparison with an authentic synthetic sample. The isolated yield of the maleimide is considered to be a minimum, since the compound is quite volatile and thus losses would be expected during removal of solvents either in uacw or in a nitrogen stream. Mesobilirubin XIII-cr (3 mg), identified by comparison of its TLC and spectral properties with those of an authentic sample, was isolated from the photolysis solution, but this tetrapyrrole is not considered to be a photoproduct of xanthobilirubic acid, since it is also detectable (by TLC)on workup of an unirradiated freshly-prepared solution of 1 in borate buffer. It is also formed in dark from 1 in acetic acid or pyridine in the presence of oxygen (Grunewald and Strope, 1973b).A second major photoproduct was isolated by preparative TLC (21% yield based on molar ratios) and its UV, IR, NMR and mass spectra were identical to those of an authentic sample of 5-formylZ,4-dimethyl-lH-pyrrole-3-propanoic acid (4). Furthermore, the methyl ester of this photoproduct (BF,/methanol) was identical to a synthetic sample of methyl 5-formyl-2,4-dimethyl-1H-pyrrole-3-propanoate. The product with Rl 0.31 (see Materials and Methods) has not yet been identified. About 20mg of it was isolated by preparative TLC from experiments starting with 220 mg of xanthobilirubic acid. The material is very water-soluble, and quite insoluble in organic solvents. All IR and NMR spectra *Dosimetry calculations (Johns, 1968), which are only approximate because of the use here of polychromatic light and a different irradiation geometry, indicate that this apparent increase in rate of photodecay with time of irradiation cannot be explained by dosimetry effects in going from a solution of high absorbance (2.0) to one of lower absorbance.

which have been taken show little resolution, although the IR indicates the presence of a carboxylic acid. The UV spectrum has a maximum (MeOH) at 266 nm. Other minor bands were detectable on the preparative TLC plates from which the maleimide and formylpyrrole were isolated. Some of these corresponded to spots detectable after work-up of an unirradiated freshly-prepared solution of xanthobilirubic acid, and were therefore not considered as photoproducts. However 3, 4 and the unidentified product with A,, 266nm were not detected upon work-up of unirradiated solutions.

a +

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CZH, 0

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CH,

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CH3

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(5)

(4) P = CH~CH~COO-

Preliminary data (UV and TLC) indicate that the maleimide and formylpyrrole are also formed on photooxidation of xanthobilirubic acid in pure methanol (J. 0. Grunewald and J. C. Walker, unpublished). Mechanistic studies. Figure 2 shows the UV-visible spectra obtained during aerobic irradiation of 250 mf of a 'freshly-prepared' xanthobilirubic acid solution (62 pM) in borate buffer (pH 8.0, A = 0.055). The magnitude of the decrease in absorbance at 410nm per 15 min increased with irradiation time.* As in the preparative-scale irradiations, there was an increase in absorbance both at 215 and at 315 nm, with the latter peak decreasing during the later stages of the irradiation.

33

. 200

300

400

500

WAVELENGTH, NM Figure 2. Absorption spectra during aerobic irradiation of a ‘freshly-prepared’ xanthobilirubic acid (1) solution in borate buffer. Numbers on curves are irradiation times in min. Initial [l] = 6 2 p M ; pH = 8; A = 0.055. All samples diluted 4:lO.

Storing a ‘freshly-prepared’ borate buffer solution of xanthobilirubic acid in the dark at 5°C for several days caused a relatively small decrease in absorbance at 410nm. The magnitude of the decrease depended upon the age of the solution; the rate was very slow compared to that of photodecay. Figure 3 shows the UV-visible spectra of samples from aerobic irradiation of such an ‘aged’ (4 days) solution. The decrease

in absorbance after the first 15 min of irradiation was 39 times larger than that observed on irradiation of a ‘freshly-prepared’ solution (Fig. 2). This rate difference cannot be interpreted as a dosimetry artifact, because the initial absorbance of the two solutions was essentially the same (2.0). Qualitatively, spectral changes were similar to those of a ‘freshly-prepared‘ solution, showing an increase in absorbance at both

07-

0.6-

4 5 .-

0.0 200

400

300

500

WAVELENGTH, N M Figure 3. Absorption spectra during aerobic irradiation of an ‘aged’ (4 days) xanthobilirubic acid (1) solution in borate buffer. Numbers on curves are irradiation times in min. Initial [l] (before aging) = 62pcM; pH = 8; A = 0.055. All samples diluted 4:lO. P A P 241

J. 0. GRUNEWALD. J. C. WALKERand E. R. STROPE

34

solution was not due to boric acid, 1 (62pM) in an unbuffered NaOH-HCl-KC1 solution (pH 8.0, A = 0.055) was similarly aged. A tube of Ascarite protected the solution from atmospheric carbon dioxide, thus maintaining the pH at 7.95-8.0 during aging. As can be seen from comparison of the initial absorbance of the ‘aged‘ solutions (Figs. 4 and 5), xanthobilirubic acid is more stable in the dark in borate buffer than in unbuffered solution of the same pH and ionic strength. Figure 5 shows a plot of the data obtained on aerobic irradiation of a ‘freshly-prepared‘ unbuffered (NaOH-HCl-KCl) solution, an ‘aged’ (4 d) unbuffered solution and the dark (control) reaction over the corresponding time period. As in borate solution, photodestruction of 1 in the unbuffered solution was faster after aging. Though the initial increase in rate TIME (MINI was somewhat less than that seen in borate, it was Figure 4. Rate of decay of xanthobilirubic acid (1) in ‘freshly- significant, indicating that the aging effect was not prepared’ and ‘aged’solutions in borate buffer. Initial [l] = due to borate buffer components. Thus all further ex62 pA4; pH = 8; A = 0.055. -u--G-- Aerobic irradiation of a ‘freshly-prepared’solution; --t-e aerobic irradia- periments were done using borate buffer solutions. Figure 6 shows the photodecay of ‘freshly-prepared’ tion of an ‘aged‘ (4 days) solution; -4-4-aeration of a ‘freshly-prepared’solution in the dark (control). xanthobilirubic acidborate solutions, to which previously irradiated solutions were added. Photolysis of 315 and 215 nm. The rate of these increases was con- a solution prepared by diluting 125 m f of a ‘freshlysistent with the more rapid decrease in absorbance prepared‘ solution (0.124 mM) with 125 ml of an aerat 410 nm. This ‘aging’ effect was oxygen dependent, obically ‘irradiated freshly-prepared’ solution was sigsince no rate-enhancement was observed on aerobic nificantly more rapid (closed circles) than photodecay irradiation of a solution which had been saturated of either a ‘freshly-prepared’ solution (62 pM) alone with nitrogen before storing in the dark at 5°C. (open circles) or a solution of the same concentration Figure 4 presents absorbance (410 nm) vs time plots prepared by addition of 125 m f of an anaerobically for irradiations of a ‘freshly-prepared‘ solution (Fig. 2) and an ‘aged’ solution (Fig. 3), along with the dark reaction (control) for the ‘freshly-prepared’ solution, 2 .o which was unchanged over the time required for complete photodecay. 5 z To ensure that the rate enhancement of xanthobili0 rubic acid photodestruction observed after aging the

*

Y

-I

w

g

2.0

a

1.0

m Q

z 0

2 m

*

a

v

W

u

5 1.0 m

0

E

m U

0

15

30

45

60

75

TIME ( M I N ) Figure 5. Rate of decay of xanthobilirubic acid (1)in ‘freshlyprepared‘ and ‘aged‘ solutions in NaOH-HCl-KCl. Initial [l] = 62 p M ; pH = 8; A = 0.055. -C-C-Aerobic irradiation of a ‘freshly-prepared‘solution; --t-e aerobic aerairradiation of an ‘aged’(4 days) solution; - m a tion of a ‘freshly-prepared’ solution in the dark (control).

15

--

45 60 TIME ( MIN 1

30

75

Figure 6. Rate of photodecay of xanthobilirubic acid (1) in borate buffer, with added ‘irradiated freshly-prepared’ solutions. Aerobic irradiation of a ‘freshly-prepared’ solution. Initial [l] = 62pM; 2 5 0 d ; pH = 8; A = 0.055 aerobic irradiation of a ‘freshly(control); -+-t pH = 8 ; A = prepared’ solution, [l] = 0.120 mM; 125 d ; 0.055, plus an aerobically ’irradiated freshly-prepared’ sohtion, [l] = 2 p M ; 125 &; pH = 8; A = 0.055. Initial [l] after mixing = 62 p M ; 44- aerobic irradiation of a ‘freshly-prepared’solution, [l] = 70 p M ; 125 d ; pH = 8; A = 0.055,plus an anaerobically ‘irradiated freshly-prepared’ solution, [l] = 52 p M ; 125 d ; pH = 8; A = 0.055. Initial [l] after mixing = 62pM.

35

Photooxidation of xanthobilirubic acid

(N,) ‘irradiated freshly-prepared‘ solution to 125 r d of a ‘freshly-prepared’ solution (squares). A ‘freshlyprepared‘ solution which was aerated in the dark for 75 min before irradiation gave a photodecay curve identical to that of the ‘freshly-prepared’ solution (open circles). These results indicate that a ‘catalyst’ for the photooxidation of xanthobilirubic acid (1) is formed from 1, and that its formation (in 75 min) requires both oxygen and light. The dependence of the rate of photodestruction of xanthobilirubic acid on catalyst concentration is shown in Fig. 7. Increasing amounts of an ‘irradiated freshly-prepared’ solution were added to ‘freshlyprepared‘ solutions, keeping the total volume and initial concentration of 1 constant for all solutions. The decrease in absorbance at 410 nm for the 6cst 15 min of irradiation showed a linear dependence on volume of ‘irradiated freshly-prepared’ solution added above a threshold amount. When aliquots of an ‘irradiatedaged‘ borate solution of 1 were added to ‘freshly-prepared’ solutions, a linear dependence of rate on volume of catalyst solution added was observed. The ‘irradiated freshly-prepared’ and ‘irradiated-aged’ solutions had the same initial concentration of 1; thus the plots in Fig. 7 indicate that more catalyst is produced by the combination of aging and irradiation than by irradiation only. To further substantiate the idea that dark oxidation of xanthobilirubic acid (aging) alone produces a catalyst for the photooxidation of 1, 7 mt of a solution (inital concentration of 1 of 0.87 mM) which had been ‘aged’ 8 months (no xanthobilirubic acid remaining by TLC and UV) was added to a ‘freshly-prepared‘ solution and the mixture

0

5 10 15 20 ADDITIONS OF CATALYTIC SOLUTIONS ( ML 1

25

Figure 7. AA410 during the first 15 min of irradiation of ‘freshly-prepared’xanthobilirubicacid (1)solutions in borate bufferwith added catalytic solutions, vs volume of catalytic solution added. --t-e Aerobic irradiation of ‘freshlyprepared‘ solutions plus ‘irradiated freshly-prepared‘ solution ([l] before irradiation = 0.835 mM), total volume = 250 d,initial 111 = 62 p M ; 44- aerobic irradiation of ’freshly-prepared’ solution plus ‘irradiated-aged’ solution ([l] before aging and irradiation = 0.835 mM, aged 52 days before irradiation), total volume = 2 5 0 f , initial [I] = 0.062 mM.

2.0

I .o

v I

0

15

I

I

I

30

45

60

75

90

TIME (MINI Figure 8. Rates of aerobic and anaerobic photodecay of xanthobilirubic acid (1) in borate buffer, with and without catalyst solution added. -o--&-Aerobic irradiation of a ‘freshly-prepared’solution, initial [l] = 62 p M , 250 mf ; 44- anaerobic irradiation of a ‘freshly-prepared‘ solution, inital [I] = 62 pM, 250 mf; -V-Vaerobic irradiation of a ‘freshly-prepared‘ solution with added (10 mt) ‘aged-irradiated‘ solution ([l] before aging and irradiation = 0.835 mM, aged 52 days before irradiation), total volume = 2 5 0 d , initial [l] = 6 2 p M ; --@--Oanaerobic irradiation of a ‘freshly-prepared’ solution with added (10 d) ‘aged-irradiated’ solution ([I] before aging and irradiation = 0.835 mM, aged 52 days before irradiation), total volume = 250 d, initial [l] = 62 p M . irradiated. The during the first 15 min was 1.05, only slightly less than the decrease predicted from Fig. 7 for 7 mt of an ‘aged-irradiated’ solution. Presented in Fig. 8 are data from the aerobic and anaerobic irradiations of ‘freshly-prepared‘ solutions alone and such solutions with catalyst added. In the absence of oxygen both the uncatalyzed (squares) and catalyzed (closed circles) photodestruction of I are markedly reduced. The slow decrease in absorbance which was observed anaerobically in both cases could be due to the residual oxygen in the nitrogen used, since no oxygen scrubbing unit was employed. To determine whether ground state or excited state oxygen was involved in the self-sensitized photooxidation of xanthobilirubic acid, DABCO was added as a singlet oxygen quencher. Table 1 presents results from irradiation of ‘freshly-prepared‘ solutions in the presence of DABCO. At 10 ph4 DABCO, the reaction was slightly inhibited during the entire irradiation period. This effect was somewhat greater using 0.1 mM DABCO. With 1 mM DABCO, inhibition was comparable to that of 10pM DABCO, but for only the first third of the reaction period, after which the photodecay rate was actually increased compared to the control. When 3mM DABCO was used, no initial inhibition was seen and the rate increase in the latter stage was enhanced. A significant increase in rate of photodestruction of 1 (compared to control

J. 0. GRUNEWALD, J. C. WALKERand E. R.

36

Table 1. Effect of DABCO on the aerobic photodecay of xanthobilirubic acid (1) in ‘freshly-prepared’ borate buffer solutions. Initial [l] = 62 p M , pH = 8, A = 0.055, 250 ml Absorbance,__

- Absorbance,.

410 nm ~

~~

DABCO Concentration, mb!

Irradiation Time, min

None

0.01

0.10

1.0

3.0

10.0

15.0

0.24

0.20

0.14

0.18

0.22

0.30

22.5

0.48

0.38

0.32

0.38

0.45

0.65

30.0

0.77

0.63

0.55

0.74

0.87

1.45

45.0

1.51

1.27

1.25

1.80

1.93

2.01

60.0

1.85

1.85

1.85

2.08

-

with no DABCO) was observed throughout the reaction period in the presence of 10 mM DABCO. When 125 d of the latter irradiated solution (10 mM in DABCO) was mixed with 1 2 5 d of a ‘freshlyprepared‘ solution of xanthobilirubic acid in borate and irradiated, photodecay was complete in 15 min. For comparison, 30 min of irradiation was required to reach the same A410using as catalyst an ‘irradiated freshly-prepared’ solution with no DABCO (Fig. 6, closed circles). The singlet oxygen quencher, DABCO, was added to ‘freshly-prepared’ solutions to which an aliquot of an ‘irradiated-age’solution had also been added, and the mixtures irradiated. The results, given in Table 2, show that DABCO at 0.01 and 0.1 mM has essentially no effect on the catalyzed photooxidation of 1. A slightly enhanced rate was observed with 1 mM DABCO. All of the solutions used in the DABCO studies had the same initial pH, volume and xanthobilirubic acid concentration. Because the results of experiments with DABCO addition to ‘freshly-prepared’solutions did not clearly indicate the presence or absence of singlet oxygen, the effect of sodium azide was studied. Azide ion has been shown to be an effective quencher of singlet oxygen, but in certain dye sensitized photooxidations, may also inhibit the reaction by quenching the dye Table 2. Effect of DABCO on the aerobic photodecay of xanthobilirubic acid (1) in ‘freshly-prepared’ borate buffer solutions with added (10 d )‘irradiated-aged‘ solution([l] before aging and irradiating = 0.835 mM, aged 5 2 days before irradiation). Initial [l] = 62 p M , pH = 8, A = 0.055, total volume = 250 I& Absorbancet,o

- Absorbancet.

410 nm

STROPE

triplet state (Hasty et al., 1972). Aerobic irradiation of a ‘freshly-prepared’ solution of 1 (62pM) which was also 1 mM in sodium azide resulted in much slower photooxidation. Only 18% of the xanthobilirubic acid was photooxidized in 45 min in the presence of sodium azide, compared to 36% in its absence in the same time period. To determine whether the catalyst was acting as an efficient triplet sensitizer for 1, experiments using photochemical filter solutions were done. The absorption spectrum‘ of a catalytically active ‘irradiatedaged’ solution showed two maxima, a broad one from 288-310nm and a sharper maximum at 215nm. Using filter solution I, which transmits no light below 330 nm, the rate of photooxidation of xanthobilirubic acid in the presence of catalyst was markedly increased compared to the control, a ‘freshly-prepared’ solution irradiated through filter solution I (Table 3). When filter solutions I and I1 were used in series so that light below 395 nm was not transmitted (Table 4),the intensity of light incident on the solution was greatly reduced, but the data given in Table 4 show that photooxidation is still significantly more rapid in the presence of catalyst than in its absence. Methylene blue, a widely used singlet oxygen sensitizer, was found to sensitize the photooxidation of xanthobilirubic acid, under conditions where 1 did not absorb any light. The decay is apparently firstorder in 1 at a methylene blue concentration of 16 nM and an initial xanthobilirubic acid concentration of 30 pM. There was no destruction of 1 in the presence of methylene blue when a similar solution was irradiated with nitrogen bubbling through it instead of air. In the presence of DABCO (1-10 mM), which has been found to inhibit the Rose Bengal photosensitized oxidation of lipoic acid in aqueous solution, presumably by quenching singlet oxygen (Schaap et al., 1974), the methylene blue sensitized photooxidation was not inhibited. At 1 mM, DABCO had essentially no effect on the first-order photosensitized decay rate. At two higher concentrations of DABCO (5 and 10 mM), the Table 3. Effect of using light of wavelengths >330 nm on the aerobic photodecay of xanthobilirubic acid (1) in a ‘freshly-prepared’ solution with added (lo&) ‘irradiated-aged’ solution ([l] before aging and irradiating = 0.835 mM,aged 52 days before irradiation). Initial [l] = 62 p M , pH = 8, A = 0.055,total volume = 250d Absorbancet=,

-

~

Absorbance,,

Irradiation time, min

Control

Catalyzed

OABCO Concentration, mM 15

0.15

1.44

30

0.40

1.76

1.04

45

0.80

1.87

1.30

1.92

1.75

Irradlatlon Time, min

None

0.01

0.10

1.0

7.5

1.00

0.97

0.94

410 nm

15.0

1.59

1.59

1.59

1.80

60

22.5

1.85

1.85

1.87

1.95

75

Photooxidation of xanthobilirubic acid Table 4. Effect of using light of wavelengths > 395 nm on the aerobic photodecay of xanthobilirubic acid (1) in a 'freshly-prepared' solution with added (0.8 me) 'irradiated-aged' solution ([l] before aging and irradiating = 0.835 mM, aged 52 days before irradiation). Initial [l] = 0.24mM. pH = 8, A = 0.055, total volume = 20 mf ~

Absorbancet=, Irradiation time, min

-

--

Absorbance+. 410 nm

Control

Catalyzed

15

0

0.05

30

0

0.54

45

0.04

0.89

60

0.15

1.27

75

0.23

1.55

rate was significanlty increased. The decrease in conafter 30 min of photosensicentration of 1 (as tized oxidation in the presence and absence of DABCO are given in Table 5. Products of the methylene blue sensitized photooxidation of xanthobilirubic acid were the same as those from the direct (self-sensitized) photooxidation, as determined by TLC and UV-visible spectral comparisons. DISCUSSION

The products which we have characterized from the aqueous photooxidation of the dipyrrylmethene xanthobilirubic acid (1) are analogous to two products identified from bilirubin photooxidation under certain conditions. Methylvinylmaleimide has been isolated from the photooxidation of bilirubin in ammoniacal methanol (Lightner and Quistad, 1972% 1972~;Bonnett and Stewart, 1975) and in chloroform (Gray et a/., 1972). However, the aldehydic product from bilirubin corresponding to the formylpyrrole (4) from xanthobilirubic acid was not found on irradiation in organic solvents, although it had been predicted as a co-product of the maleimide (McDonagh, 1971). We reported (Grunewald and Strope, 1973a) the first isolation of such an aldehyde from the photooxidation of a dipyrrylmethene chromophore, in aqueous solution, and since then Bonnett and Stewart (1975) have found the corresponding dipyrrole-dialdehyde as a minor product from the photooxidation of bilirubin in aqueous solution, but not in methanolic solution. Our results indicate that both methylethylmaleimide (3) and the formylpyrrole 4 are major photooxidation products of xanthobilirubic acid, irradiated either as *Recent preliminary experiments on the methylene blue photosensitized oxidation of the formylpyrrole indicate that the unidentified compound (k,,, 266nm) can be formed from 4. However we have no evidence as yet that it could not also be formed directly from xanthobilirubic acid.

37

its sodium salt in aqueous solution or as the neutral molecule in methanol. Mechanistically, a maleimide and a formylpyrrole would be predicted to form concomitantly (McDonagh, 1971, and Scheme 1, below) from either bilirubin or dipyrrylmethenes. The fact that a maleimide, but not a diformyldipyrrole, has been isolated from bilirubin photooxidation in methanol (Bonnett and Stewart, 1975) may be due to the instability of the dialdehyde in the work-up procedure. Also pyrrole carboxaldehydes have been shown to undergo dye-sensitized photooxidation in methanol to yield deformylated products (Lightner and Quistad, 1973).This may in part account for the fact that the aldehydes have not been isolated from the dye-sensitized or self-sensitized photooxidation of dipyrrylmethene-type compounds in organic solvents, while compounds which could arise from aldehyde photooxidation have been found (Lightner and Quistad, 1972a; Lightner and Crandall, 1973). We have observed a decrease in the absorbance due to 4 during the last stages of the photooxidation of 1 but this decrease is not prevented by filtering out light (