Photochemistry and Photobiology, Vol. 59, No. 5, pp. 503-505, 1994 Printed in the United States. All rights reserved 003 1-8655/93 $05.00+0.00

0 1994 American Society for Photobiology

RESEARCH NOTE

A NEW PHOTOPRODUCI' FROM FUROCOUMARIN PHOTOLYSIS IN DILUTE AQUEOUS SOLUTION:

5-FORMYL6-HYDROXYBENZOFURAN

KAREN A. MARLEY* and RICHARD A. LARSON Institute for Environmental Studies, University of Illinois, 1 101 West Peabody Drive, Urbana, IL 6 1801, USA

(Received 16 September 1993; accepted 17 January 1994)

Abstract-This paper presents the analysis and identification of an unstable product previously undescribed from furocoumarin photolysis, 5-formyl-6-hydroxybenzofuran. Reverse-phase chromatography and solid-phase extraction techniques allowed its isolation.

INTRODUCTION

In the characterization of furocoumarin photooxidation products there is a general consensus that coumarins are formed; that is, the 4',5'-double bond of the furan ring is disrupted and a ring-opened product, such as 6-formyl-7- hydroxycoumarin (FHC)? (1) is formed from psoralen (2).'" Theoretical calculations predict more photoreactivity a t the 3,4-double bond of the pyrone ring'; however, there are no reports of any ring-opened products at this position, although the formation of the corresponding furocoumaric acid (3) does O C C U ~ . ~ Of additional interest are the observations by several investigators of the formation of unstable interme- diates during the photooxidation of psoraled and related f u r o c ~ u m a r i n s . ~ ~ ~

We report o n the formation of a previously undescribed pyrone ring-opened product from the photolysis of psoralen i n dilute aqueous solution. This product is easily oxidized and was found to be unstable during some isolation proce- dures. Additional photoproducts were observed including the aforementioned FHC and furocoumaric acid a forthcoming paper will present the kinetics of formation of these products as well as the solvent dependence of the reaction.

MATERIALS AND METHODS

Chemicals. Psoraien, 8-methoxypsoralen (8-MOP) and coumarin were obtained from Sigma Chemical Company (St. Louis, MO) and used as received (by high-performance liquid chromatography [HPLq and gas chromatography [GC], < 0.1% 8-MOP was found in psor- alen; no impurities were found in 8-MOP or coumarin). The HPLC and GC-mass spectroscopy (MS) solvents were high purity, spectro- scopic grade from Burdick & Jackson (Muskegon, MI). Glass-dis- tilled water was prepared by distillation of deionized water over alkaline potassium permanganate. Air-saturated aqueous solutions were prepared by suspending a small amount of the compound in

*To whom correspondence should be addressed. $4bbrevzutions: %MOP, 8-methoxypsoralen; FHBF, 5-formyl-6-hy-

droxybenzofuran; FHC, 6-formyl-7-hydroxycoumarin; GC, gas chromatography; HPLC, high-performance liquid chromatogra- phy; IRD, infrared detector; MS, mass spectroscopy; PDAD, pho- todiode array; SDB, polystyrenedivinylbenzene; SPE, solid-phase extraction.

water, stimng for 24 h, followed by filtration with a 0.45 fim nylon filter. Concentration of the stock solution was determined by ab- sorbance measurement using the molar extinction coefficient. To eliminate screening effects of high substrate concentration, solutions were diluted so that the final solution had an optical density no greater than 0.3 in the region of irradiance; for example, psoralen concentration was no more than 20 fiM.

Irradiution. Initial experiments were performed at room tem- perature in 1 x 10 cm screw-cap Pyrex tubes with irradiation by an SS2500 solar simulator system equipped with a 2500 W xenon arc lamp, sunlens diffuser and AM-I filter (Spectral Energy Corp., West- wood, NJ). The UVA intensity (measured with a UVX radiometer, San Gabriel, CA) was ca 23 W m-2, equivalent to a bright, sunny day in midsummer at 40"N latitude. For product isolation, 1 L batch reactions were irradiated for 2 h with a medium pressure 200 W mercury arc lamp (UVA intensity ca 58 W m-2, no X <290 nm) enclosed within a water-cooled borosilicate immersion well (Ace Glass Co., Vineland, NJ).

Solid-phase extraction (SPE). 3M EmporeTM extraction disks (47 mm diameter) were obtained from Varian Analytical (San Jose, CA). Reverse-phase extraction, using either the CIS or polystyrenedivi- nylbenzene (SDB) disk was used; more efficient product recovery was found for the SDB disks. One liter of the irradiated solution was filtered through a precleaned extraction disk; the disk was then eluted with either methanol (for HPLC analysis or semipreparative pun- fication) or methylene chloride (for GC analysis).

HPLC separation. Direct analyses of the reaction mixtures were made after irradiation using an analytical SDB PRP-1 (Hamilton Co., Reno, NV) reverse-phase column (250 mm x 4.1 mm, 10 fi pore size) with 50-50 (voVvol) acetonitrile-water. Detection was by UV absorbance: single wavelength by a Kratos 757 spectroflow (Ramsey, NJ); photodiode array (PDAD) by a PFI (Groton Tech- nology, Inc., Groton, MA).

Semipreparutive purification. A PRP- 1 semipreparative column (305 mm x 7 mm) was used for product isolation using a two step purification: (1) The methanol SPE extract (see above) of a reaction mixture was separated using 50-50 (voVvol) acetonitrile-water; (2) the desired fraction was collected and then reinjected using 1000/0 methanol as the mobile phase to remove water from the desired product. After the removal of water, the fraction could then be dried using a rotary evaporator operated with a water vacuum aspirator (20°C) without significant loss of the product. For spectral charac- terization the methanol extracts from four batch reactions (initial concentration before irradiation: 4 mg psoralen per liter of water) were prepared and combined to give enough product for IR and NMR analysis.

Instrumentation. The GC separations were made on a DB-5 cap- illary column (30 m x 0.32 mm internal diameter J&W Scientific, Placerville, CA) with an initial temperature of 40°C for 5 min, then 40-280°C at 5T/min, with a final hold of 10 min at 280°C. The

503

504 KAREN A. MARLEY and RICHARD A. LARSON

L

4

Figure 1. Direct HPLC analysis of a psoralen reaction mixture fol- lowing 90 min of irradiation by a solar simulator. Ultraviolet ab- sorbance detection at 240 nm: 1 = 6-formyl-7-hydroxycoumarin; 2 = psoralen, 80% degradation from initial 20 pM concentration; 4 =

late-eluting product, 5-formyl-6-hydroxybenzofuran.

GC-FID was a Varian 3300 (Varian Corp., Walnut Creek, CA); GC- MS was a Hewlett Packard 5890A GC coupled with a Finnigan 800 Ion Trap mass spectrometer (Finnigan MAT, San Jose, CA). Infrared absorption spectra were measured on a GC-FTIR (Hewlett Packard, Avondale, PA) system: HP5890A GC coupled to a 5965B infrared detector (IRD), which employs a light pipe-based interface and a narrow-band mercury-cadmium-telluride detector. Spectra were ob- tained in the vapor phase after elution from the GC column operating with an 8 cm-' resolution and a useful range of 4000 to 700 cm-l. Proton NMR spectra in CD,OD were recorded on a 300 MHz QE- 300 (General Electric Co., Schenectady, NY) narrow bore instrument with a pulse width of 5.00 ps and a delay time of 3.35 s. Ultraviolet absorption spectra were recorded on a Model U-2000 double-beam UV/vis spectrophotometer (Hitachi Instruments, Inc., Tokyo, Ja- pan).

RESULTS AND DISCUSSION

During irradiation, the concentration of psoralen dissolved in water rapidly diminished (half-life of approximately 10 min), as followed by HPLC. As the photolysis proceeded, several small product peaks including FHC became evident as well as a major product eluting after psoralen (Fig. 1). The HPLC-PDAD analysis of this reaction mixture was used to obtain the UV absorption spectrum of this product (Fig. 2). Instability of the late-eluting product was noted when anal- ysis was delayed after irradiation; that is, the HPLC peak was no longer present if analysis was performed the suc- ceeding day.

Attempts were made to extract the reaction mixture from water into an appropriate solvent for analysis-identification by G C coupled to a mass spectrometer. Any extracts that were subjected to heat, such as the increased temperature (30°C) needed to remove water at reduced pressure by rotary evanoration. led to low recoverv of the Droduct(s\. TJsine an

II_\ 1.00 - I

0.80 7 I I

I 0.20 - //

I

I- -, 0.00 u' --+ I

I I

200 250 300 350 400 Wavelength (nm)

Figure. 2. Ultraviolet absorption spectrum of 4. Nearly identical spectra were obtained from the isolated product and (PDAD) from

the late-eluting product in Fig. 1.

SPE method for isolating hydrophobic compounds from wa- ter (an extraction method that is rapid and can be performed at room temperature), one major product by GC-FID and GC-MS was found that was initially identified as 5-formyl- 6-hydroxybenzofuran (FHBF) (4).

Attempts at large-scale isolation of this product using open- column liquid chromatography were not successful. Addi- tional instability of FHBF was noted for samples that had been successfully extracted by SPE into methylene chloride- sample loss occurred after a period of several weeks even when stored at 0°C. Although additional solvents have not been examined, FHBF was found to be reasonably stable in methanol (at room and storage temperatures) and allowed further characterization to proceed. Semipreparative purifi- cation with final isolation of the product in methanol was made and the spectral data obtained are consistent with the assignment of the proposed structure (see Table 1).

In our photolysis studies similar pyrone ring-opened prod- ucts were found for both 8-MOP and coumarin, although the rates of formation and yields were lower. From 8-MOP (5), a tentative identification of 5-formyl-6-hydroxy-7-methoxy- benzofuran (6) based on mass spectra was made: an M+ ion of 192 with a strong M- 1 (indicative of loss of the aldehyde hydrogen). From coumarin (7), salicylaldehyde (8) was con-

Table 1. Spectral characteristics of 5-formyl-6-hydroxybenzofuran*

m/z (intensity)

MS CgH603 162 (100) 161 (95) 133 (29) 116 (25) 105 (45)

NMRt AH (ppm) 10.02 (s, 'H, CHO), 7.95 (s, IH, H,,), 7.72 (d, IH,

H(2),,,,4. J = 2 Hz), 6.99 (s, 'H, H,,), 6.86 (d, 'H, H(3)v,n,l, J = 2 Hz)

IR 3400 to 3200 (YOH) , 2741 (uC.Hcho), 1675 ( ~ c - 0 )

*After semipreparative isolation. ?In agreement with Worden et aL9 for methoxy analog of compound

(4).

Research Note 505

OH 0 1

R = H = 2 R = OCH3 = 5

3

a 0 7

k R = H = 4

-OH 8

firmed (HPLC-PDAD and GC-MS data matched an authen- tic standard by retention time and spectra). In each case- psoralen, 8-MOP and coumarin-it is important to note in the HPLC analysis that the hydroxyaldehyde product emerged after the parent lactone compound under the reverse-phase conditions used for elution; if the percentage of mobile phase composition was varied to higher amounts of acetonitrile, the parent compound and product coeluted. During the pho- tolysis of psoralen it was interesting to find that dimerization reactions did not occur in dilute aqueous solution; at higher concentrations (2.5 mM) in methylene chloride, psoralen di- mer formation was detected. The FHBF has been used as an intermediate in the chemical synthesis of p s ~ r a l e n , ~ J ~ but was not present in the commercial material received in our laboratory.

The formation of the benzofuran photoproducts might be initiated by attack of water or oxygen at the 3,4-double bond of an excited state of psoralen. The intermediate oxygenated species could then decay by routes that include cleavage of the 3,4-double bond and hydrolysis of the ester function to afford FHBF.

CONCLUSIONS

5-Formyl-6-hydroxybenzofuran is a product previously unreported from the photooxidation of psoralen. This com- pound is somewhat unstable during some isolation proce- dures and may, therefore, have been overlooked in previous studies. The phototoxic activity of FHBF is currently under investigation. The carbonyl functional group of FHBF cer- tainlv indicates the Dossibilitv for both dark and light toxicitv:

for example, aldehydes are generally reactive with proteins and have also been found to form adducts with DNA.'IJ2 Compounds with carbonyl chromophores that absorb light in the solar UV region have demonstrated phototoxicity (in addition to dark toxicity), such as a closely related methylke- tone benzofuran, 6-metho~yeuparin'~ and a monoterpene aldehyde, citral.I4

Acknowledgements- We thank Mark Nanny for help in obtaining the NMR spectrum and Tim Collette for the IR spectrum. We also thank Fei-Wen Chuang, Robin DiNardo, Gary Epling and Penney Miller for helpful discussions. This work was supported in part by USDA grant AG-89-372804-897.

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