J. Nutr, Sci. Vitaminol., 23, 281-290, 1977

EFFECT OF WAVELENGTH ON THE PHOTOCHEMICAL REACTION OF ( D2) IRRADIATED BY MONOCHROMATIC LIGHT1,2

Tadashi KOBAYASHI, Sachiko YOSHIMOTO, and Mitsue YASUMURA3

Department of Hygienic Chemistry, Kobe Women's College of Pharmacy, Higashinada-ku, Kobe 658, Japan (Received February 21, 1977)

Summary The effect of wavelength on the photochemical reaction of ergocalciferol (vitamin D2) was investigated. An ergocalciferol solution in ethanol was irradiated by monochromatic UV light of various wave lengths with a fixed quantum (8.0•~108erg/cm2), trimethylsilylated and then applied to a capillary column GLC as described previously (10) in order to estimate the reaction products. The results showed that UV light in a range of 295-312nm was most effective in the photo chemical transformation of ergocalciferol into suprasterol2 I and II. The formation of compounds I, I2, II1 and II2, which had been found as novel reaction products by BARKER et al. (8), was not exactly confirmed because the former two were thermally isomerized into gyro- and isopyro - D2 via ergocalciferol while the latter two were isomerized into 5,6-tans-D2

at the temperature for GLC analysis. However, since the peaks caused by

gyro and isopyro-D2 were observed to be rather large in the gas chromato grams of long-term irradiated solutions, although no information on the existence of ergocalciferol was obtained from the UV spectra and TLC of the solutions, significant amounts of the compounds h and 12 might be formed by irradiation of ergocalciferol. On the other hand, the peak due to 5,6-taans-D2 was observed to be very small in the gas chromato

grams of irradiated solutions with different wavelengths although no information on the existence of 5,6-taans-D2 was also obtained from the results of TLC. Therefore, the photochemical transformation of ergo calciferol into compounds II1 and II2 might occur to only a small extent regardless of irradiated wavelengths.

1 Studies on the ultraviolet irradiation of . Part I. 2 Following abbreviations are used: Pyro-D2 and D 3, pyroergocalciferol and pyrochole calciferol; isopyro-D2 and D3, isopyroergocalciferol and isopyrocholecalciferol; 5,6-trans-D2 and D3, 5,6-trans-ergocalciferol and 5,6-trans-; CA, cholesteryl acetate; UV, ultra violet; TLC, thin-layer chromatography; GLC, gas-liquid chromatography; TMS, trimethylsilyl. 3 小 林 正 ,吉 本 佐 雅 子,康 村満 枝

281 282 T. KOBAYASHI, S. YOSHIMOTO, and M. YASUMURA

It was reported in previous papers (1, 2) that the effect of wavelength on the photochemical transformation of D into vitamin D had been inves tigated. An ethanol solution of either or 7-dehydrocholesterol was irradiated by monochromatic UV light with a spectroirradiator in a range of 230-400nm, and the yield of either ergocalciferol (vitamin D2) or cholecalciferol (vitamin D3) was estimated by the GLC method as described previously (3). The results showed that the monochromatic UV light of 295nm was most effective on the formation of vitamin D in both cases of ergosterol and 7-dehydrochole (1, 2). In the present study, a method similar to one described previously was applied to the photochemical reaction of ergocalciferol by UV irradiation. WINDAUSet al. (4) first isolated suprasterol2 I and II4 as crystalline allophanates from a long-term irradiated mixture of ergosterol. WESTERHOFand KEVERLING BUISMAN(5) found that the direct origin of suprasterol2 I and II in the photo chemical reaction was not ergosterol but ergocalciferol because the UV irradiation of ergocalciferol gave suprasterol2 I and II as the main products. The chemical structure of suprasterol2 II was confirmed to be as shown in Fig. 1 both by a

Fig. 1. Photoreaction of vitamin D series (9). chemical method (6) and by X-ray analysis (7). Since suprasterol2 I is an optical isomer of suprasterol2 II, its chemical structure can be shown as that in Fig. 1. 4 Suprasterol2 I and II indicate the compounds of the vitamin D2 series derived from ergo calciferol, whereas suprasterol3 I and II indicate the compounds of vitamin D3 series from chole calciferol. IRRADIATION OF ERGOCALCIFEROL BY UV LIGHT 283

Recently, BAKRERet al. (8) isolated four novel reaction products, tentatively denoted as compounds I1, I2, IIl and II2, besides suprasterol3 I and II from the UV irradiated mixture of cholecalciferol in ethanol. Their chemical structures confirm ed that the first two were stereoisomeric vinylallene derivatives while the last two were stereoisomeric cyclobutene derivatives as shown in Fig. 1. Compounds Il and I2 were confirmed to be converted into cholecalciferol by heating because the gas chromatograms of their TMS ethers gave peaks corresponding to pyro and isopyro-D3 derived from the thermal cyclization of cholecalciferol, whereas compounds II1 and II2 were confirmed to be converted into 5,6-traps-D3 by heating in octane (8). It was also reported that the composition of the irradiated products were 19, 62, 2, 6 and 11% for suprasterol3 I, suprasterol3 II, compound I1, com pound I2 and the sum of compounds IIl and II2, respectively (8). Figure 1 is the scheme of the photochemical reaction of vitamin D summarized by HAVINGA(9). On the basis of the results and considerations mentioned above, we investi gated the effect of wavelength on the photochemical reaction of ergocalciferol chosen as a representative of vitamin D. Monochromatic UV light for irradiating ergocalciferol solutions was obtained by using a spectroirradiator, and the photo chemical reaction products were determined by the GLC method on a wall-coated open tubular glass capillary column (capillary column) as described in a previous paper (10).

EXPERIMENTAL

1. Materials and reagents. Ergocalciferol. A commercial grade from

Philips-Duphar Co. was recrystallized from -water (4:1): mp 115-116•Ž,

E1%1cm,265nm 485 (in ethanol). acetate (CA). Cholesterol, Wako Chem. Ind. Co., was acetylated according to a conventional method and recrystallized from acetone: mp 115•Ž. Other reagents were used according to a previous paper (3).

2. Spectroirradiator. A CRM-FA-type spectroirradiator (Japan Spectro scopic Co.) with a xenon arc lamp as a light source, a grating to obtain a mono chromatic light and an integrator to count quantum of irradiated energy was used.

Irradiation by monochromatic light in the range of 200-700nm was possible with this apparatus.

3. Ultraviolet irradiation of ergocalciferol solution. A solution of ergocalci ferol in ethanol (2.0 or 10.0mg/ml) was used in this work. Exactly 4ml of the solution was taken in a quartz (10•~10•~50mm) placed in a spectroirradiator. After setting wavelength and integrator to obtain a desired monochromatic UV light and quantum of energy, irradiation was carried out under nitrogen flushing. The slit width was always fixed at 10mm. The irradiated solution was trans ferred to a round bottom flask and the cell was washed several times with 2-3ml of ethanol to join the flask. The solvent was evaporated to dryness under reduced 284 T. KOBAYASHI, S. YOSHIMOTO, and M. YASUMURA

pressure below 40•Ž and the residue was dissolved in 1.0ml of pyridine. After adding 1.0ml of an internal standard solution (3.0 or 15.0mg/ml of CA solution in pyridine) to the solution, trimethylsilylation was carried out by mixing with 0.5ml of hexamethyldisilazane and 0.1ml of trimethylchlorosilane and then the solution was allowed to stand for 10 min at room temperature. The precipi tation formed by the addition of TMS reagents was eliminated by centrifugation for 15 min and the supernatant was denoted as a GLC sample solution.

4. Capillary column gas-liquid chromatography (GLC). A glass capillary

(internal diameter 0.25mm, length 56m) was prepared with use of a Shimadzu GDM-1 glass drawing machine according to the direction of SAITO and FURU

KAWA (11). The inside wall of the capillary was coated with OV-17 as a stationary

phase with use of a Shimadzu MCT-1A micro-column treating stand and then dried by nitrogen flushing. The resulting wall-coated capillary column was divided

into three parts of 14, 28 and 14m length. The middle part (28m) was used as a capillary column for the following GLC.

Five ƒÊl of the GLC sample solution obtained above was taken by a micro

syringe and then applied to GLC using the following apparatus and analytical conditions:

Apparatus. A Shimadzu GC-4BPF gas chromatograph equipped with a hydrogen flame ionization detector.

Column. The capillary column obtained above (0.25mm•~28m) was set in a column holder and then connected with a gas chromatograph.

Operating parameters. Temperature: column 240•Ž, detector 280•Ž, injec tion port 280•Ž; flow rate of a carrier gas (N2) 1ml/min.

5. Thin-layer chromatography (TLC). A sample solution in a suitable or

ganic solvent was spotted on a Kieselgel GF254 plate of E. Merck Co. (250ƒÊ-thick, 20•~20cm) activated at 110•Ž for 1 hr and then developed with a mixed solvent

of benzene-acetone (95:5). Spots were visualized as brown spots by spraying 20% p-toluenesulfonic acid in ethanol and then heating.

RESULTS AND DISCUSSIONS

1. GLC on a photochemical reaction mixture of ergocalciferol

A solution of ergocalciferol in ethanol (2.0mg/ml) was irradiated by mono

chromatic UV light of 295nm with a quantum of 8.0•~108erg/cm2 and then a GLC sample solution was prepared according to EXPERIMENTAL. The solution was

applied to both the capillary column GLC as described in EXPERIMENTAL and the

packed column GLC (1.5% OV-17 packed on Shimalite W, 0.3•~260cm) as described in a previous paper (3). Figure 2 shows the gas chromatograms resulting

from the two GLC methods and each peak was assigned from the results similarly

performed on the respective authentic compounds. The separation between TMS ethers of suprasterol2 I and II could not be achieved by the packed column GLC IRRADIATION OF ERGOCALCIFEROL BY UV LIGHT 285

Fig. 2. Gas chromatograms of the TMS ether of an irradiated mixture of ergocalciferol. The numbers in the gas chromatograms mean the TMS ethers of the following compounds

while CA means cholesteryl acetate (internal standard): 1, gyro-D2; 2, suprasterol2 II; 3, suprasterol2 I; 4, 5,6-trans-D2; 5, isopyro-D2. Concentration of ergocalciferol at

irradiation and quantum of irradiated energy were 2.0mg/ml and 8.0•~108erg/cm2 , respectively. whereas the two peaks were successfully separated from one another by the capillary column GLC. Therefore, the capillary column GLC was used in this work.

2. Effect of wavelength on photochemical reaction of ergocalciferol Each 4.0ml of an ergocalciferol solution in ethanol (2.0mg/ml) was individual ly irradiated by one wavelength of the monochromatic UV light, 242, 268, 295,

321, 347 and 373nm, according to EXPERIMENTAL. Quanta of irradiated energy were fixed at 8.0•~108erg/cm2 in all wavelengths by changing irradiation times.

As shown in Table 1, all the irradiated solutions gave a same absorption maximum

Table 1. UV spectral data on the photochemical reaction mixtures of ergocalciferol irradiated by monochromatic UV lights of various wavelengths.

Note: Concentration of ergocalciferol in EtOH at irradiation of all wavelengths was 2.0mg/ml. 286 T. KOBAYASHI, S. YOSHIMOTO, and M. YASUMURA at 265nm in their UV spectra, but the absorbances decreased from that of ergo calciferol. The smallest absorbance was observed in the UV spectrum of the irradiated solution with a light of 295nm, which suggested that the wavelength might be most effective on the photochemical transformation of ergocalciferol. When the solution was further irradiated by the same light, the absorption maxi mum finally disappeared. Figure 3 shows the thin-layer chromatograms of the irradiated solutions. The irradiated solutions with the lights of 347 and 373nm gave one spot cor responding to ergocalciferol, whereas the other solutions gave the four spots, including one unknown spot (Rf, 0.59) and the three corresponding to suprasterol2 I (Rf, 0.37), ergocalciferol (Rf, 0.41) and suprasterol2 II (Rf, 0.50).

Fig. 3. Thin-layer chromatograms of ergocalciferol, 5,6-trans-D2, suprasterol2 I and II and photochemical reaction mixtures of ergocalciferol. D2, ergocalciferol; t-D2, 5,6 -trans-D2; SI, suprasterol2 I; SII, suprasterol2 II; reaction mixture, photochemical reaction mixtures of ergocalciferol irradiated by monochromatic UV light of various wavelengths (the numbers in the chromatograms mean the irradiated wavelengths).

The irradiated solutions were individually treated according to EXPERIMENTAL to prepare each GLC sample solution (30mg/ml of CA solution in pyridine was used as an internal standard solution in these cases) and then applied to the capillary column GLC. A mixture of each 1.0ml of an ergocalciferol standard solution

(8.0mg/ml in pyridine) and of the internal standard solution was similarly tri methyisilylated and then applied to the capillary column GLC. The peak area ratio of each peak to CA was calculated in each gas chromatogram and the relative peak area ratio was calculated by the following formula:

Relative peak area ratio (%)=b/a•~100 IRRADIATION OF ERGOCALCIFEROL BY UV LIGHT 287

a: Peak area ratio of pyro-D2 TMS ether to CA in the gas chromatogram of an ergocalciferol standard solution. b: Peak area ratio of each peak to CA in the gas chromatogram of a GLC sample solution. Figure 4 shows the gas chromatograms of the irradiated solutions with monochromatic UV light in wavelengths of 242, 268, 321 and 347nm (Since that

Fig. 4. Gas chromatograms of photochemical reaction mixtures of ergocalciferol irradiated by monochromatic UV light in wavelengths of 242, 268, 321 and 347nm . The numbers in the gas chromatograms mean the TMS ethers of the following compounds while CA means cholesteryl acetate (internal standard): 1, pyro-D2; 2, suprasterol2 II; 3, supra sterol2 I; 4, 5,6-trans-D2; 5, isopyro-D2. with the light of 295nm has been already shown in Fig. 2 and that with the light of 373nm gave only the peaks of pyro- and isopyro-D2 TMS ethers besides the peak of CA, they were omitted from Fig. 4). The relationship between the irradiated 288 T. KOBAYASHI, S. YOSHIMOTO, and M. YASUMURA wavelengths and the relative peak area ratios is shown in Fig. 5. Since the gas chromatograms of the irradiated solutions with the light of 347 and 373nm were almost identical with that of an ergocalciferol standard

Fig. 5. Relationship between irradiated wavelengths and relative peak are ratios in the gas chromatograms of photochemical reaction mixtures of ergocalciferol. solution, the light was confirmed to have little effect on the photochemical trans formation of ergocalciferol. On the other hand, the gas chromatograms shown in Figs. 2 and 4 indicated that the UV light in a range of 242-321nm brought a similar type of reaction although the extents of reaction were different among the irradiated wavelengths. As shown in Fig. 5, the relative peak area ratios of the irradiated solutions with the light of 295 and 321nm indicated greater decrease on the pyro-D2 values and greater increase on the suprasterol2 I and II values than the respective values of the other irradiated solutions. The results gave a con clusion that the light in the range of 295-321nm must be most effective on the photochemical transformation of ergocalciferol into suprasterol2 I and II. The conclusion on the effect of irradiated wavelength is quite similar to that on the photochemical transformation of provitamin D into vitamin D as reported pre viously (1, 2). A peak corresponding to 5,6-trans-D2 TMS ether was observed in the gas chromatograms shown in Figs. 2 and 4 although the spot of 5,6-trans-D2 could not be detected in the thin-layer chromatograms shown in Fig. 3. Therefore, the peak observed in the gas chromatograms might be derived from the thermal isomerization of the compound II1 and II2. Since the peak area ratios of 5,6 -trans-D2 were very small in all the cases as shown in Fig. 5, the photochemical transformation of ergocalciferol into the compound II1 and II2 might occur to only a small extent regardless of irradiated wavelengths. IRRADIATION OF ERGOCALCIFEROL BY UV LIGHT 289

3. Effect of quanta of irradiated energy on photochemical reaction of ergocalciferol Each 4.0ml of an ergocalciferol solution in ethanol (10.0mg/ml) was indi vidually irradiated by the monochromatic UV light of 295nm with one of the quanta of 7.13, 14.25, 21.38, 28.50 and 35.63•~108erg/cm2. The UV spectra of the three irradiated solutions with the quanta of 7.13, 14.25 and 21.38•~108erg/cm2 gave a maximum at 265nm, whereas those of the other two solutions gave no absorption maximum in the region higher than 210nm. The thin-layer chromato grams of the former three solutions gave the four spots identical with the case of 295nm shown in Fig. 3 (Rf, 0.37, 0.41, 0.50 and 0.59), whereas the latter two solutions gave the three spots (Rf, 0.37, 0.50 and 0.59) with lack of a spot corres ponding to ergocalciferol (Rf, 0.41). The relationship between the quanta of irradiated energy and the relative peak area ratios is shown in Table 2. Decrease of pyro-D2 values with increase of

Table 2. Relative peak area ratios of isomers in the gas chromatograms of photo chemical reaction mixtures of ergocalciferol irradiated by the monochromatic UV light of 295nm with various quanta of irradiated energy.

Note: Concentration of ergocalciferol in EtOH at irradiation was 10.0mg/ml.

suprasterol2 I and II values were observed according to the increase of irradiated energy until the irradiation with the quantum of 21.38•~108erg/cm2, which showed

that the photochemical transformation of ergocalciferol into suprasterol2 I and II

gradually occurred. On the other hand, large deviations on the values were not observed between the irradiated solutions with the quanta of 28.50 and 35,63 •~108erg/cm2, which suggested that the photochemical transformation might be

completed by the irradiation with the quantum of 28.50•~108erg/cm2. Since compounds I1 and I2 which were isolated as the novel photochemical

reaction products of cholecalciferol by BAKKER et al. (8) are thermally isomerized

into pyro- and isopyro-D3 via cholecalciferol at a temperature of GLC analysis, remaining cholecalciferol and the produced compounds Il and I2 in an irradiated cholecalciferol solution can not be distinguished by the GLC analysis. However,

the peaks due to pyro- and isopyro-D2 TMS ethers detected in the gas chromato

grams of the irradiated solutions with the quanta of 28.50 and 35.63•~108erg/cm2 were thought to be derived from only the compounds I1 and I2, because their UV spectra gave no absorption maximum and the spot corresponding to ergocalciferol

was not observed in their thin-layer chromatograms. Therefore, the relative peak 290 T. KOBAYASHI, S. YOSHIMOTO, and M. YASUMURA

area ratios of pyro-D2 on the irradiated solutions might show the sum of the

compounds I1 and I2. The peak area ratios of 5,6-traps-D2 were small and almost constant among

the irradiated solutions with the quanta of 14.25, 21.38, 28.50 and 35.63•~108 erg/cm2. The results suggested that the photochemical transformation of ergo

calciferol into the compounds II1 and II2 might occur to only small extents even though irradiation was prolonged.

REFERENCES

1) KOBAYASHI.T., and YASUMURA,M. (1973): Studies on the ultraviolet irradiation of pro vitamin D and its related compounds. III. Effect of wavelength on the formation of potential vitamin D2 in the irradiation of ergosterol by monochromatic ultraviolet rays. J. Nutr. Sci. Vitaminol., 19, 123-128. 2) KOBAYASHI,T., HIROOKA, M., and YASUMURA,M. (1976): Effect of wavelength on the ultraviolet irradiation of 7-dehydrocholesterol. (in Japanese), 50,185-189. 3) KOBAYASHI,T., and YASUMURA,M. (1972): Studies on the ultraviolet irradiation of pro vitamin D and its related compounds. II. Determination of potential vitamin D2 in ultra violet irradiated products of ergosterol by gas-liquid chromatography. J. Vitaminol., 18, 78. 83. 4) WINDAUS,A., GAEDE,J., KOSER,J., and STEIN,G. (1930): Uber einige krystallisierte Bes trahlungsprodukte aus Ergosterin and Dehydro-ergosterin. Justus Liebigs Ann . Chem., 483, 17-30. 5) WESTERHOF,P., and KEVEELINGBUISMAN, J. A. (1956): Investigations on . VIII . Some hitherto unknown irradiation products of ergosterol. Rec. Tray. Chim., 75, 1243-1251. 6) DAUBEN, W. G., and BAUMANN,P. (1961): Photochemical transformations. IX. Total structure of suprasterol. II. Tetrahedron Letters, 565-572. 7) SAUNDERSON,C. P., and CROWFOOTHODGKIN, D. (1961): The crystal structure of supra sterol. II. Tetrahedron Letters, 573-578. 8) BAKKER,S. A., LUGTENBURG,J., and HAVINGA,E. (1972): Studies on vitamin D and related compounds. XXII. New reactions and products in vitamin D3 . .Rec. Tray. Chim., 91,1459-1464. 9) HAVINGA, E. (1973): Vitamin D, example and challenge. Experientia, 29, 1181-1193; (1976): Photochemistry of trienes-Novel irradiation products in vitamin D field. Chimia, 30, 27-30. 10) KOBAYASHI,T., YOSHIMOTO,S., and YASUMURA,M. (1976): Gas-liquid chromatographic separation of irradiated mixtures of provitamin D and vitamin D on glass capillary column. Vitamins (in Japanese), 50,157-162. 11) SAITO, H., and FURUKAWA, O. (1974): A study on glass open tubular columns used in gas chromatography. Bunseki kagaku, 23, 339-347.