Supplementary material Facile transformation of the five-membered exocyclic E-ring in 13 2 -demethoxycarbonyl chlorophyll derivatives by molecular oxygen with titanium oxide in the dark Naoya Takahashi, Keiya Hirota and Yoshitaka Saga* Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka, Osaka 577-8502, Japan
EXPERIMENTAL DETAILS Synthesis Synthesis of methyl pyropheophorbide a (1) Chl a was extracted from Spirulina geitleri with a mixture of methanol and petroleum ether (2/1, vol/vol). Then, 2% HCl was added to the extract solution, and an organic phase was neutralized with aqueous 4% NaHCO 3. The organic phase was washed with distilled water and dried over anhydrous Na 2 SO 4. After evaporation, the residue was dissolved in methanol, followed by addition of a small amount of H 2 SO 4. The solution was stirred overnight under nitrogen in the dark. Then, the reaction mixture was poured into distilled water, and extracted with CH 2 Cl 2. The CH 2 Cl 2 solution was neutralized with saturated NaHCO 3 aq., washed with distilled water, and dried over anhydrous Na 2 SO 4. After evaporation, the residue was dissolved in g-collidine and refluxed for 3.5 h under nitrogen in the dark. After removal of g-collidine under the reduced pressure, the residue was purified by silica-gel flash column chromatography (FCC) and recrystallization to give methyl pyropheophorbide a (1). 1 H NMR (CDCl 3 ): d, ppm 9.40, 9.30, 8.55 (each 1H, s, 5-H, 10-H, 20-H), 7.95 (1H, dd, J=12, 18 Hz, 3-CH), 6.26 (1H, dd, J=1, 18 Hz, 3 1 -CH-trans to 3-CH), 6.15 (1H, dd, J=1, 12 Hz, 3 1 -CH-cis to 3-CH), 5.26, 5.11 (each 1H, d, J=20 Hz, 13 2 -H 2 ), 4.48 (1H, dq, J=2, 8 Hz, 18-H), 4.30-4.28 (1H, m, 17-H), 3.63, 3.63 3.39, 3.18 (each 3H, s, 2-CH 3, 7-CH 3, 12-CH 3, COOCH 3 ), 3.67-3.59 (2H, m, 8-CH 2 ), 2.74-2.66, 2.61-2.54, 2.34-2.26 (1H + 1H + 2H, m, 17- CH 2 CH 2 ), 1.82 (3H, d, J=7 Hz, 18-CH 3 ), 1.66 (3H, t, J=8 Hz, 8 1 -CH 3 ), 0.42, -1.75 (each 1H, s, NH). 13 C NMR (CDCl 3 ): d, ppm 196.20, 173.50, 171.33, 160.24, 155.04, 150.62, 148.95, 144.88, 141.49, 137.76, 136.12, 135.95, 135.75, 131.50, 130.39, 129.14, 128.27, 122.47, 105.96, 103.98, 97.08, 92.98, 51.67, 51.63, 49.94, 48.00, 30.91, 29.82, 23.11, 19.38, 17.39, 12.07, 12.00, 11.15. MS (MALDI): m/z found 548.4, calcd. for C 34 H 36 N 4 O 3 [M] +, 548.3. UV-vis (CH 2 Cl 2 ): l max, nm 667 (relative intensity, 0.43), 610 (0.08), 539 (0.09), 508 (0.10), 413 (1.00). IR (CH 2 Cl 2 ): n, cm -1 1734 (17 2 -C=O), 1691 (13-C=O), 1621 (C=C). Synthesis of pyropheophytin a (2) Chl a was treated with 2% HCl, followed by neutralization with aqueous 4% NaHCO 3, and dryness over anhydrous Na 2 SO 4. After evaporation, the residue was purified by FCC to give pheophytin a. Pheophytin a was dissolved in g- collidine and refluxed for 3.5 h under nitrogen in the dark. After removal of g-collidine under the reduced pressure, the residue was purified by FCC and recrystallization to give pyropheophytin a (2). 1 H NMR (CDCl 3 ): d, ppm 9.32, 9.22, 8.55 (each 1H, s, 5-H, 10-H, 20-H), 7.91 (1H, dd, J=12, 18 Hz, 3-CH), 6.23 (1H, dd, J=2, 18 Hz, 3 1 -CH-trans to 3-CH), 6.13 (1H, dd, J=2, 12 Hz, 3 1 -CH-cis to 3-CH), 5.28, 5.11 (each 1H, d, J=20 Hz, 13 2 -H 2 ), 5.27 (1H, t, J=7 Hz, P2-H), 4.64-4.55 (2H, m, P1-H 2 ), 4.50 (1H, dq, J=2, 7 Hz, 18-H), 4.30 (1H, dt, J=8, 3 Hz, 17-H), 3.60, 3.39, 3.12 (each 3H, s, 2-CH 3, 7-CH 3, 12-CH 3 ), 3.55 (2H, q, J=8 Hz, 8-CH 2 ), 2.77-2.69, 2.64-2.56, 2.36-2.25 (1H + 1H + 2H, m, 17-CH 2 CH 2 ), 2.01-1.92 (2H, m, P4-H 2 ), 1.84 (3H, d, J=7 Hz, 18-CH 3 ), 1.67 (3H, s, P3-CH 3 ), 1.64 (3H, t, J=8 Hz, 8 1 -CH 3 ), 1.58-0.98 (19H, m, P5-H 2, P6-H 2, P7-H, P8-H 2, P9-H 2, P10-H 2, P11-H, P12-H 2, P13-H 2, P14-H 2, P15-H), 0.88, 0.83, 0.81 (6H + 3H + 3H, d, J=7 Hz, P7-CH 3, P11-CH 3, P15-(CH 3 ) 2 ), 0.29, -1.82 (each 1H, s, NH). MS (MALDI): m/z found 812.1, calcd. for C 53 H 72 N 4 O 3 [M] +, 812.6. UV-vis (CH 2 Cl 2 ): l max, nm 667 (relative intensity, 0.43), 610 (0.07), 539 (0.09), 508 (0.10), 414 (1.00).
Synthesis of Zn pyropheophytin a (3) A methanol solution saturated by Zn acetate was added to an organic solution of 2 and stirred for 2 h under nitrogen in the dark. Aliquot of 4% aqueous NaHCO 3 was added to the reaction solution, and pigments were extracted with diethyl ether. The organic phase was washed with distilled water, and dried over anhydrous Na 2 SO 4. After evaporation, the residue was purified by recrystallization to give Zn pyropheophytin a (3). 1 H NMR (CDCl 3 ): d, ppm 8.70, 8.64, 8.30 (each 1H, s, 5-H, 10-H, 20-H), 7.77 (1H, dd, J=12, 18 Hz, 3-CH), 6.08 (1H, d, J=18 Hz, 3 1 -CH-trans to 3-CH), 5.99 (1H, d, J=12 Hz, 3 1 -CH-cis to 3-CH), 5.07 (1H, t, J=7 Hz, P2-H), 4.46, 4.38 (each 1H, d, J=20 Hz, 13 2 -H 2 ), 4.31 (1H, dq, J=2, 7 Hz, 18-H), 4.19-4.10 (2H, m, P1-H 2 ), 3.99 (1H, d, J=9 Hz, 17-H), 3.28, 3.19, 2.76 (each 3H, s, 2-CH 3, 7-CH 3, 12-CH 3 ), 3.15 (2H, q, J=8 Hz, 8-CH 2 ), 2.37-2.29, 2.16-2.08, 2.00-1.95 (2H + 1H + 1H, m, 17-CH 2 CH 2 ), 1.92-1.87 (2H, m, P4-H 2 ), 1.83 (3H, d, J=7 Hz, 18-CH 3 ), 1.56 (3H, s, P3-CH 3 ), 1.54-0.97 (19H, m, P5-H 2, P6-H 2, P7-H, P8-H 2, P9-H 2, P10-H 2, P11-H, P12-H 2, P13-H 2, P14-H 2, P15-H), 1.41 (3H, t, J=8 Hz, 8 1 -CH 3 ), 0.87, 0.82, 0.80 (6H + 3H + 3H, d, J=7 Hz, P7-CH 3, P11-CH 3, P15-(CH 3 ) 2 ). MS (MALDI): m/z found 874.9, calcd. for C 53 H 70 N 4 O 3 Zn [M] +, 874.5. UV-vis (CH 2 Cl 2 ): l max, nm 655 (relative intensity, 0.71), 606 (0.12), 557 (0.07), 516 (0.05), 424 (1.00). Synthesis of methyl 13 2 -oxo-pyropheophorbide a by LiOH-promoted allomerization LiOH (326 mg) was dissolved in a mixture of distilled water (4.0 ml) and methanol (1.4 ml), and was poured into the THF solution (5 ml) of 1 (52.6 mg). The mixture was stirred vigorously for 3 h in the dark. Then, the reaction mixture was concentrated under reduced pressure and neutralized with acetic acid. The products were extracted with CHCl 3, washed with distilled water, and dried over anhydrous Na 2 SO 4. After evaporation, the residue was dissolved in methanol containing a small amount of H 2 SO 4, and stirred overnight under nitrogen in the dark. The reaction mixture was poured into distilled water, and extracted with CH 2 Cl 2. The CH 2 Cl 2 solution was neutralized with saturated NaHCO 3 aq., washed with distilled water, and dried over anhydrous Na 2 SO 4. After evaporation, the residue was purified by FCC and recrystallization to give methyl 13 2 -oxo-pyropheophorbide a. 1 H NMR (CDCl 3 ): d, ppm 9.77, 9.75, 8.99 (each 1H, s, 5-H, 10-H, 20-H), 8.08 (1H, dd, J=12, 18 Hz, 3-CH), 6.34 (1H, dd, J=1, 18 Hz, 3 1 -CH-trans to 3-CH), 6.26 (1H, d, J=1, 12 Hz, 3 1 -CH-cis to 3-CH), 5.18-5.16 (1H, m, 17-H), 4.69 (1H, q, J=7 Hz, 18-H), 3.80, 3.59, 3.51, 3.29 (each 3H, s, 2-CH 3, 7-CH 3, 12-CH 3, COOCH 3 ), 3.72 (2H, q, J=7 Hz, 8-CH 2 ), 2.86-2.78, 2.74-2.66, 2.41-2.28 (1H + 1H + 2H, m, 17-CH 2 CH 2 ), 1.89 (3H, d, J=7 Hz, 18-CH 3 ), 1.71 (3H, t, J=7 Hz, 8 1 -CH 3 ), 0.12, -2.42 (each 1H, s, NH). 13 C NMR (CDCl 3 ): d, ppm 192.75, 185.19, 174.83, 173.66, 166.85, 153.80, 152.40, 151.12, 144.57, 142.29, 137.42, 137.35, 136.20, 134.43, 131.25, 130.23, 128.90, 126.37, 123.61, 104.88, 104.39, 101.40, 95.56, 52.68, 51.61, 49.28, 31.88, 31.51, 23.84, 19.38, 17.44, 12.51, 12.18, 11.22. HRMS (FAB): m/z found 562.2576, calcd. for C 34 H 34 N 4 O 4 [M] +, 562.2580. UV-vis (CH 2 Cl 2 ): l max, nm 677 (relative intensity, 0.62), 621 (0.05), 518 (0.14), 421 (0.81), 388 (1.00). IR (CH 2 Cl 2 ): n, cm -1 1733 (17 2 -C=O), 1705 (13-C=O, 13 1 -C=O), 1621 (C=C). Estimation of the conversion efficiencies The conversion efficiencies from 1/2 to 1 /2 by contact with TiO 2 particles in the presence of O 2 was estimated by fitting visible absorption spectra after incubation for 4 h with two Gaussian curves possessing the 667-nm and 677-nm peaks, which were parallel to the Q y absorption bands of 1/2 and 1 /2, respectively. The Q y molar extinction coefficients, e=4.71 10 4 and 2.90 10 4 M -1 cm -1, for 1/2 [S1] and 1 /2 [S2] were used for estimation of their concentrations. The conversion efficiency from 3 to 3 was estimated in essentially the same manner as in the case of 1
and 2 on the hypothesis that the relative ratio of the Q y molecular extintion coefficients of 3 /3 was the same as those of 1 /1. REFERENCES S1. Smith KM, Goff DA, Simpson DJ. J. Am. Chem. Soc., 1985; 107: 4946. S2. Ma L, Dolphin D. J. Org. Chem., 1996; 61: 2501.
Fig. S1. 1 H NMR spectrum of purified 1 in CDCl 3. denoted CHCl 3 and impurities in the solvent. Fig. S2. 13 C NMR spectrum of purified 1 in CDCl 3. denoted CHCl 3. Fig. S3. 13 C NMR spectra of purified 1 (A), synthesized methyl 13 2 -oxo-pyropheophorbide a (B), and 1 (C) in CDCl 3 between 180 and 200 ppm.
Fig. S4. FT-IR spectra of purified 1 (A), methyl 13 2 -oxo-pyropheophorbide a (B), and 1 (C) in CH 2 Cl 2. Fig. S5. FT-IR spectra of purified 1 (A), synthesized methyl 13 2 -oxo-pyropheophorbide a (B), and 1 (C) in CH 2 Cl 2 between 1500 and 1800 cm -1.
Fig. S6. On-line visible absorption spectra of major (A: 2 eluted at 86 min) and minor products (B: eluted at 91 min) from 2 by contact with TiO 2 particles in the presence of O 2. Fig. S7. 1 H NMR spectrum of purified 2 in CDCl 3. denoted CHCl 3 and impurities in the solvent.
Fig. S8. 13 C NMR spectrum of purified 2 in CDCl 3. denoted CHCl 3. Fig. S9. 1 H NMR spectrum of the purified minor product from 2 in CDCl 3. denoted CHCl 3 and impurities in the solvent.
Fig. S10. An on-line visible absorption spectrum of 3 (eluted at 28 min) by contact with TiO 2 particles in the presence of O 2. Fig. S11. 1 H NMR spectrum of purified 3 in CDCl 3. denoted CHCl 3 and impurities in the solvent.
Fig. S12. 13 C NMR spectrum of purified 3 in CDCl 3. denoted CHCl 3.