obtained almost quantitatively from the transformation of
HA.22 There have been some investigations on the metal ion-
complexed derivatives of hypocrellins; however, systematic
and, especially, quantitative studies on the photodynamic
actions of these complexes have not yet been performed. In
this work, we chose HB as the model compound and synthe-
sized the complex of HB with Al3`. Full experimental data on
the synthesis and characterization of Al3`ÈHB (3) is reported.
In addition, we investigated the generation of superoxide
radical anions (O ~~) and singlet oxygen (1O ) through photo-
recorded on a Hitachi F-4500 Ñuorescence spectrophotometer.
The Ñuorescence quantum yield was obtained by the relative
method using HB in DMSO as a standard. The Ñuorescence
quantum yield of HB was assumed to be 0.0058 and the exci-
tation wavelength used was 570 nm.23 In all cases, the absorb-
ance values at the excitation wavelength were lower than 0.1
for a 1 cm pathlength. IR spectra were measured with a
BIO-RAD FTS 165 grating spectrometer. 1HNMR spectra
were measured with a Varian XL-400 instrument (300 MHz).
Elemental analyses (C, H, O) were performed on a Carlo
Erba-1106 elemental analyzer. The percentage of chlorine was
measured by the Hg titration method. The percentage of alu-
minium was measured on an IRIS Advatage ICP emission
spectrophotometer.
2
2
sensitization of this new material using electron paramagnetic
resonance (EPR) and spectrophotometric methods. To quan-
tify the formation of O ~~ during Al3`ÈHB-mediated photo-
2
sensitization, we have utilized the cytochrome c reduction
method. The quantum yield of 1O generated by Al3`ÈHB
2
was also determined.
Characterization of the Al3‘–HB complex
The complexation of HB with increasing amounts of Al3` can
be recorded spectrophotometrically. The addition of Al3`
causes a bathochromic shift and an increase in the intensity of
the absorption bands of HB (Fig. 1). The changes in the
absorption spectra imply that a complex of HB with Al3` is
produced. When the concentration of Al3` added to the solu-
tion reached 100 lM, which was equal to the initial HB con-
centration, the changes in the absorption spectra tended to
tail o†.
During complexation, a single group of isosbestic points at
315, 349, 384 and 462 nm was maintained within the spectral
region (300È700 nm) under study, indicating that only one
species is produced in the system. The absorption data for the
Al3`ÈHB complex are shown in Table 1.
It may be observed that the absorption spectrum of the
Al3`ÈHB complex is somewhat similar to that of the dianion
of HB. Fig. 2 shows the absorption spectrum of HB in NaOH
solution. This suggests that Al3` bonds with the phenolic
hydroxy oxygen and the carbonyl oxygen of HB, through
removal of the phenolic hydroxy proton.
Experimental
Chemicals
Hypocrellin B was prepared as described previously.22 5,5-
Dimethyl-1-pyrroline-N-oxide (DMPO), 2,2,6,6-tetramethyl-4-
piperidone (TEMP) and 9,10-diphenylanthracene (DPA) were
purchased from Aldrich Chemical Company. Superoxide dis-
mutase (SOD) and cytochrome c were purchased from Sigma
Chemical Company. Reduced nicotinamide adenine dinucleo-
tide (NADH) was obtained from Biochem. Technology Cor-
poration, the Chinese Academy of Sciences. 1,4-
Diazabicyclo[2.2.2]octane (DABCO) and dimethylsulfoxide
(DMSO) were purchased from Merck Chemical Company.
Anhydrous AlCl , sodium azide (NaN ), deuterated solvents
3
3
and other agents of analytical grades were purchased from
Beijing Chemical Plant. The working stock solutions were
prepared immediately before use.
Synthesis of the complex of HB with Al3‘
AlCl (100 mg) and HB (95 mg) were mixed and vigorously
3
stirred in CHCl (20 ml) for 5 min at rt in the dark. The
3
mixture was Ðltered and the solvent evaporated from the Ðl-
trate under reduced pressure. The residue obtained was re-
dissolved in deionized water and dialyzed against deionized
water using a Spectrapor membrane with a molecular weight
cut-o† of 6000È8000. The low molecular weight components
(\6000È8000) di†usive across the Spectrapor, while the
higher molecular weight materials are left inside the dialysis
bag. After dialysis, the retained solution was dried and the
desired complex (dark-red, amorphous powder) was obtained
(108 mg, yield: 91%) (Anal. calc. for Cl~ É Al3`ÈHB
(C
H
AlClO ): C, 61.22; H, 3.74; O, 24.49; Cl, 5.95; Al,
30 22
9
4.60%. Found: C, 60.78; H, 3.87; O, 24.78; Cl, 5.93; Al,
4.64%.).
Spectroscopic measurements
Fig. 1 Changes in the absorption spectrum of HB on addition of
various amounts of Al3` to an ethanol solution of HB (100 lM).
[Al3`]/[HB] \ 0, 0.2, 0.5, 1, 4. Arrows indicate the directions of
change.
The UV-Vis absorption spectra were recorded on a Shimadzu
UV-1601 spectrophotometer. Fluorescence spectra were
Table 1 Spectral parameters of HB and Al3`ÈHB complex
Compound
jA /nm (DMSO)
jF /nm (DMSO)
U ] 103
d [(CD ) SO]
3 2
max
max
F
H
2
468, 545, 590
618
5.8
16.27, 16.20 (2H, s); 6.65, 6.63
(2H, s); 4.11, 4.08, 4.07, 3.97
(12H, s); 3.94, 3.18 (2H, dd, J \
11 Hz); 2.28 (3H, s); 1.83 (3H, s)
3
503, 568, 614
621
2.3
6.75, 6.66 (2H, s); 4.18, 4.12,
4.09, 4.01 (12H, s); 3.98, 3.22
(2H, dd, J \ 11 Hz); 2.35 (3H, s);
1.92 (3H, s)
848
New J. Chem., 2001, 25, 847È852