Photophysical studies of pheophorbide a and pheophytin a. Phosphorescence and photosensitized singlet oxygen luminescence

Journal of Photochemistry and Photobiology, B: Biology, 5 (1990) 245 - 254

245

PHOTOPHYSICAL STUDIES OF PHEOPHORBIDE a AND P H E O P H Y T I N a. P H O S P H O R E S C E N C E AND PHOTOSENSITIZED S I N G L E T O X Y G E N L U M I N E S C E N C E A. A. KRASNOVSKY, Jr., K. V. NEVEROV and S. YU. EGOROV

Department of Biology, M. V. Lomonosov Moscow State University, Moscow 119899 (U.S.S.R.) B. ROEDER and T. LEVALD

Department of Physics, Humboldt-University, Invalidenstr. 42, Berlin 1040 (G.D.R.) (Received May 19, 1989; accepted August 19, 1989)

Keywords. Pheophorbide, pheophytin, chlorophyll, triplet state, phosphorescence, singlet oxygen.

Summary The triplet states of pheophorbide a and pheophytin a were studied in several environments by direct measurement of the phosphorescence of the pigments and photosensitized singlet oxygen (102) luminescence. The spectra, lifetimes and quantum yields of phosphorescence and the quantum yields of 102 generation were determined. These parameters are similar for monomeric molecules of both pigments in all the environments studied. Aggregation of the pigment molecules leads to a strong decrease in the phosphorescence and 102 luminescence intensities, which is probably due to a large decrease in the triplet lifetime and triplet quantum yield in the aggregates. The results obtained for pheophorbide a and pheophytin a are compared with those previously' reported for chlorophyll a. The data suggest that the photodynamic activity of the pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures. Aggregated molecules seem to have a much lower activity.

1. Introduction The application of plant pigments for photodynamic therapy (PDT) of tumours is now receiving considerable attention [ 1]. One of these pigments is pheophorbide a which has been reported to be a promising drug for photodynamic treatment of some forms of cancer and psoriasis [2 - 5]. The chemical structure of pheophorbide is similar to that of the main pigments of plant p h o t o s y n t h e s i s - pheophytin and chlorophyll (Scheme 1). There is Elsevier Sequoia/Printed in The Netherlands

246

,C=Cg2 g

CW~

W3C~

C2Ws

113C~

CW3

O=C I

OCll~

0-!~]

Pheophodoide a : R:H Pheoph~jlin a : R= C2oI~39 Scheme i.

convincing experimental evidence that the photodestructive effects of these pigments in tumours, plants and model systems are due to the same photophysical processes i.e: pigment triplet state (3p) and singlet molecular oxygen (102) formation [1, 6, 7]. Therefore the investigation of 3p and IO 2 generation by the pigments is of considerable interest for photosynthesis and PDT research. Reliable information on these processes can be obtained b y direct measurement of pigment phosphorescence and photosensitized 102 luminescence. The existence of pigment phosphorescence and photosensitized 102 luminescence has been observed previously for solutions of chlorophyll a and pheophytin a in organic solvents [ 8 - 11]. We have observed these phenomena in solutions of pheophorbide a [12]. This paper is an extension of that work and summarizes the results of our studies comparing the behaviour of pheophytin a and pheophorbide a in organic solvents and heterogeneous aqueous media.

2. Materials and methods Phosphorescence was measured on the apparatus described previously [11]. Using this equipment we could measure delayed light emission with a lifetime exceeding 0.5 ms. The phosphorescence measurements were carried o u t at 77 K in special metal cuvettes (optical path, 5 mm). The phosphorescence spectra (see Figs. 1 and 2) were corrected for the wavelength response of the detecting system which was determined using calibrated band incandescent lamps. Relative values of the phosphorescence quantum yields ¢Pp were measured using excitation by red light passed through a cutoff filter (KS-15) (~/> 650 nm). The relative ¢Pp values were calculated as the ratio of the phosphorescence intensity to the area under the absorption

247

o.s

\',~.,

I~

//

-'

'~

~

~,...~,

,oo

.

~oo

// I/

~o ~ e°ml

,oo

///

o850J / 9()0

9SO

'tO00

10'50

tt()O

tlg0

~ l"nm 3

Fig. i. Phosphorescence emission spectra (curves 1- 3), phosphorescence excitation spectrum (curve 4) and absorption spectrum (curve 5) of pheophorbide a in diethyl ether (curve 1), ethanol (curves 2, 4 and 5) and in aqueous solution of Triton X-IO0 (curve 3) at 77 K. Values of the monochromator slit width were 2 n m and 8 n m during measurements of the excitation and emission spectra respectively.

tO-J/ i li~~\ ~:I'~'.~ I

,//\~

.....

2 3

111 lli

ili l s

II

SS0

~0

~0

\'-~.

..~

~00

10'S0

400

ttbo

M

~,'-,"~J,V 500

.~0

600

k, ,

700

~ Into ]

~ Enml

Fig. 2. Phosphorescence emission spectra (curves I - 3), phosphorescence excitation

spectrum (curve 4) and absorption spectrum (curve 5) of pheophytin a in diethyl ether (curve 1), ethanol (curves 2, 4 and 5) and in aqueous solution of Triton X-100 (curve 3) at 77 K.

248 spectra of the solutions in the region of excitation. The maximum optical density of the solutions used for Op measurements did not exceed 0.4 at the red absorption band. The IO 2 luminescence was studied at 293 K in air-saturated solutions. For experiments in CC14, in which the 102 lifetime T~ is about 30 ms [11], the apparatus described above was used with a phosphoroscope and monochromatic light excitation. The intensity of the exciting light in these experiments was 0.1 - 0.3 mW cm -2. For experiments in other solvents, in which r~ is shorter than 0.5 ms, pulsed laser excitation and a p h o t o n counting system were used as in refs. 13 and 14. In this case a nitrogen laser was used (single flash energy, 0.04 mJ; flash duration, 10 ns; flash repetition rate, 100 Hz, wavelength, 337 nm). The luminescence was focused onto the entrance slit of a high intensity grating m o n o c h r o m a t o r and was detected with a photomultiplier (S-l) cooled by dry ice. Using this apparatus we could measure the decay kinetics of luminescence with a lifetime greater than 0.5 ps. The total luminescence intensity was obtained by integrating the kinetic curves. Absorption spectra were measured on SF-18 and Specord UV-visible spectrophotometers (U.S.S.R. and G.D.R.). For low temperature measure: ments a small Dewar vessel containing the samples was placed inside the integrating sphere of the SF-18. This allowed us to measure the low temperature spectra of strongly scattering solutions frozen as snows. Pheophytin and pheophorbide were obtained according to the procedure described in ref. 15 with final chromatography on saccharose. The preparation of the pigments was carried out at the A.N. Bach Biochemistry Institute of the U.S.S.R. Academy of Science and the Moscow Institute of Fine Chemical Technology. Pigments were studied in organic solvents (reagent grade) purified by additional distillation, bidistilled H20 and deuterium oxide (D20) containing 0.3% of H20 ("Isotope", U.S.S.R). The non-ionic detergent Triton X-100 was obtained from Loba-Chemie, Austria. The aqueous systems were prepared by mixing water with ethanolic solutions of the pigments. The final concentration of ethanol in water was 5%. The detergent concentration in the aqueous micellar system was 2%. The pigment concentration was 0.1 - 5 #M.

3. Results

3.1. Pigment phosphorescence At 77 K pigment phosphorescence with a lifetime of 1.00 +- 0.05 ms and two spectral maxima at 930 - 938 and 1055 - 1070 nm are observed in all solutions. The intensity ratio of the maxima is 1:0.3 (Table 1; Figs. 1 and 2). In organic solvents and aqueous detergent solutions the phosphorescence excitation spectra coincide with the low temperature absorption spectra of the pigment solutions (Figs. 1 and 2). In addition, the absorption spectra of these solutions are similar at room and liquid nitrogen tern-

249 TABLE 1 Parameters of pigment phosphorescence

Pigment

Solvent

Maxima o f emission spectra a

Tp (+5%) (ms)

~Pp ( + 2 0 % ) (a.u. d)

~p/Tpb (+25%) (a.u.d)

(nm) Pheophorbide a

Pheophytin a

Chlorophyll a [16 18] -

Ethanol Diethyl ether H20 + Triton + 5% e t h a n o l H 2 0 + 5% ethanol

937, 1065 932, 1060 931, 1060

1.0 1.0 1.0

1.0 b 0.6 b 1.1 b

1.0 0.6 1.1

936, 1065

1.0

0.20 b

--

Ethanol Diethyl ether H20 + Triton + 5% e t h a n o l H 2 0 + 5% ethanol

938, 1065 930, 1060 931, 1 0 6 0

1.0 1.0 1.0

1.1 b 0.7 b 1.0 b

1.1 0.7 1.0

932, 1 0 6 5

1.0

0.01 b

--

Ethanol Diethyl ether

980, 1110 929, 1060

1.8 2.7

1.0 c 4.2 c

0.6 1.6

a T h e p o s i t i o n s o f t h e m a x i m a are d e t e r m i n e d w i t h a n a c c u r a c y o f +1 n m a n d +5 n m for t h e s h o r t a n d l o n g w a v e l e n g t h b a n d s respectively. b T h e d a t a were m e a s u r e d using p h e o p h o r b i d e a in e t h a n o l as a s t a n d a r d . CThe d a t a are c a l c u l a t e d o n t h e basis o f t h e results p r e s e n t e d in refs. 17 a n d 18 a s s u m i n g t h a t ¢bp o f c h l o r o p h y l l a in e t h a n o l is e q u a l t o u n i t y . O u r p r e l i m i n a r y m e a s u r e m e n t s have s h o w n t h a t t h e ¢bp values are e q u a l f o r c h l o r o p h y l l a n d p h e o p h o r b i d e in e t h a n o l i c solutions. da.u., a r b i t r a r y units.

peratures (Figs. 1 - 3) [8, 9]. It is known that at room temperature pheophytin and pheophorbide are monomeric in organic solvents and detergent solutions [8 - 10, 12, 19 - 24]. Hence, we can conclude t h a t at 77 K these pigments are monomeric and the phosphorescence detected in our experiments originates from monomeric pigment molecules. Measurement of the phosphorescence quantum yields ~p shows t h a t the ~p values are equal for monomeric pheophytin and pheophorbide {Table 1). These data enable us to estimate the q u a n t u m yields of pigment triplet state formation ¢bt . It is k n o w n [25] that (~p = ~ t T p / T r

where Tp and rr are the real and radiative lifetimes of the phosphorescence respectively. Taking this formula and the data presented in Table 1, we can conclude that the values of Ct/Tr, and hence the ~ t values, are equal for the monomeric molecules of the t w o pigments in all the solvents used. This conclusion agrees with the results obtained by flash photolysis where ~t values of 80% were measured for both pigments in organic solvents [23, 24].

250

t.o-

*

rl ~

1.o

0.5"

0

~

--I .....

P'

2

O.g

~0

6so

7~0

~'[nm]

6~0

650

700

~ ['nm]

Fig. 3. Absorption spectra of pheophorbide (A) and pheophytin (B) in detergent-free water (curves 1 and 2) and ethanol (curve 3) at room (curves 1 and 3) and liquid nitrogen (curve 2) temperatures.

The ~p and ~t/Tr values of monomeric chlorophyll a are more strongly solvent dependent than those of pheophorbide a and pheophytin a (Table 1). This probably means that the solvation state of the chlorophyll molecules strongly influences their ~t values. It is known that in aqueous detergent-free solutions containing 5% ethanol, absorption spectra correspond to aggregated (dimeric and oligomeric) pigment molecules [ 1 9 - 22]. At room temperature, the long wavelength absorption maximum of aggregated pheophorbide lies at 686 nm. At 77 K this band remains dominant b u t an additional band appears at 670 nm i.e. in the region of the maximum of the monomeric pigment molecules (Fig. 3) [12]. This may indicate that partial deaggregation of the pigment occurs on cooling. In aqueous pheophytin solution the position of the red absorption maximum at room temperature varies between 680 and 690 nm, which is a result of the superposition of the t w o bands at 670 and 695 nm. At 77 K these bands become more pronounced, b u t the short wavelength band does n o t increase (Fig. 3). This means that deaggregation of pheophytin does n o t take place in frozen solutions. The phosphorescence spectra and lifetimes in the aggregate-containing aqueous solutions are close to those of the pigment in ethanol. However, the (I)p values in detergent-free solutions are much lower than those of the monomeric pigments (Table 1). Phosphorescence is n o t observed for selective excitation of the aggregates in the region ~ ~ 680 rim. A comparison of the data leads to the conclusion that the phosphorescence is due to monomeric pigment molecules and aggregation results in a large decrease in the phosphorescence, probably due to a large reduction in ~bt or Tp.

3.2. Photosensitized 102 luminescence According to the data reported previously [11, 12], illumination of air-saturated pigment solutions at room temperature induces photosensitized 102 luminescence with a maximum at approximately 1270 nm (Fig. 4). The excitation spectra of luminescence coincide with the absorption spectra of

251

! LO-

O O

O

O

-¢~--~

o

O

O

O

O

O

~2 0 --i

0.5"

~[nm]

0

o

20

40

60

t,[#,]

Fig. 4. Kinetic curves and spectrum of photosensitized :02 luminescence after flash-laser excitation of pheophorbide a in CC14 (curves 1 and 5), D20 plus Triton X-100 (curve 2), diethyl ether (curve 3) and ethanol (curve 4).

the solutions [11, 12]. At low sensitizer concentrations when IO 2 quenching is negligible, the following luminescence lifetimes are obtained: 28 ms in CCla; 35 /~s in D20 containing 2% Triton X-100; 32 /~s in diethyl ether; 14 ps in e t h a n o l . In order to determine the quantum yields ¢ a of 102 production, the relative quantum yields of oxygen luminescence were measured in solutions containing about 10 #M of the pigments (about 0.1 pM in CC14). Solutions of meso-tetraphenylporphyrin (TPP) and mesotetra(p-sulphophenyl)porphyrin (TPPS) were used as standards. TPP was used in organic solvents and TPPS in water and ethanol. Absolute ~pa values were calculated by assuming that the ~ value for TPP and TPPS was 70% -+ 5%. This value is based on an analysis of the data presented in ref. 14. Table 2 shows that the ~ a values of monomeric pheophytin and pheophorbide are approximately equal in organic solvents and aqueous detergent solutions. A comparison with the data of ref. 11 indicates that monomeric chlorophyll has a slightly lower activity. Pheophorbide activity in aqueous detergent solutions is dependent on ethanol concentration. An increase in alcohol concentration from 5% to 10% causes an increase in (I)a from 56% to 70%. It is possible that alcohol enhances pigment solubilization b y detergent molecules. This effect is n o t observed in pheophytin solutions. In detergent-free water, both pigments display low activity. This indicates a strong decrease in 10 2 production on aggregation.

252 TABLE

2

dPA values in pigment solutions Pigments

Solvents

dPA Relative values

Absolute values

(+1o%)

(%)

dPta

Pheophorbide a

CC14 Diethyl ether Ethanol D 2 0 + T r i t o n + 5% e t h a n o l D 2 0 + T r i t o n + 10% e t h a n o l D 2 0 + 5% e t h a n o l

1.15 1.05 0.85 0.70 1.00 ~ 0.02

80 74 60 50 70 <2

80 [24]

Pheophytin a

CC14 Diethyl ether Ethanol D 2 0 + T r i t o n + 5% e t h a n o l D 2 0 + 5% e t h a n o l

1.15 0.95 0.85 1.00 < 0.02

80 70 60 70 <2

8 0 [ 23 ]

Chlorophyll a b

CCI4

0.82

57

60 [ 2 3 ]

a T h e d a t a were o b t a i n e d using t h e flash p h o t o l y s i s m e t h o d in organic solvents at 20 °C. b T h e d a t a are c a l c u l a t e d o n t h e basis o f t h e results p r e s e n t e d in ref. 11.

4. Discussion

The phosphorescence measurements show that the energy, lifetime and quantum yield of the triplet state are similar for monomeric pheophytin and pheophorbide in all the environments studied (Table 1). This means that the phytol tail does not influence the kinetic and spectroscopic properties of the pigment triplet state. However, it changes the type and efficiency of pigment aggregation and probably influences solubilization of the pigments by detergent miceUes. The phosphorescence of aggregated pheophorbide and pheophytin is not observed in our experiments. This is consistent with an assumption that aggregation decreases the ~t and Tp values of pigment molecules. The results of the phosphorescence study are in complete agreement with those of photosensitized 10 2 luminescence measurements. It is apparent from the phosphorescence analysis that the energy, lifetime and quantum yield of the pigment triplet state are consistent with efficient 10 2 production according to the following mechanism p +hv > 1p 3p + 0 2

> 3p > P + 102

253 w h e r e P, 1p a n d 3p are t h e p i g m e n t m o l e c u l e s in t h e g r o u n d , e x c i t e d singlet and t r i p l e t states. I t is k n o w n t h a t if t h e l i f e t i m e o f 3p u n d e r a n a e r o b i c c o n d i t i o n s e x c e e d s 0.1 ms, kinetic analysis o f this m e c h a n i s m gives t h e f o l l o w i n g e q u a t i o n f o r t h e ¢P~ values in a i r - s a t u r a t e d s o l u t i o n s [11, 26] : ~p~ = ~pt(kA/ko~ ) = S ~

t

w h e r e kA is t h e r a t e c o n s t a n t f o r e n e r g y t r a n s f e r f r o m 3p t o 0 2 a n d k o : is t h e t o t a l rate c o n s t a n t f o r 3p q u e n c h i n g b y o x y g e n . I t is also k n o w n t h a t f o r p o r p h y r i n s t h e S~ c o e f f i c i e n t is close t o u n i t y [ 2 6 ] . O u r e x p e r i m e n t a l d a t a p r e s e n t e d in T a b l e 2 are c o n s i s t e n t w i t h this s c h e m e . T h e ~A a n d q~t values are similar a n d t h e SA c o e f f i c i e n t c a n be e s t i m a t e d t o be 0.75 - 1. A g g r e g a t i o n o f p h e o p h y t i n a n d p h e o p h o r b i d e m o l e c u l e s leads t o a s h a r p decrease in t h e q~A values. This is c o n s i s t e n t w i t h t h e results o f o u r p h o s p h o r e s c e n c e s t u d y a n d p o i n t s t o a decrease in q~t a n d / o r t h e t r i p l e t state lifetime rt on aggregation. In a g r e e m e n t w i t h this c o n c l u s i o n a decrease in ~ t a n d Tt has b e e n o b s e r v e d d u r i n g an investigation o f t h e c o n c e n t r a t i o n q u e n c h i n g o f p i g m e n t t r i p l e t s t a t e f o r m a t i o n [ 2 7 ] . A similar decrease in p h o t o s e n s i t i z i n g a c t i v i t y has b e e n o b s e r v e d on aggregation o f c h l o r o p h y l l , p r o t o c h l o r o p h y l l , p r o t o c h l o r o p h y l l i d e , p r o t o p o r p h y r i n IX, m e s o p o r p h y r i n I X a n d t h e i r derivatives [14, 2 8 ] . A discussion o f t h e e x p e r i m e n t a l d a t a enables us t o p r o p o s e t h a t the p h o t o d y n a m i c activity o f p h e o p h o r b i d e , p h e o p h y t i n a n d c h l o r o p h y l l a n d t h e i r p r e c u r s o r s in p l a n t a n d a n i m a l tissues is d u e m a i n l y t o m o n o m e r i c p i g m e n t m o l e c u l e s ; a g g r e g a t e d m o l e c u l e s are likely to be m u c h less active.

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