Radiat Environ Biophys (1990) 29:225-239 Radiation and Environmental Biophysics © Springer-Verlag 1990

Photoproducts generated from hematoporphyrin under high-intensity picosecond flash photolysis Evidences for involvement of biophotonic processes

T. Gantchev 1, E. Keskinova 2, M. Kaltchev 3, and D. Angelov 2 10ncochromosome Laboratory, National Center of Oncology, Bulgarian Medical Academy, BG-1156 Sofia, Bulgaria 2 Institute of Solid State Physics, and 3 Institute of Kinetics and Catalysis, Bulgarian Academy of Sciences, Sofia, Bulgaria

Received December 20, 1989 / Accepted in revised form March 19, 1990

Summary. Hematoporphyrin (Hp) solutions were subjected to a wide range high intensity (0.2-10.0 GW/cm 2) near-UV laser pulse radiation (2exc = 355 nm, pulse duration 30 ps). The formation of stable Hp photoproducts was followed by UV-VIS absorption spectroscopy and liquid-gel column chromatography. As judged by the influence of free radial scavengers, a significant part of the products is assigned to arise from the reaction of Hp with OH- (and H-) radicals. Using nitroxide radicals (TEMPO and TEMPONE) and the spin trap DMPO the generation of primary transient photoproducts, hydrated electrons (e~-q), OH- and H- radicals, was studied varying the pulse intensity at a constant absorbed light energy. The results showed that bi-photonic processes are responsible for the observed product generation (different for e~q photoejection and OH.(H.) formation). A tentative diagram of the Hp excitation routes involved in the present high intensity laser flash photolysis is suggested. According to it, OH. and H. radicals are supposed to be generated in a resonance energy transfer reaction from highly excited Hp** to water molecules (H20 sensitization).

Introduction

Porphyrins (e.g. hematoporphyrin, Hp) are known to be efficient sensitizers for photooxidation of various of biological molecules [29]. Many studies carried out using homogeneous and organized media, and applying continuous wave (CW)-low intensity laser (lamp) light irradiation show that two main photoreac- tion mechanisms sensitized by Hp in its lowest triplet state are involved, i.e. electron transfer to oxygen and/or substrate with the generation of O~-. and/or radical species [6, 10, 13], and energy transfer to ground-state oyxgen (302) yielding singlet oxygen (102) [26, 28, 31]. On the other hand, it is well established that light absorption by various aromatic compounds under flash photolysis

226

conditions (high intensity pulse illumination) is followed by other excitation- deactivation pathways responsible for direct free radical generation and/or pho- toejection of electrons (e2q formation). In many cases these processes involve consecutive biphotonic light absorption via lowest excited singlet [-9, 16] or triplet [-4, 32] states. Several studies aimed at elucidation of high intensity laser pulse excitation of Hp (or its derivative HpD, used in photochemiotherapy of tumors [8]) were recently performed [3, 20]. However, the type of generated transient products was not clearly demonstrated. In general, reactive intermedi- ates, such as solvated electrons (e2q), hydroxyl radicals (OH.) and hydrogen atoms (H.) may result from the deactivation of high-energy excited Hp. In our previous study applying nanosecond (10 ns) or picosecond (30 ps) laser flash irradiation (2exc = 355 rim) and direct optical detection of intermediates formed we have found that electrons can be photoejected from excited Hp molecules by a biphotonic process [17]. The maximum values of the quantum yields were determined to be 0.02 and 0.17 for ns and ps pulse excitation, respectively. We have continued these investigations applying the same high intensity ps laser pulse irradiation (varying pulse intensity and total absorbed light energy) and following the type and yield of different transient or stable products formed. The production of highly reactive intermediates (e.g. OH. and e~-q) was followed by EPR and spin-trapping techniques. The formation of stable Hp photopro- ducts resulting from porphyrin "self"-sensitization reactions was observed by absorption spectroscopy and liquid-gel column chromatography.

Materials and methods

Materials

Hematoporphyrin IX diHC1 (Hp, approx. 85% pure) was purchased from Por- phyrin Products (Utah, USA) and used without further purification. The main impurities were specified as Protoporphyrin (PP) and Hydroxyethylvinyl-deuter- oporphyrin (HVD). Hp solutions were prepared by dissolving known amounts Hp in 0.1 N NaOH and adjusting to the desired pH value by 0.1 N HC1 or 0.01 M phosphate buffer (pH 7.4). Flash photolysis experiments were performed using alkaline (pH 11), neutral (pH 7.4) and acidic (pH 3) solutions. In solutions of differern pH two basic hematoporphyrin equilibria exist, monomer/dimer/ aggregate and protolytic which may alter the light excitation/deactivation Hp processes [-19, 24, 25]. Furthermore, the lifetime and the reactivity of the tran- sients may be different at different solution pH.

The nitroxide radicals 2,2,6,6-tetramethylpiperidine-l-N-oxyl (TEMPO), 2,2,6,6-tetramethyl-4-oxopiperidine-l-N-oxyl (TEMPONE) were synthesized in the Institute of Organic Chem. (Bulg. Acad. Sci.). The spin trap: 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) was purchased from Aldrich Chem. Co. (Milwaukee, USA) and purfied removing coloured impurities before use. Lipophilic Sephadex LH 20 was a Pharmacia Biotechnology product (Uppsala, Sweden). All other chemicals used were of reagent grade quality. Most of the experiments were

227

mode t ocked Nd : YAG 2~ 3~

KDP KDP 3 (2)

m SRS ~=L700nm •

3~ uvf~lett e

laser crossection [amptifier s~ variation : ] ( I= 106 +1010 W/cm 2 ) Jpeak detectors[

I I A°c I

/

[Ap~te lie I

Fig. 1. Principal optical scheme for the laser flash photolysis with high-intensity 30 ps pulses (used for excitation and probing): SRS, stimulated Raman scattering cell (medium CH3CN); EMS, electromechanical switch; (1), calibrated thermopile energy detector; (2M), photodiodes: E(2)/E(1) ~ transmission at 355 nm; E(4) /E (3 )~ transmission at 700 nm (for e~q-detection)

performed in air-saturated solutions, unless otherwise stated. Deaeration was performed by solution bubbling with high purity argon or nitrogen oxide (NaO). All other chemicals used were of reagent grade purity. Bidistilled deionized water was used for solution preparations. Hp concentration was determined spectrophotometrically using Hp absorptivity value e4o 1 n m = 4.23 x 105 M- 1 cm- 1 in 0.25 M H2SO 4.

Methods

Near-UV light irradiation of Hp solutions was performed by mode locked Nd- YAG pulse laser: 2~xc= 355 nm, pulse duration 30 ps, pulse energy 3-5 mJ and pulse intensity variation: 108-101° W/cm 2. The intensity of the laser radiation was varied by changing the excitation beam cross-section (Fig. 1). The pulse energy was controlled by a calibrated energy meter connected to an Apple II-computer allowing automatic control of the total irradiation dose. The given optical scheme, separation of the excitation (2=355 nm) and monitoring (2 = 700 nm) beams was also applied for direct observation of hydrated electron formation under the present irradiation conditions [17]. Two other light sources were also used: N2-pulse laser (2exc= 337 nm, pulse duration and energy 10 ns and 3 mJ, respectively and pulse intensity variation: 1 0 7 - 108 W/cm2; For CW irradiation argon-dye laser system ("Spectra Physics", Argon laser, model 171 and dye laser, model 375 with maximum power output of 0.5 W/cm 2 at 632

228

+2 nm) was used. Solutions were irradiated in quartz cells of different optical path (0.2, 0.5 and 1.0 cm). Since, some of the DMPO-spin adducts were expected to be short lived, when spin-trapping experiments were performed, after irradia- tion the solutions were rapidly chilled in liquid nitrogen and unfrozen immediate- ly before the EPR measurements. The duration of all manipulations after the irradiation, incl. sample freezing, thawing and EPR cell adjustment was not longer than 2-3 min. No significant EPR spectra changes were observed in repetitive scans after 5-10min. The main spin adducts detected (e.g. D M P O - O H ) showed decay half lifetime of c.a. 90-100 min, consistent with previous observations [-11]. EPR measurements were performed using 200 D SRC Bruker instrument equipped with an Aspect computer system. Spin adduct concentrations were calculated using nitroxide radicals as a standard. Hp UV- VIS spectra were obtained on Shimadzu 3000 double beam-dual wavelength spectrophotometer in 1.0 or 0.2 cm optical path quartz cells. Liquid-gel chroma- tography of Hp was performed on Sephadex LH 20 column (2 x 25 cm) equili- brated with an elution system appropriate for Hp disaggregation-dioxane:meth- anol:0.01 M phosphate buffer (pH 7.4)= 2:1:1 (v/v). The column was calibrated with porphyrins of known structure: PP, HVD and dihematoporphyrin ether (ester), DHE. Elution profiles were obtained applying continuous registration at 2 = 395 nm and flow rate of 0.3 ml/min, all experiments were performed at room temperature (200-22 ° C).

Results

a) Changes of the optical properties and Hp products formation with variation of the pulse intensity and absorbed energy

Different activated oxygen species (102, 02 ", OH-, etc.) were reported to inter- act with porphyrin macrocycle resulting in damage of the conjugated system and/or irreversible modification of its periphery [2, 7, 18]. Figure 2a shows the absorption spectrum changes of Hp solutions (air saturated) irradiated under 355 nm laser pulses of different intensities and total absorbed energies. The main observed differences are: i) decreased intensity of B-absorption band (Soret band); ii) slight increase in the region of 450 nm, and occurrence of a new absorption band at 650.680 nm; and iii) relative changes of Q~, (0-1) and Qy (0-1) vibronics (lowest porphyrin electronically excited state). In order to avoid certain aggregation effects which can mask the real spectrum changes within the Soret band region we have measured the relative absorption changes at 2 = 401 nm of the irradiated Hp solutions subsequently dissolved in 0.25 M H2SO 4 (disaggregated porphyrins in a dicationic form). Figure 2b shows the obtained data as a function of laser pulse intensity (at a constant absorbed energy) for several different irradiation conditions: optically thin ( O . D . 3 5 5 n m

= 0.21 cm-1) and thick (O.D. 35s nm= 1.1 cm-1) Hp solutions; and in the pres- ence of 102-quencher (2 mM NAN3). As can be seen, in all cases in the low intensity range the absorptivity decreases linearly with the pulse intensity increase. At higher intensities (> 1 GW/cm 2) the absorptivity drop is saturated.

229

The absence of N a N 3 effect on the O.D. change should suggest negligible partici- pation of tO2 in the above processes (no triplet state mediated photosensitizing reactions). Further evidience that the generation of Hp photoproducts is pulse intensity dependent was obtained by liquid-gel chromatography of the irradiated solutions on Sephadex LH 20 column. Several typical elution profiles are shown in Fig. 3. In the left column a) the chromatogram of the starting compound is shown. Arrows 1) and 2) indicate the Hp peak and the unresolved peak from more hydrophobic porphyrins (PP and HVD, present as impurities), respec- tively. Column b) shows the chromatograms obtained after irradiation of air- saturated Hp solutions at 0.5 G W / c m 2 pulse intensities and different absorbed light energy (50-400 m J). Column c) shows the chromatograms obtained under the same conditions as in b), but applying pulses with 8.5 G W / c m 2 intensity. As can be seen the amounts and/or the type of the products are irradiation dose and pulse intensity dependent. It is also notable that at high doses and/or intensities a dimeric (oligomeric) photoproduct is generated (arrows-3). In gener- al, the identification of hematoporphyr in photoproducts is a difficult problem. Our technique did not allow the exact types of photoproducts to be determined

Fig. 2. a Absorption spectrum of 22 pM Hp in alkaline (pH 11) solution before irradiation (1, - ) ; and after irradiation at: total absorbed energy E= 100 mJ/ml, pulse intensity 1=0.5 GW/cm 2) (2, -- --); E=800 mJ/ml, I=0.5 GW/cm 2 (3, --.) and E = 800 mJ/ml, 1= 8.0 GW/cm 2 (4, - " --). Qx{Y) and B, spectroscopic symbols for S1 and $2 Hp excited states, respectively. b Changes in absorptivity at 401 nm vs. irradiation pulse intensity, measured in 0.25 M H2SO 4 (10 x dilution of the irradiated solution, 0.2 cm optical path cell): optically thick solutions (OD355n m =1.1 cm -i, pH 11), E=0.4J/ml (--©--) and E=0.4 J/ml in presence of 2 mM NaN3 (-- • --), E = 1.0 J/ml (-- i --); and optically thin solution (OD355n m =0.21 cm -1, pH 11), E=0.4J/ml (--A--)

..? .~,'

a)

I A= 0,025

I I I I 4.00 500 600 70C

1,0

0,8

0,6

E~ 0,4

o 0,2

X (nm)

b) Q

-&

A

~ A • • •

2 4 6 8 10 12 I (GW / cm 2)

230

(due to the fact that some of the peaks are unresolved and/or lacking of appro- priate standards). In Fig. 3, (column-d), chromatograms obtained after irradia- tion of Hp solutions under different conditions are also shown for comparison: i) CW-light irradiation by argon-dye laser, 2 = 632 nm and total absorbed energy 10 J (dl); ii) high-intensity pulse irradiation, but in the presence of OH. radical scavengers: 10% methanol, pulse intensity 8.5 GW/cm 2 and dose 200 mJ, (d2); and iii) the spin trap DMPO (50 mM), pulse intensity 8.5 GW/cm z and dose 400 mJ (d3). As it was shown previously for PP [7], when CW irradiation is applied (d0 the main photoproducts are expected to arise exclusively after Hp interaction with activated oxygen species (tO 2 or 0 2 "). In the presence of OH. (or other radicals) scavengers, the yield of products formation is decreased (d2 and d3). Thus, it can be suggested that the major part of the products formed when Hp is irradiated by high intensity light, arise under free radical attack from the bulk aqueous phase, but not from Hp fragmentation [1, 25].

b) EPR and spin trapping experiments

Nitroxide radicals are known to act as hydrated electron (ejq) and hydroxyl radical (OH.) scavengers [22, 31]. The irradiation of Hp solutions in the presence

8,5 GW/cm 2

lV 0,5 GW/cm 2 l

dos l /~f ]

02 20 2 2 0 0 ~

I

lh r retention time ---,-- ~, = 395 nm reg

d 1

Fig. 3. Chromatograms of equal Hp amounts obtained on Sephadex LH 20 column (2 x 25 cm) using elution system: dioxane: MeOH: 0.01 M buffer (pH 7.4)=2:1:1. Non-irradiated solution (column a)); after ps-laser pulse photolysis at 0.5 GW/cm 2 per pulse (column b)) and 8.5 GW/cm 2 per pulse (column c)). Total absorbed energies as indicated (E = 50400 m J). Chromatograms in column d) sample irradiated by CW-Argon/ dye laser 2ox~=632 nm, I=200 mW/cm 2, E= 10 J (dl); samples after ps-laser photolysis (I = 8.5 GW/cm 2) in the presence of free radical scavengers: 10% MeOH, E=200 mJ (d2); and 50 mM DMPO, E=400 mJ (d3). Arrows indicate: (1) Hp; (2) PP and HVD; and (3) Hp-oligomers

231

of TEMP O or T E M P O N E resulted in reduction of the nitroxide radicals as indicated by the decrease in the low field EPR spectrum line intensity (A_ 1, Fig. 4). As can be seen a more efficient reduction of the nitroxide radicals takes place at intensity variations in the region below 0.5 GW/cm 2. Solution irradia- tion with higher pulse intensities (> 2 GW/cm 2) resulted in plateau shaped A_ 1 change. When the solutions contained additionally 2 m M ferricyanide (e2q scav- enger), the A_ 1 amplitude decrease was negligible in the low intensity region (not shown), which indicates predominant e2q participation in the nitroxide radi- cal reduction process. Further nitroxide reduction should arise from the involve- ment in the overall process of other free radicals (e.g. OH-, see below). It is to be noted that the experiments performed under the same irradiation condi- tions and measuring the transient optical density (A O.D. V00nm) also indicated high rate ef~ photoejection (quantum yield <0.17) in the intensity region below 0.5 GW/cm [17].

Figure 5 shows the EPR spectra obtained after irradiation of Hp-DMPO air-saturated solutions at different pH values. In acidic solution (pH 3, spectrum a) a typical D M P O - O H spin-adduct spectrum is obtained (aN=a/ /= 14.9 G). In neutral (pH 7.4) and alkaline solutions (pH 12), spectra b and c, respectively, the DMP O-OH spectrum is superimposed with another 9 line spectrum (aN =16.7 G and azr/=22.4 G). According to the pulse radiolysis data [30], the DMPO nitroxide, exhibiting coupling to two equivalent protons, must arise from tapping of hydrogen atoms (reaction 1) or electrons followed by protona- tion (reaction 2):

DMPO + H" , D M P O - - H (1)

H +

DMPO + e L , [DMPO] - , DMPO -- H (2)

Several arguments can be adducted in support of the statement that under the present conditions of high intenstity flash photolysis reaction (1) is mostly operative: i) the pulse intensity response of the quantum yield of D M P O - H and D M P O - OH is found to follow a significantly different dependence as corn-

100-

80 - Fig. 4. Reduction of nitroxide radicals with variation of pulse intensity at a o~ 6 O- constant absorbed energy (500 mJ/ml) followed by the height change of high < ' ~ 0 field EPR spectrum line (A_ i)- Solutions contained 80 ~tM HP and 32 gM TEMPO ( - • - - ) or 20 TEMPONE (-- © --), pH lt. Averaged data from 5 independent measurements

o o

o%'"

o

I

0 2

• o e o o o

o

t" I-

o o (°) (o)

i I I i I

4 6 8 lO ] (GW/cm 2 )

232

lOG

c)

DMPO -H

I

DMPO-OH

I [ L I 1 I

Fig. 5a-e. Spin-adduct EPR spectra observed after irradiation of 0.11 mM Hp and 33 mM DMPO air-saturatd solutions at: a pH 3.0; b pH 7.4; and c pH 11. Irradiation conditions: sample volume 400 gl; OD35~nm= 1.0 in 0.2 cm cell; E=200 mJ and pulse intensity 4 GW./cm 2. EPR spectrometer settings: CF 3470 G; SW 200 G; M 0.8 G; TC 100 ms; ST 200 s; P 6.4 mW; and Gain 2 x 105

pared to the e2q one [17] (compare also Figs. 4 and 7); ii) the generation D M P O - - H adduct is also effective in alkaline solutions (Fig. 5c); iii) solution bubbling with N20 (e~q scavenger) during irradiation with high intensity (> 2 GW/cm 2) pulses does not affect significantly D M P O - OH and D M P O - H EPR spectra intensities (to be published); and iv) the irradiation of D M P O / H p solutions by 10 ns pulses derived by N2-1aser with intensity range 50-100 ~MW/cm 2 does not result in D M P O - H (and D M P O - OH) spin adducts although at these light intensities hydrated electrons can be effectively generated [17].

Several other control experiments were also performed. No EPR signals were observed when DMPO alone was irradiated under wide range pulse intensi- ties (0.5,-10 GW/cm2). Evidence that the observed D M P O - O H spectrum arises from direct OH. free radicals trapping was obtained by performing experiments in the presence of OH. radicals scavenger (5% methanol). The obtained EPR spectrum is shown in Fig. 6 a. This spectrum is a superimposed one involving three ~tifferent spin adducts: D M P O - O H (aN=aN= 14.9 G), D M P O - - H (aN = 16.6 G, an=22.4 G) and D M P O - CH2OH (aN= 16.1 G, an=22.7 G). As can be seen the D M P O - O H spectrum intensity is much lower than that obtained

X X X X

0 0 + + +

233

X X X X

O O ÷ + ÷

Fig. 6a, b. Spin adducts observed after irradiation of Hp/DMPO air-saturated solutions (pH 7.4) containing also: 5% MeOH (a); or 2% v/v SDS (b); DMPO--H (x); DMPO--OH (traces) (©); DMPO-CHzOH (+). All other conditions as in Fig. 5

lOG

under the same irradiation conditions but without CH3OH. Thus it can be assumed that the major part of D M P O - O H is primarily formed spin-adduct and does not originate from any secondary reaction (perioxy-radicals mediated) or DMPO oxidation/addition reaction (at least in neutral solutions). No spin- adducts were registered when Hp/DMPO flash photolysis was performed by N2-1aser (2exo = 337 nm, intensity range 50-100 MW/cm2). From this it follows that for the generation of free radicals (OH. and H.), Hp excitation by high photon flux density is required (pulse intensity higher than ~ 1 GW/cm2).

Further evidence that the observed formation of OH- radicals was not due to any secondary process involving participation of peroxyradicals generated for example after interaction of H. (or eeq) with 02 was obtained by illuminating of Hp-DMPO solutions in the presence of superoxide dismutase (SOD). Using SOD concentration up to 1000 units/ml resulted only in subtle attenuation of the DMPO-OH signal (less than 30%). The same weak effect on DMPO-OH yield was observed after performing flash photolysis in deaerated solutions. How- ever, under the latter conditions the concentration of D M P O - H adduct was significantly increased (to be published). This is an evidence that spin-trapping of H" radicals by DMPO in air-saturated solution is a competitive reaction with the high rate reaction between H. and molecular oxygen (k~2 x 101° M -1 "s -1, [-5]).

Several other experiments demonstrated that Hp triplet states are not involved in OH. and H. generation processes. It is well known that triplet state photosensitizing reactions are very sensitive to the Hp aggregation state in solution. The aggregation results in increased excited-ground state quenching and decreases the yield of transients formation (e.g. Hp triplets, activated oxygen species, etc.) [25, 31]. Under the present irradiation conditions, however, Hp disaggregation obtained by adding 2% SDS did not result in marked change of D M P O - - O H yield (Fig. 6b). It is noteworthy that under these conditions D M P O - - H adduct was not registered (probably H- was scavenged by SDS, or other environmental effects were involved, e.g. high 02 local concentration).

234

1,0- ×

o,8- , . -- .

~0,6- C ) el_

~- 0,~- EEl

0,2-

. ~ - - cr - - 5 ? -

i / 5 / / ~

/ ~

i I ' I [ ~ I

2 4 6 8 10

I (5W/cm 2 )

Fig. 7. D M P O - O H spin-adduct concentration vs. pulse intensity at a constant observed energy (200 mJ/200 gl) in solutions of pH 3 ( - A --); pH 7.4 (-- e --); and pH 11 ( - o - - ) . Solutions absorptivity at 355 nm in 0.2 cm optical path cell: 0.65 (pH 3.0) and 2.0 (pH 7.4 and pH 11.0). Averaged data from 3-5 independent measurements

The result that OH. radical formation is not apparently dependent on Hp aggre- gation state should imply involvement of a photosensitizing process which avoids Hp triplet state population. Under the same flash phototysis conditions (I-- 8 GW/cm2), when the solution contained also 20 m M EDTA (capable to reduce Hp via its excited triplet state) no differences in the spin-adducts spectra intensities were found (not shown).

In order to obtain more complete information about the photoprocesses taking place when Hp is irradiated by high-intensity laser pulses, a set of experi- ments was performed applying constant absorbed light energy (200 m J/200 gl) and varied pulse intensity (0.2-10.0 GW/cm 2) with all other conditions equal, and measuring the yields of trapped spin-adducts. Figure 7 shows the D M P O - - O H yields obtained for solutions with different pH as a function of pulse intensity. As can be seen the intensity response of the DMPO-OH adduct concentration follows a non-linear dependence: below 2 GW/cm 2, the DMPO- OH yield is very low (~0 .1=0.2 ~tM), increasing rapidly several times when the pulse intensity is higher than 2-3 GW/cm 2, and remaining constant with further intensity increase (plateaus in Fig. 7). The obtained sigmoidal curves show clearly that the OH. generation process is not monophotonic [21]. The calculated maximum quantum yields (Q = number of D M P O - OH adducts/total number of absorbed photons) are 3.9 x 10 -4, 2.4 x 10 -4 and 3.7 x 10 -4, for solu- tions with pH = 3.0, 7.4 and 11.0, respectively. Similar DMPO - H concentration dependence with the increase of pulse intensity at a constant absorbed energy was also observed, however, showing lower absolute yields, which probably reflects the loss of H. radicals due to their interaction with 0 2 (to be published). It is to be noted that peroxyradicals (if formed) can also be trapped by DMPO, but the reaction is very slow [11]. The lifetime of DMPO-O2 ( D M P O - H O 2 ) is shorter than that of D M P O - OH, and peroxy-radical spin adducts can further be converted to D M P O - O H ones [27]. Other reasons for lower D M P O - H yields might be differences between the DMPO reaction rates with OH. and H-, and/or different efficiencies of the parallel reactions ( H p - OH and H p - H adducts formation [2, 25]).

235

Discussion

The results obtained in the present study show that various Hp final products are formed under high intensity near UV laser flash photolysis. It is expected that complete hematoporphyrin photodestruction (e.g. ring opening) as well as irreversible modification of its periphery (generation of porphyrin derivatives) take place. The number of the products and their concentration is increased with the intensity or absorbed energy increase. Possible participation of the primarily generated Hp products and the involvement of the initially present impurities (PP and HVD) in the subsequent photoexcitation/deactivation pro- cesses as well as further products formation cannot be ruled out. It was shown previously, that several protoporphyrin photoproducts are still efficient photo- sensitizers (at least with respect to energy transfer reactions to 02, [7]). As it follows from the results obtained in Sect. a), the applied light energies are still too low to obtain complete conversion (destruction) of the initial substance. The experiments performed in the presence of free radical scavengers (methanol and DMPO, Fig. 3) indicate that a large part of the Hp photoproducts are formed under free radical attack from the bulk aqueous phase, but not from porphyrin macrocycle fragmentation. The primarily formed H p - O H (and H p - H ) adducts were previously shown to yield strongly aggregated di-, tetra- and hexahydro-derivatives as final products [-2]. The occurrence in the chro- matograms of peaks due to high molecular weight (MW>1200) products and their dissapperance in the presence of OH. (and H.) scavengers should be assigned to H p - OH (and/or H p - H) products. The reduced hematoporphyrin ( H p - H and Hp-e£-q adducts in the presence of O 2 can further initiate other side chain reactions [-18].

The excitation/deactivation mechanism resulting in OH- (and H-) generation detected by spin-trapping measurements is suggested to involve two (multi)-step Hp excitation which apparently follows from the non-linear intensity response of the measured D M P O - - O H (H) adduct yields and occurrence of a saturation intensity region in Fig. 7 (curve sigmoidality, [,21]). It is to be mentioned that a possible decrease of the apparent adduct yields at low pulse intensity (< 2 GW/cm 2) due to destruction of the formed pyrroline nitroxide (as shown for TEMPO and TEMPONE, Fig. 4) should be negligible and would not signifi- cantly change the sigmoidality of the curves shown in Fig. 7. It is to be expected also that at pulse intensities lower than 0.5 GW/cm 2, the participation of perox- yradicals in the overall adduct yield observed, should be more pronounced due to higher hydrated electron quantum yield under these conditions (interac- tion of e~-q with dissolved oxygen) [-5, 17]. When higher intensities are applied ( I> 1 GW/cm 2) the adduct formation should be bi(multi)-photonic as follows from the curve shapes in Fig. 7. Further evidence of the participation of bi(multi)- photonic photoprocess was obtained irradiating Hp - DMPO solutions by nano- second (10 ns) laser flashes of same pulse energy, but lower intensity (N2-1aser, 2exc= 337 nm). No DMPO spin adducts were registered in this case which is an indication that high photon density flux (pulse intensity) is required for initia- tion of OH. and H- radical generation by excited Hp molecules. Direct subtrac- tion of - O H and - H groups from highly excited Hp (if occurs) does not seem to play any appreciable role in the observed products formation. Experi- ments performed with other similar dyes but lacking in - O H side chain groups

236

E (eV)

8

7 - -

6 - M y

- - M x

5 - Ly

4 - - Lx

--- N x y

3 - - B x y

- O y

2 - - Qx

I

Sm

h~

S 3 l S 2 -

h~

+H20 , , H20 - ' - " 'OH* H '

Ss h ~ ~ egq

Fig. 8. A tentative diagram of Hp excitation routes involved in the present high-intensity ps-pulse photolysis. Two- step excitation pathways: So

$2(3)~.$1 --.. S 5 and So ~ $2(3~ --* S,. are supposed to the responsible for e~-q and OH" (H') generation, respectively. Spectroscopic Hp excited state symbols (Q, B, N, L and M) and their energies (in eV) are taken from relevant literature El4]

(e.g. tetrasulfophthallocyanine) also showed similar generation rates of D M P O - O H and D M P O - H spin-adducts under the present irradiation condi- tions (to be published). From the previous study [17] it follows that biphotonic e;-q formation under ps pulse irradiation has high quantum yield in the pulse intensity region below 0.5 GW/cm 2 and drops rapidly when Hp solutions are irradiated with higher intensity light pulses. Similar results were obtained in the present study using TEMPO and TEMPONE as e]q traps. This additionally supports the assumption that D M P O - H adduct is formed (at high intensities) by direct H. trapping (reaction (1)).

The unequivocal interpretation of the obtained results is in agreement with the suggestion of involvement of different higher Hp electronically excited states responsible for predominant formation of e£-q and OH. (H.) transients: S o

$ 3 ( 2 ) ~ . S 1 ---+ S 5 and So - - ' $3 (2 ) ~ Sm pathways, respectively (see Fig. 8). The spectroscopic symbols used in this figure are taken from the relevant literature [14]. The predominant excited states population is a function of the absorptivity values, corresponding lifetimes and photon flux densities. The lifetime of the fluorescent ($1) state is ~15ns [19], while the lifetimes of the highly excited states are very low (< 1 ps). At low pulse intensities after the depletion of the gound state (So), the comparatively longlived $1 state should play the role of the "newly opened" ground state and the S1 ~ $ 5 excitation pathway should be mostly operative. Further, it can be assumed that when the photon flux density is enough or immediate second photon absorption from $3(2) excited state(s), the Sm state (hypothetical) can be excited. The energy of the Hp molecule under these conditions is high enough (~ 7 eV) to reach any lower H20 electron- ic excited state [15]. Then the following energy transfer reaction from highly excited Hp (Hp**) to H20 can be suggested to be favourable:

h v = h v =

Hp ~ Hp* ~ Hp** 3 . 5 e V 3 . 5 e V

237

H p * * + H 2 0 , [-Hp... H20]* , H p + H 2 0 * (3)

HaO* ~ OH" + H-

Difficulties arise in the interpretation of the data obtained in acidic Hp - D M P O solutions (no D M P O - H adducts were observed). Parallel to the pos- sible influence of DMPO and Hp protonation effects, certain direct electron transfer reactions can not be ruled out, i.e. reactions involving participation of Hp free radicals in a water splitting process (4) and/or direct addition of OH to DMPO (5):

Hp** --+Hp + • + e~-q

Hp** + Hp --, [Hp.. + .. Hp]* ~ H p - " + Hp +.

Hp +" + H20 ~ [Hp.... H20 ] +" ~ Hp + OH" + H +

[Hp ... H20] + • + DMPO ~ Hp + D M P O - O H + H +

(4)

(5)

The proposed excitation diagram (Fig. 8) gives reasonable evidence of the occurrence of different routes of e2q and OH" (H o) generation from highly excited Hp molecules depending on the excitation light intensity. The lack of further information about higher Hp excited states (two step) optical absorption parame- ters, however, does not allow theoretical modelling of the studied processes to be performed at this stage.

Water sensitization by other organic compounds under similar high intensity laser pulse irradiation conditions was previously suggested, too [20, 23]. How- ever, direct registration of OH. and H. radicals has not been achieved till now. It is notable that this process is "switched on" only when high intensity irradia- tion (I > 2 GW/cm 2) is applied, resulting in second photon absorption by highly excited ($2, $3) states, rather than $1 singlet (fluorescent) state. The results of the present study also show that reactive free radical species (eaq, OH. and H.) can be generated not only by far-UV and X-ray radiation or pulse radiolysis, but also using near-UV light of high intensity. We suppose that further elucida- tion of this new Hp photosensitizing mechanism (direct participation of singlet excited states, in contrast to well known Hp triplet photoreactions) may be particularly important in situations where selective photodamage of biological substartes is required (e.g. studying protein-DNA crosslinking), since the OH. radicals generated by chromophores bound to macromolecules will not be ran- domly distributed in the bulk aqueous phase, as in the case when ionizing radiation is applied.

Acknowledgements. Ts.G. acknowledges the financial support of Bulgarian Ministry of Science and Higher Education (through grant No. 387). The research described herein was supported, in part, by grant No. 212 of the same ministry.

238

References

1. Amigarova MI, Makarenkova I (1980) Radiolysis of protoporphyrin in 2 N HzSO~ I. Radiation-chemical yields of irreversible transformations. High Energy Chem (USSR) 14:494499 (in Russian)

2. Amigarova MI (1986) Radiolysis of hematoporphyrin in aqueous solutions. In: Hedvig P, Nyikos L, Schiller R (eds) Proceedings of the 6th Tihany Symposium on Radiat. Chem. vol 2. Akademiai Kiado, Budapest, pp 855-862

3. Andreoni A, Cubeddu R, de Silvestri S, Laporta P, Svelto O (1982) Two-step laser activation of hematoporphyrin derivative. Chem Phys Lett 88:37-39

4. Bent DV, Hayon E (1975) Excited state chemistry of aromatic amino acids and related peptides. I Tyrosine. J Am Chem Soc 97:2599-2606

5. Boyd AW, Carver MB, Dixon RS (1980) Computed and experimental product concentration in the radiolysis of water. Radiat Phys Chem 15:177-185

6. Cox GS, Whitten DG, Giannotti C (1979) Interaction of porphyrin and metalloporphyrin excited states with molecular oxygen, energy-transfer versus electron-transfer quenching mechanisms in photo oxidations. Chem Phys Lett 67:511-515

7. Cox GS, Bobillier C, Whitten DG (1982) Photooxidation and singlet oxygen sensitization by protoporphyrin IX and its photooxidation products, Photochem Photobiol 36:401-407

8. Dougherty TJ (1985) Photodynamic therapy. In: Jori G, Perria C (eds) Photodynamic thera- py of tumors and other diseases. Libreria Progetto, Padova, pp 267-279

9. Feitelson J, Hayon E, Treinin A (1973) Photoionization of phenols in water. Effect of light intensity, oxygen, pH and temperature. J Chem Soc 95:1025-1029

10. Felix CC, Reszka K, Scaly RC (1983) Free radicals from photoreduction of hematoporphyr- in in aqueous solution. Photochem Photobiol 37:141-147

11. Finkelstein R, Rosen GM, Rauckman EJ (1980) Spin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitrones. J Am Chem Soc 102:4994-4999

12. Fuhrhop JH, Mauzerall D (1971) The photooxygenation of magnesium-octaethyl porphin. Photochem Photobiol 13:453-458

13. Gantchev T, Gotchev G, Shopova M (1989) Hematoporphyrin type I (free radical) photo- sensitizing reactions in liposomes. Stud Biophys 131:7-20

14. Gaughey WS, Deal RM, Weiss C, Goutermann M (1965) Electronic spectra of substituted metal deuteroporphyrins. J Mol Spectra 16:451-463

15. Ghormley JA, Hachanadel CJ (1971) Production of H, OH and H202 in the flash photolysis of ice. J Phys Chem 75:40M3

16. Grabner G, K6hler G, Zechner J, Getoff N (1977) Pathways for formation of hydrated electrons from excited phenol and related compounds. Photochem Photobiol 26:449-458

17. Grabner G, Getoff N, Gantchev T, Angelov D, Shopova M (1990) Hydrated electron forma- tion in nanosecond and picosecond laser flash photolysis of hematoporphyrin in alkaline aqueous solution, Photochem Photobiol (in press)

18. Hopf FR, Whitten DG (1975) Photochemistry of porphyrins and metalloporphyrins. In: Smith KM (ed) Porphyrins and metalloporphyrins. Elsevier, Amsterdam New York Oxford, pp 667-695

19. K6hler G, Getoff N, Shopova M, Gantchev T (1989) Photophysical properties of hemato- porphyrin and dihematoporphyrin ether (ester) in homogeneous and microheterogeneous environments. Z Naturforsch 44 a: 307-312

20. Kozlov AA, Lobko VV, Matveetz YA, Nikogosyan DN, Letokhov LS (1987) Decomposi- tion of thymine sensitized by hematoporphyrin and its derivative at one- and two-step laser excitation. Lasers Life Sci 1 : 247-264

21. Lachish V, Shafferman A, Stein G (1976) Intensity dependence in laser flash photolysis experiments: hydrated electron formation from ferrocyanide, tyrosine and tryptophan. J Chem Phys 64:4205~4211

22. Nigan S, Asmus KD, Willson RL (1976) Electron transfer and addition reactions of free nitroxyl radicals with radition induced radicals. Chem Soc Faraday Trans I 72:2324-2340

23. Oraevsky AA, Nikogasyan DN (1985) Picosecond two-quanta photochemistry of thymine in aqueous solution. Chem Phys 100:429 445

239

24. Pottier R, Laplante JP, Chow Y-Fa, Kennedy J (1985) Photofrins: a spectral study. Can J Chem 63:1463 1467

25. Quint R, Quint RM, Getoff N, Gantchev T, Shopova M (1989) Flash photolysis and ESR studies of hematoporphyrin and Photofrin II in polar and microheterogeneous environ- ment. Z Naturforsch 44a:283-290

26. Reddi E, Jori G, Rodgers MAJ, Spikes JD (1983) Flash photolysis studies of hemato- and copro-porphyrins in homogeneous and microheterogeneous aqueous dispersions. Pho- tochem Photobiol 38: 639-645

27. Rosen GM, Rauckman EJ (1981) The spin trapping of biologically generated free radicals in: Rodgers MAJ, Powers E1 (eds) Oxygen and oxy-radicals in chemistry and biology. Academic Press, New York, pp 97-108

28. Rossi E, Van de Vorst A, Jori G (1981) Competition between singlet oxygen and electron transfer mechanisms in the porphyrin-sensitization of 1-tryptophan and tryptamine in aque- ous micellar dispersions. Photochem Photobiol 34:447454

29. Spikes JD (1984) Photobiology of Porphyrins. In: Doiron DR and Gomer CJ (eds) Porphyrin localization and treatment of tumors. Liss, New York, pp 19-39

30. Sargent FP, Gardy EM (1976) Spin trapping of radicals formed during radiolysis of aqueous solutions. Direct electron spin resonance observations Can J Chem 90:275 279

31. Shopova M, Gantchev T (1988) Influence of medium polarity on the photosensitizing prop- erties of hematoporphyrin and hematoporphyrin derivative (Photofrin-II). J Photochem Photobiol B (Biol) 2:365 371

32. Steen HB (1974) Wavelength dependence of the quantum yield of fluorescence and photoion- ization ofindoles. J Chem Phys 61:39974002