Singlet Oxygen Trap in Biological Systems

Dimethyl 3,3'-(4-Methyl-l,3=naphthylene)dipropionate Oxygen Trap in Biological Systems Klaus Muller* and Klaus Ziereis

Institute of Pharmacy, University of Regensburg, P.O. Box 101042, D-8400 Regensburg, Gemany

Received February 2, 1993

369

as a Singlet

The utility of dimethyl 3,3'-(4-methyl-1,3-naphthylene)dipropionate (DMNDP) as a singlet oxygen trap in biological systems is described. The determination of the rate constant for the reaction of DMNDP with singlet oxygen in acetonitrile gave a value of 9.8 . M-ls-'; the half-life of the corresponding endoperoxide at 37°C in phosphate buffered saline is 412 min. DMNDP does not possess sensitizing properties, and its usefulness for the detection of singlet oxygen is demonstrated in the photooxidation of liposomes by 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP).

Dimethyl-3,3'-(4-methyl-1,3-naphthylen)-dipropionat als Singulett- Sauerstoff-Fanger in hiologischen Systemen

Die Verwendung von Dimethyl-3,3'-(4-methyl- I ,3-naphthylen)-dipropio- nat (DMNDP) als Singulett-Sauerstoff-Fanger in biologischen Systemen wird beschrieben. Die Bestimmung der Gescbwindigkeitskonstante fur die Reaktion von DMNDP mit Singulett-Sauerstoff in Acetonitril gab einen Wert von 9.8 lo-' M-'s". , di e Halbwertszeit des entsprechenden Endo- peroxids bei 37°C in Phosphat-Puffer betragt 412 min. DMNDP weist keine Sensibilisator-Eigenschaften auf. Die Verwendung als Singulett- Sauerstoff-Detektor wird mittels der Photooxidation von Liposomen durch 5,10,15,20-Tetraphenyl-21H,23H-porphin (TPP) gezeigt.

1,4-Endoperoxides derived from 3-(4-methyl-I-naphthyl)propionic acid (MNP) and 3,3'-( 1,4-naphthylene)dipropionic acid (NDP) have been used for mechanistic studies of '0, reactions in aqueous systems, because they are known to release '0, In a reverse Diels-Alder reaction under mild con- ditions'.'.'). On the other hand, their parent naphthalenes react slowly with '0, '). In addition, the produced endoperoxides are unstable, with a half- life of 23 min at 35°C'). Therefore, these compounds are not ideally suited for the trapping of '0, in biological systems. Although a number of tech- niques have been developed for this purpose, many of the chemical traps employed are not specific for '0,. but may react with other oxidizing spe- cies in the system5). Endoperoxides of anthracenes are quite stable and can be made soluble in the biological system by suitable substituents6). Unfor- tunately, these compounds are photosensitizers by themselves7). Thus a more reliable trap is highly desirable.

CH, 0

As a check on sensitizing properties of MNDPA and DMNDP, we photooxygenated the '02-acceptor 2,3-dime- thyl-2-butene (3) in the presence of these compounds. Since neither the allylic hydroperoxide 4 (Schenck-reaction9), Scheme 2) was produced by the ene-reaction nor endoper- oxides of MNDPA and DMNDP were formed in a sensi- tized process, adventitious photooxidation can be ruled out when using MNDPA and DMNDP as a probe for lo2.

3 4

Scheme 2: Schenck-reaction

O'OR

1 a: R = H 2 b: R = C H 3

Scheme 1: Trapping of '0, by TMNDPA (la) and DMNDP (lh)

When preparing a series of endoperoxides of substituted naphthalenes, we found that the endoperoxides 2a and 2b of 3,3 '-(4-methyl- 1,3-naphthyIene)dipropionic acid (la, MNDPA) and dimethyl 3,3'-(4-methyl-1,3-naphthylene)- dipropionate (lb, DMNDP, Scheme 1) were substantially more stable than MNP and NDP in PBS at physiological temp., corresponding to half-lives of 398 and 412 mid) . Therefore, their parent naphthalenes MNDPA and DMNDP may provide useful chemical traps of '02 for mechanistic studies in biological system.

As a measure of reactivity toward '02 we determined the rate of reaction by a technique that yields the sum of reac- tion and quenching rates (kR + kq)"). The kinetic scheme for reaction of DMNDP, which can either react with (kR) or quench (kQ) '02 is given by eqs. (1)-(3), where kD is the decay rate of '02 in the solvent:

kR

kQ DMNDP + '02 --+ DMNDP02 (1)

(2) DMNDP + lo2 + DMNDP + 302 k D

lo2 kD --+ 302 (3)

The expression for the rate of direct disappearance of DMNDPis:

Arch. Pharm. (Weinheim) 326,369-371 (1993) 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993 0365-6233/93/0606-0369 $5.00 + .25/0

370 Muller and Ziereis

A plot of the reciprocal of the rate of DMNDP disappear- ance against the reciprocal of DMNDP concentration will give a linear plot. The ratio of slope to intercept is kD/(kR + kQ). The decay rate (k,) for '02 in acetonitrile is reported to be 1.8 . lo4 M - k ' ' I ) , substitution gives the sum of k, + kQIO). The values obtained from these experiments are 1.85

M-'s-' for DMNDP. For comparison, the reactivities of the known traps 2,3-dimethyl-2-butene, 2,5-dimethylfuran, and rubrene are 2.97 . M-'s-' 9 . 23 3 . M'ls-' and 4.16 . 10-7 M-ls-1 , respective1yl2).

0.9 . 10-7 ~ - 1 s - 1 for MNDPA and 9.8 k 2.4 .

I b

1 . I . . . . . , . , .

0 5 10 15 20 25 min Figure 1: RP-HPLC-chromatogram of the endoperoxide 2b pro- duced by the TPP-sensitized M) photooxidation of DMNDP ( lb, 2 . M) in liposomes. Eluent: Methanol/water/acetic acid (72 + 28 + 0.1, vol.), pH 5.5, UV detection at 260 nm.

The utility of DMNDP as a '02 trap was tested in physio- logical buffer system and in a study of the photooxidation of phospholipids by TPP at 37°C. Fig. 1 shows the reversed-phase HPLC-chromatogram of the photooxidation of DMNDP. The peak of the resulting endoperoxide is well separated from DMNDP and can easily be detected and quantitatively determined in the micromolar range.

Experimental Part

Muteriuls

MNDPA and DMNDP were prepared as describeds). Methylene blue was from Merck (Darmstadt, Germany), 2,3-dimethyl-2-butene and S,lO,l5,2O-tetrapheny1-2 lH,23H-porphine (TPP) were from Aldrich (Stein- heim, Germany). Bovine brain was obtained from the local slaughter- house.. HPLC: Kontron 420, Kontron Uvikon 735 LC UV detector equipped with a Nucleosil 7 C 18 (250 x 4 mm) column. Data: MacLab data acquisition system (WissTech, Germany), analysis: software Peaks on "I Apple Macintosh Quadra 700 computer.

Studies on sensitizer properties

The Schenck-reaction was performed as described13): 2,3-Dimethyl-2- butene (200 mg) and naphthalene derivative M) in CH3CN (30 mL) were irradiated under O2 with 200 W Osram Halostar halogen lamps in an ethanol-cooled immersion well at -10°C for 4 h. The formation of 2.3- dimethyl-1-butene-3-hydroperoxide was followed by TLC (SiO2/CH2Cl2); detection: KVacetic acid at 50°C.

Determination of rate constants

The photooxidation of MNDPA and DMNDP has been studied by the direct disappearance technique as describedi4). Solutions of MNDPA and DMNDP (2-10 . M) and methylene blue (1.5 . M) in CH,CN (10 mL) were irradiated in special glass cylinders equipped with a side neck for sampling with 200 W Osram Halostar halogen lamps in an ethanol- cooled immersion well at -10°C for 5 min. The samples were saturated with O2 before irradiation. The concentrations of MNDPA and DMNDP before and after the photooxidation were monitored by HPLC using meth- anol/water/acetic acid (77 + 23 + 0.1) and UV detection at 228 nm for MNDPA and 223 nm for DMNDP. The values reflect the average of five sets of experiments.

Detection of ' 0 2 in aqueous system

The detection of '02 in aqueous system by MNDPA was examined by irradiation of MNDPA (5 . M) with 200 W Osram Halostar lamps in phosphate buffered saline (50 mL), pH 7.4 at 37OC in a shaking water bath. The appearance of the endoperoxide 2a was monitored by HPLC using methanol/water/acetic acid (77 + 23 + 0.1, vol.) and UV detection at 260 nm.

Preparation of phospholipids from bovine hruin

Phospholipids were prepared essentiaIIy as described by Gutteridge'". Bovine brain, cooled on ice, was freed from blood vessels and washed repeatedly with 0.15 M NaC1. pH 7.4. It was then macerated with a homogenizer and extracted three times with four times the volume of ace- tone. The extraction mixture was filtered by suction, the residue was dried under vacuum, and then repeatedly extracted with petroleum ether (40- 60°C). The extracts were filtered, dried at 45OC, and dissolved in ether. The mixture was treated with five times the volume of acetone, and the resulting precipitate was collected by centrifugation, dried and stored under N2 at -70°C in the dark.

Preparation of liposomes

Liposomes were prepared by mixing CHC13 solutions (0.5 mL) of bovine brain phospholipids (SO mg/mL), TPP (1.29 mg/mL) and DMNDP (1.26 mg/mL). The solvent was removed by N, and to the resulting lipid film were added five small glass beads and 0.15 M NaC1, pH 7.4. Final concentrations were 5 mg phospholipids/mL, 2 . M DMNDP and 106 M TPP. The mixture was purged with N2 for 1 min and vigorously dis- persed in a vortex mixer (Heidolph) for 5 min. The liposomes were allowed to swell for 1 h at 4"C, and vesicles with a mean size of 1-10 pn were obtained"). Controls were performed in the absence of TPP.

Detection of ' 0 2 in liposomes

The samples obtained above were irradiated with water-cooled 200 W Osram Halostar halogen lamps at 37°C in a shaking water bath. The appearance of the endoperoxide 2b was monitored by HPLC using a mix- ture of methanol/water/acetic acid (72 + 28 + 0.1, vol.), pH 5.5, as eluent and UV detection at 260 nm.

Arch. Pharm. (Weinheim) 326,369-371 (19931

Singlet Oxygen Trap in Biological Systems 37 1

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Arch. Pharm. (Weinheim) 326,369-371 (1993)