Lactoperoxidase-catalyzed lodination of arachidonic acid: Formation of macrolides

Lactoperoxidase-Catalyzed Iodination of Arachidonic Acid: Formation of Macrolides J.M. B O E Y N A E M S , 1 D. R E A G A N 2 and W.C. H U B B A R D , Departments of Pharmacology and Chemistry 2 , Vanderbilt University, Nashville, TN 37232 ABSTRACT

In the presence of iodide and hydrogen peroxide, lactoperoxidase catalyzed the conversion of arachidonic acid into several iodinated products; the major one was previously identified as an iodo-6lactone. Two minor and less polar products have now been characterized as 15-iodo-14-hydroxyeicosatrienoic acid, to-lactone and 14-iodo-15-hydroxyeicosatrienoic acid, toqactone, on the basis of 12sI incorporation, mass spectrometry, proton magnetic resonance spectroscopy and chemical modifications. Incubation of Arachidonic Acid with Lactoperoxidase

INTRODUCTION

Lipid iodination has been the subject of a number of studies with two different emphases. It has been shown that the thyroid lipids of dogs on high iodine intake contain olefin diiodides ( 1 - 3 ) . Other studies demonstrated that lactoperoxidase-catalyzed iodination of intact cells labels several classes of membrane lipids in addition to cell surface proteins ( 4 - 9 ) . In both cases, iodination presumably involves the covalent binding of iodine to fatty acids either by addition to double bonds or substitution for hydrogen. Arachidonic acid is metabolized through a variety of pathways, all of which involve primarily dioxygenation reactions. In tissues such as the thyroid gland, which contain an iodide peroxidase, iodination might provide an alternative pathway for arachidonic acid metabolism. We have thus investigated the iodination of arachidonic acid catalyzed by lactoperoxidase and identified the major product as an iodo-~-lactone (10). In this paper, we report the structure of two less abundant products of this iodination reaction. MATERIALS AND METHODS Materials

(5, 6, 8, 9, 11, 12, 14, 15-3H)Arachidonic acid ( 6 0 - 1 0 0 Ci/mmol) and 12s I (15.8 mCi//ag) were purchased from New England Nuclear, Boston, MA, and from Amersham, Arlington Heights, IL, respectively. Arachidonic acid (purity >99%) was obtained from Nu-ChekPrep, Elysian, MN. Lactoperoxidase (EC 1.11.1.7) from milk (58 purpurogallin units/ mg) and methimazole (1-methylimidazole-2thiol) were purchased from Sigma Chemical Co., St. Louis, MO. 1Present address: Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels. Brussels, Belgium.

Lactoperoxidase (2.9 /ag/ml or 0.17 purpurogallin units/ml), [ 3 HI arachidonic acid ( 5 - 5 0 / a g / m l , 0.05/aCi/ml), [12si] KI (0.4 mM, 0.05 /aCi/ml) and H202 (0.36 mM) were stirred in phosphate buffer (0.1 M, pH 7.4) for 30 min at 20 C. After addition of sodium thiosulfate, the reaction mixture was extracted with two vol of ethyl acetate. Liquid Chromatography

Silicic acid column chromatography was performed in 1.5 cm-diameter glass columns packed with a slurry in chloroform of Porasil A ( 3 5 - 7 5 p particles: Waters Associates, Milford, MA) (column height: 18 cm). Elution was performed with chloroform (90 ml), chloroform/methanol (95:5, v/v: 60 ml) and methanol (30 ml). Three-ml fractions were coUected and aliquots were counted in solid and liquid scintillation counters for 125I and 3H, respectively. Reversed phase-high pressure liquid chromatography (RP-HPLC) was performed on a /aBondapak C18 column (3.9 x 300 ram, 10 /am particles: Waters Associates, Milford, MA). The injector (model U6K) and the pump (model 6000A) also were from Waters Associates. The samples were injected, dissolved in 50 pl methanol. Elution was performed with methanol/water (80:20, v/v) and the flow rate was 1 or 2 ml/min. Gas Chromatography and Mass Spectrometry

Gas chromatographic analysis was performed on a Varian 2100 instrument with flame ionization detection, using a 1% OV-1 column isothermally at 185 C. Equivalent chain lengths were determined by reference to methyl esters of saturated fatty acids. Mass spectra scanning and selected ion monitoring were performed on a Hewlett-Packard combined gas chromato-

246

IODINATED MACROLIDES

graph-quadrupole mass spectrometer (Model 5982A); 1% OV-1 columns (1 m • 2 mm) were used at 190 C with helium as carrier gas (flow rate: 30 ml/min). The injection port temperature was 250 C and the electron energy was 70 eV. Chemical Modifications and Derivatizations

Reduction by lithium aluminum hydride (LiAIH4): 10 /ag of material was dissolved in 0.1 ml THF and 1 mg LiA1H4 was added; after flushing with N2, the reaction mixture was heated at 60 C for 72 hr and then diluted with water and extracted with ethyl acetate. Catalytic hydrogenation: 10 /ag of m a t e r i a l was dissolved in 0.5 ml ethanol to which 1 mg platinum oxide was added; hydrogen gas was bubbled for 2 min, after which the reaction mixture was diluted with water and extracted with diethyl ether. Trimethylsilyl ether derivatives were obtained by reaction with excess bis-trimethylsilyl-trifluoro-acetamide (BSTFA) in pyridine. Proton Magnetic Resonance Spectroscopy

Proton magnetic resonance spectra were recorded on a JEOL FX-90Q Fourier transform spectrometer operated at 90 MHz. The sample was dissolved in deuterochloroform and tetramethylsilane was used as internal reference. R ESU LTS

Incubation of arachidonic acid with lactoperoxidase in the presence of iodide and hydrogen peroxide resulted in the formation of several iodinated products; five peaks of coeluting 3 H-and lZSI-radioactivities were resolved by silicic acid column chromatography (10). The major product was identified previously as 6-iodo-5-hydroxy-eicosatrienoic acid, ~-lactone (10). A minor peak of radioactivity (X) eluted earlier than the iodo-~-lactone in the void volume of the column. The yield of this product represented 10% of the yield of the iodo-5-1actone, which itself amounted to 1020% of the added arachidonic acid. During further purification by RP-HPLC on a/aBondapak 'Cls column, all- and 125 I-radioactivities coeluted again. The retention volume was 44.2 + 2.8 ml (mean + SD, n = 5) compared to 23 ml for the iodo~-lactone (solvent: methanol/ water, 80:20, v/v; flowrate: 1 or 2 ml/min). The peroxidase inhibitor methimazole inhibited the generation of both the iodo-~-lactone and compound X in a similar range of concentrations ( I 0 - 1 O0/aM; not shown). Gas chromatographic analysis of underivatized component X showed a single peak of

247

equivalent chain length C-21.8 (OV-I), compared to C-23.9 for the iodo-~-lactone. The electron ionization mass spectrum of this material was indistinguishable from that of the iodo-~-lactone (Fig. 1A) (10); it showed a molecular ion at m/z 430 and a prominent peak at m/z 303 (M-127), produced by the loss of iodine, which is the typical fragmentation of alkyl iodides (1 I). This suggested that component X and the iodo-~-lactone could be isomers. After catalytic hydrogenation, a single peak of equivalent chain length C-19 (OV-1) was observed during gas chromatography. Its electron ionization mass spectrum showed a molecular ion at m/z 310, like the spectrum of the hydrogenated iodo-5-1actone (Fig. I B) (10). The shift from a prominent peak at m/z 303 to a molecular ion at m/z 3 I0 could be explained by the saturation of three double bonds and the substitution of hydrogen for iodine. This substitution would be expected to increase the volatility of the compound and would thus explain the decrease of the GC-retention time observed after hydrogenation. The mass spectrum of hydrogenated material X differed from that of the hydrogenated iodo~-lactone by the lack of a base peak at m/z 99, characteristic of 6-1actones (12), and by the presence of ions at m/z 239 (M-71) and m/z 225 (M-85) (10). Ions at m/z 125, 111, 97, 83 and 69 are likely to be generated by the sequential loss of CH2 units from a saturated hydrocarbon chain. After reduction of the hydrogenated material X with LiA1 H4 followed by silylation, gas chromatography revealed a major peak of equivalent chain length C-22.5 (OV-1). The mass spectrum showed characteristic ions at m/z 387, 373, 187 and 173 (base peak) and was thus consistent with the expected fragmentation pattern of a mixture of 1, 14 and 1, 15 diols, resulting from the reduction by LiA1H4 of a mixture of 14-hydroxy- and 15-hydroxy-colactones (Fig. 2). The relative intensity of these ions suggested that the 15-hydroxy-eo-lactone was slightly more abundant than its 14-isomer. By comparison, a similar treatment (hydrogenation, LiA1H4, silylation) of the iodo-~-lactone provided a compound having the same equivalent chain length C-22.5 (OV-1); its mass spectrum showing major fragment ions at m/z 313 and m/z 247 was consistent with the structure of a 1, 5 diol. The proton magnetic resonance spectrum of material X purified by RP-HPLC was consistent with a mixture of 14-iodo-15-hydroxyeicosatrienoic acid, 6oqactone and 15-iodo-14hydroxyeicosatrienoic acid, eo-lactone. It revealed the following peaks: 0.89 ppm (tr, 3H, C20), 1.28 ppm (brs, 6H, C17, Cls, C19), 1.65 LIPIDS, VOL. 16, NO. 4 (1981)

J.M. BOEYNAEMS, D. REAGAN AND W.C. HUBBARD

248

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FIG. 1. (A) Electron ionization mass spectrum of component X, the minor product of lactoperoxidase-catalyzed iodination of arachidonic acid, eluted in the void volume of the silicic acid column. (B) Electron ionization mass spectrum of hydrogenated component X. An explanation for the existence of ions at m/z 225 and 239 is depicted in Fig. 2. ppm (m, 4H, C3, C16), 2 . 0 - 2 . 5 ppm (m, 6H, C2, C4, C]3), 2 . 5 - 2 . 8 ppm (m, 4H, C7, C]0), 4.14 ppm (tr of d, C14 or Cls, proton on iodine-bearing carbon), 4.46 ppm (tr of d, Cls, proton on alcohol-bearing carbon), 4.87 ppm (d of tr, C14, proton on alcohol-bearing carbon) and 5.39 ppm (m, 6H, Cs, C 6 , C 8 , C 9 , C l i , C12). An homonuclear decoupling study supported the presence of the iodine and the hydroxyl function on vicinal carbons.

-OH

DISCUSSION -]~ODO- 14 - HyOeOXV EICOSATRZEN~C AC[D, W-LAGTON[

The major product of lactoperoxidasecatalyzed iodination of arachidonic acid is 6-iodo-5-hydroxyeicosatrienoic acid, 6-1actone (10). This transformation is analogous to the well-known reaction of alkaline iodolactonization ( 1 3 - 1 5 ) . It is known that /~, 3' and 7, 6 unsaturated carboxylic acids can be converted into iodo-T-lactones and 6, e unsaturated acids into iodo-~-lactones (14). We have now observed the formation of 15-iodo-14-hydroxyeicosatrienoic acid, 6o-lactone and 14-iodo-15hydroxyeicosatrienoic acid, w-lac-tone. These compounds are macrolides which could also be called 15-iodo-eicosatrien-14-olide and 14-iodoeicosatrien-15-olide. It is likely that these macrolides are formed by a mechanism similar to the formation of the iodo-6-1actone (Fig. 3). LIPIDS, VOL. 16, NO. 4 (1981)

'4 -XO00 - ~S- HyoI~oxY [ICOSATRIENCqC ACZO, ~-LACTON[

FIG. 3. Tentative scheme of arachidonic acid transformation into iodinated macrolides.

FIG. 2. L i A 1 H 4 reduction of hydrogenated component X: gas chromatographic-mass spectrometric analysis of the main product. The major fragmentation pattern is described and interpreted.

249

IODINATED MACROLIDES C o r e y et al. ( 1 5 ) h a v e p r e v i o u s l y o b s e r v e d t h e spontaneous transformation of peroxyarachid o n i c acid i n t o 1 4 , 1 5 - e p o x y e i c o s a t r i e n o i c acid by intramolecular oxygen transfer. These investigators concluded that a 15-membered ring m a y be e n e r g e t i c a l l y f a v o r a b l e c o m p a r e d t o s m a l l e r s t r u c t u r e s . T h e f o r m a t i o n o f 15- a n d 1 6 - m e m b e r e d i o d i n a t e d m a c r o l i d e s c o u l d be e x p l a i n e d b y similar c o n s i d e r a t i o n s a n d p r o b a b l y i l l u s t r a t e s a t e n d e n c y o f a r a c h i d o n i c acid to a d o p t a J-like c o n f i g u r a t i o n . F u r t h e r s t u d i e s will d e t e r m i n e if t h e s e m a c r o l i d e s are f o r m e d in i n t a c t cells a n d w h a t c o u l d be t h e i r biological activity.

2. 3. 4. 5. 6. 7. 8. 9. 10.

ACKNOWLEDGMENTS We thank Dr. D. Taber and Dr. T. Burka for stimulating discussions and Dr. J. A. Oates for his continuous interest. This work was supported by NIH grants GM 15431 and BRSG-RR-05424 and by Public Health Service International Research Fellowship TW 02685. J. M. Boeynaems is Fellow of the Fogarty International Center and Aspirant of the Fonds National de la Recherche Scientifique (Belgium).

11. 12. 13. 14. 15.

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