mixture of docosane and tetracosane disulfates, one of which corresponds to

A NEW CLASS OF LIPIDS: CHLOROSULFOLIPIDS* BY JOHN ELOVSON AND P. R. VAGELOS DEPARTMENT OF BIOLOGICAL CHEMISTRY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI Communicated by Herbert E. Carter, January 15, 1969 Abstract.-Acid hydrolysis of sulfolipids from Ochromonas danica liberates 13-chloro-docosan-1,14-diol; 11,15-dichloro-docosan-1,14-diol; and other docosan-diols with three to six chlorine atoms per molecule. In stationary cells grown on 1.47 mm Cl-, the majority of sulfolipids are chlorinated, with the hexachloro species being the most abundant. In a preliminary study of fatty acid synthesis in Ochromonas danica, it was found not only that cell-free extracts of that organism were completely unable to incorporate labeled malonyl-coa into long-chain fatty acids, but also that they inhibited fatty acid synthesis in other systems. The inhibitory principle, quantitatively recovered in the methanol:water phase of a Folchl extract of 0. danica homogenates, also inhibited glucose-6-phosphate dehydrogenase in a manner suggestive of the detergent effects of palmityl CoA described by Srere2 and Taketa.3 Unlike a thioester, however, it was stable to hot 0.1 M alkali and rapidly destroyed by hot 0.1 M acid. This suggested that it might be related to the docosan-1, 14-disulfate recently isolated from 0. danica by Mayers and Haines.4 This paper describes the isolation and partial characterization of the inhibitory material from 0. danica. It is shown to be a heterogeneous mixture of docosane and tetracosane disulfates, one of which corresponds to the compound described by Mayer and Haines, the others differing from this by having one, two, three, four, five, or six chlorine atoms substituted for the same number of hydrogens. Materials and Methods.-O. danica was from the American Type Culture collection. Silica Gel G was supplied by Brinkman; Adsorbosil 3, SE-30 silicone gum, GasChrom Q hexamethyldisilazanet (HMDS), and trimethylsilylchloride (TMSC1) by Applied Science. divbis(trimethylsilyl)acetamide (BSA) was a gift of Dr. W. Sherman. H2SB04 and HC136 were obtained from New England Nuclear Corp. 0. danica was grown heterotrophically at room temperature, with shaking, in ambient light on a defined medium. S35-labeled cells were grown on 0.1 mm Na23504 (50 mc/ mmole), substituting the equivalent chlorides for other sulfates. For growth on 0.47 and 1.47 mm NaCl36 (0.048 mc/mmole), sulfates were substituted for other chlorides. Cells were harvested by centrifugation in stationary phase, ruptured with a French press, and the lipids were extracted with 20 volumes of chloroform: methanol 2: 1. The extract was shaken with 0.4 volume of water. The aqueous phase was washed free of pigments with chloroform, reduced in vacuo, and extracted with n-butanol. The extract was washed with water and the butanol was removed in vacuo. The residue was taken up in methanol, filtered, and taken to dryness. This residue will be referred to as "crude sulfolipid." Sulfolipids were hydrolyzed in 1 M HBr at 950 for 1 hr. The mixture was extracted twice with ether. The ether was washed to neutrality with water, dried (MgSO4), and evaporated. This residue will be referred to as "crude diols." Trimethylsilyl (TMS) derivatives were prepared by treatment with pyridine: HMDS: TMSC1 5:5: 1, or pyridine: BSA 1: 1. Methyl ethers were prepared as described by Hakomori.6 Analytical gas-liquid chromatography (GLC) of TMS derivatives was performed on an F 957

958 BIOCHEMISTRY: ELOVSON AND VAGELOS PROc. N. A. S. and M Model 402 instrument with a 6-ft glass column packed with 1% SE-30 on (las- Chrom Q, programmed to 2 min isothermal at 2120 followed by a linear inerease of 50/min up to 2670 and held there. GLC-inass spectrometry was performed on an LKB- 9000 instrument with a 6-ft glass column packed with 0.2% SE-30 on GasChrom Q and operated up to 2100. Thin-layer chromatography (TLC) of sulfolipids was performed on 0.25-mm plates of Adsorbosil 3 with chloroform: methanol: water 100:55:4. Plates were activated at 1500 for 2 hr. Compounds were visualized by being sprayed with 5% phosphomolybdic acid in methanol and then heated at 1500. For autoradiography of labeled lipids, the plates were first sprayed with 2,5-diphenyloxazole (PPO) in isopropanol before being placed on Kodak X-ray film. The labeled sulfolipids were quantitatively eluted with 10 ml methanoll:chloroform:water 70:30:10. TLC of the etherextractable material after acid hydrolysis of sulfolipids was performed on Silica G plates with benzene: chloroform: methanol 100:60:5. For preparative purposes the bands were localized by being sprayed with 0.2% 2,7-dichlorofluoresceine. The long-chain diols were eluted with 10 ml 10% methanol in ether. For substitution with sodium methyl mercaptide, 150 mg sodium methoxide in a heavy-wall culture tube was cooled in a dry ice-acetone bath and an excess of methyl mercaptan condensed into the tube. The material to be treated was added to the cold mixture in 0.1 ml methanol, and the tube was tightly closed with a Teflon-lined screw cap and incubated at 800 overnight. When cool, the tubes were carefully vented in the fume hood, 1 ml 4 M acetic acid was added, and the products were recovered by ether extraction. The mixed isomers of 9(10)-chloro-octadecan-1,10(9)-diol were prepared by LiAlH4 reduction of oleic acid to the alcohol, epoxidation with peracetic acid, and the formation of the chlorohydrin with dry HOl in ether. 9(10),12-diehloro-octadecan-1,10(9)-diol was similarly prepared by treatment of ricinoleic acid with thionyl chloride at room temperature, reduction with LiAlH4, epoxidation, and opening of the oxirane with dry HCl. The products were purified by TLC, and their identity was confirmed by GLC-mass spectrometry. Results.-GLC of the TMS derivatives of the ether-extractable material after acid hydrolysis of the crude sulfolipids (a) showed at least 12 compounds (Fig. lb). When mass spectrometric analysis of some of these prompted the conclusion that they contain organic chlorine, 0. danica cells were grown on radioactive sulfate or chloride. The sulfate-labeled material 5 I0 5 MIN. contained five major bands at Rf about 0.2-0.5 (b) (Fig. 2); the slowest-moving of these was absent CE in the material from Cl3-labeled cells. The crude diols from unfractionated sulfolipids were separated G H into five fractions by preparative TLC. GLC A al t A of their TMS derivatives showed the following rela- D 2 tionship between these bands and compounds A-I. (Fig. lb): Band 1 (fastest-moving): compound I. 5 10 15 MIN Band 2: compounds F2, G2, and H. Band 3: com- FIG. L.-GLC of TMS- pounds D, F1, Gi. Band 4: compounds B and E. derivatives of ether-extract- Band 5 (slowest-moving): compounds A and C. able material from acid In material from C136-labeled cells the slowesthydrolysis of crude sulfo- I lipids. Details in Materials moving band, 5, contains no radioactivity. These and Methods. 0. danica fractions were used directly for GLC mass specwas grown on (a) 0.47 mm Cl- and (b) 1.47 mm Cl-. trometry.

VO L. 62, 1969 BIOCHEMISTRY: ELOVSON AND VAGELOS 959 GLC mass spectrometry: Compounds A and C: The TMS derivatives have highest fragments at m/e 471 and 499, which are consistent with the typical (M-15)7 fragments arising by loss of a methyl radical from the molecular ions of a TMS-docosane- and tetracosane-diol, respectively. The former is presumably identical to the docosan-1,14-diol described by Mayers and Haines,4 as shown by a base peak at m/e 215 (C-22 through C-14-OTMS) and the abundant fragment at m/e 373 (C-1-OTMS through C-14-OTMS); the tetracosane derivative has the same distribution of intensities at m/e 229 and 387, which unequivocally places the secondary TMSO group at carbon 15. A B C...~~~~~~ ~~ ~ ~~ ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... FIG. '2. TLC of crude sulfolipids. Autoradiograms: details in.iiaterials and Methods. (A) Lipids from 0. danica grown on S35042-. (B) Lipids from 0. danica grown on C136-. (C) Rechromatography of separated fractions from B. Compound B (Fig. 3a): The TAIS derivative has a split peak at highest m/e 505/507 in a ratio of about 2: 1, i.e., 34 and 36 m/e units heavier than that found for compound A. The base peak at m/e 215 is the same as for compound A, but peakim/e 373 in compound A is replaced by another, less abundant, split peak 34 and 36 m/e units further out at 407/409. Deuterium labeling of the TAIS groups confirmed that the fragments at 215 and 407/409 carried one and two TMS units each, respectively. The simplest explanation for these findings is that compound B arises by substitution of a chlorine atom for a hydrogen atom on a carbon proximal to the secondary hydroxyl in docosan-1,14- diol. The new fragment at m/e 397 lacks chlorine and retains only two out of the original six TMIS methyls. It is suggested that it arises by the elimination of TMSCI from the M-15 ion and that such interaction between the C1 and TAMS groups is most likely if they occupy adjacent carbons. Treatment of fraction 4 with 0.1 M KOH at room temperature transformed compound B into a compound whose TMS derivative gave highest fragments at mn/e 412 (M) and 397 (AI-15) and significant m/e 155 and 299 (a-cleavage at oxirane ring),

960 BIOCHEMISTRY: ELOVSON AND VAGELOS PROC. N. A. S. (a) 215 d9 M520 73 d9 xio 397 d4 505 dm5 359di[ 407 d,8 100 200 300 400 560 600 700 (b) 69 243 X10 157 157 ~~~~~M.01 ~22IMM44-3~~ -me (C) 100 200 300 460 SOO 600 700 73 Lat. ~~~~~~~~~341 vo, Ṯ X~~~~~~~~~~~0 44949 (d) 100 200 300 400 500 600 73~~~~~~~0 73 S4 M4 407 249 100 2 0 300-500 600 0 (e) 201 303TOC 129 129 303 372 x10 282 ~~~~~~503 in. a.: IJL. l.-le.1. 232303 53 557 593 2~ 4 100 260 360 400 500 600 70 consistent with the TMS derivative of 13,14-epoxy-docosail-l-ol. Treatment of this compound with 1 Ml H2SO4 at 1000 for one hour to open the oxirane ring, followed by methylation, gave the spectrum in Figure 3b. Highest mass at m/e 368 (M-MeOH) and the two abundant fragments at m/e 157 and 243, corresponding to cleavage between the two methoxy groups, demonstrate that the original compound B has a 13,14-chlorohydrin structure. The spectrum of synthetic 9(10)-chloro-octadecan-1,10(9)-diol (Fig. 3c) shows the expected chlorine isotope split at m/e 449 (M-15) and 351 (C-1-OTMIS through C-10-OTMS), the expected chlorine-free fragments at m/e 341 (M-15- TMSCl), and the abundant m/e 215 (C-18 through C-10-OTMS of the 10-

VOL. 62, 1969 BIOCHEMISTRY: ELOVSON AND VAGELOS 961 ( M 522 73 303 1 ~~~~~~~~~~~~~~~~~~522 145 x1io 507 385 MeS 213 281 I, A, IL 100 200 a00 460 56i 500 -r 600 700 a 100 gm 578 261 261 22_,5-,4 DIVOT 419 Me SI sme 73 LM!J 441 578 159 213 5 IL I,ILh ' I' 1 1 \, L i L i E L. 61I, 5 675 100 200 300 400 500 600 700 289 0 73 (h) LM 690 I ~~~~283 401 4 22-I11 iot C2 41 Il~~~kL~~,1LL~~~ ~~509 247 ~ 965467 160 260 300 400 500 600 7600 FIG. 3.-Mass spectra of diols and derivatives. Abscissa: m/e. Ordinate: intensity relative to most abundant fragment. Proposed structure and fragmentation of the analyzed compound is shown schematically. T = TMS group. MeS = methyl mercaptan group. The number of deuterium atoms introduced into the fragments from di8-bsa is shown in part a. See Materials and Methods and Results for experimental details and identification of compounds: Compounds are labeled as in Figure lb. (a) TMS-B: 13-chloro-docosan-1, 14-diol. (b) Product of compound B after treatment with alkali, acid, and methylation: 1,13,14-trimethoxy-docosane. (c) TMS-9(10)-chloro-octadecan-1, 10(9)-diol. (d) TMS-D: 11,15-dichloro-docosan-1,14-diol. (e) TMS derivative of product from compound D after treatment with alkali and acid: 1 l-chloro-docosan-1,14,15-triol. (f) TMS derivative of product from 9(10),12-dichloro-octadecan-1,10(9)-diol after treatment with sodium methyl mercaptide: 9(10),12-di(methylthio)-octadecan-1,10(9)-diol. (g) TMS derivative of product from compound D after treatment with sodium methyl mercaptide: 11,15-di(methvlthio)-doccsan-1,14-diol. (h) TMS-I: hexachloro-docostan-1,14-diol. hydroxy isomer). The abundant peak at. mr/e 303 (C-1-OTMS through C-9- OTMS of the 9-hydroxy isomer) has an intensity of 56 per cent relative to m/e 215 arising from the other isomer. In compound B, on the other hand, the equivalent fragment at mrne 359 represents less than 0.5 per cent of m/e 215, indicating that 99 per cent is the 13-chloro-docosan-1,14-diol. Compound E: Chlorine isotope clusters at m/e 533 (M-15) and 421, a peak at 425 (M-15-TMSCl), and a base peak at 229 strongly suggest the 14-chlorotetracosan-1,15-diol homolog of compound B. Compound D: A Cl2 isotope cluster at highest m/e 539 (M-15) (Fig. 3d)

9629IiOCHEMISTRY: ELOVSON AND VAGELOS Piltoc. N. A. S. suggests an elementary composition derived from compound B by substitution with a second chlorine. The abundant Cl1 cluster at m/e 249 indicates that a chlorine atom is situated distally to the secondary hydroxyl on C-14, while the same isotope cluster at m/e 407, as in compound B, indicates another Cl atom between C-I and C-14. The Cl1 cluster at m/e 431 (MI-I5-TM1SCl) may be typical of a chlorohydrin. The fragment at m/e 503 (M-15-HCl) has no equivalent in the monochloro chlorohydrins. Fraction 3 was treated with alkali and acid to convert a chlorohydrin group in compound D to a vicinal glycol. The spectrum of the TMS derivative of the product (Fig. 3e) shows Cl1 isotope clusters at m/e 593 and 503 as expected for the (MI-15) and (M-15- TMSOH) of a TMS-chloro-docosan-triol, as well as a fragment at m/e ;557 (M-15-HCl). The new abundant fragment at m/e 201 (C-22 through C-1i- OTMS) and the less intense m/e 303 (C-22 through C-14-OTiMS) demonstrate that the original chlorohydrin has its chlorine on C-1i, rather than C-13 as in compound B. Thus the second chlorine must be between the two hydroxyl functions at C-1 and C-14, probably not on C-13 where it could compete for oxirane formation with the hydroxyl on C-14. The structures of the prominent even-electron ions at m/e 372 and 282 (372-TMSOH) are unknown. No diagnostic fragments show the position of the nonchlorohydrin chlorine atoms in compound D or TMIS-9(10),12-dichloro-octadecan-1,10(9)-diol. The latter has the expected Cl2 isotope cluster at m/e 483 (M-15); Cl1 clusters at m/e 447 (M-15-HCl), 375 (M-15-TMSCl), 351 (C-1-OTMS through C-10- OTMlS); and the abundant m/e 249 (C-22 through C-10-OTMS of the 10-hydroxy isomer), as well as the chlorine-free fragment, at m/e 303 (C-1-OTMS through C-9-OTMIS of the 9-hydroxy isomer). Attempts to substitute a hydroxyl or methoxyl group for the chlorine in this compound resulted in elimination only, but introduction of the methyl mercapto group under much less basic conditions was successful. The TMS derivative of the product (Fig. 3f) shows a prominent molecular ion at m/e 522, and the expected fragments at m/e 507 (M-15), :303 (C-I-OTM\1S through C-9-OTMIS of the 9-hydroxy isomer), and 261 (C-18 through C-10-OTM\JS of the 10-hydroxy isomer). The peak at m/e 145 represents C-18 through C-12-SCH3 and shows the position of the original chlorine on C-12. The same product from compound D (Fig. 3g) shows the expected molecular ion at m/e 578, m/e 563 (M-15), 441 (M-CH3S-TMSOH),. 419 (C-1-OTM'NIS through C-14-OTiMS), and 261 (C-22 through C-14-OTMS). The peak at m/e 159 confirms the position of the chlorohydrin chlorine on C-15, and the peaks at m/e 289 (C-1-OTMS through C-11-SCH3) and 199 (289-TM\ISOH) support the assignment of C-11 to the isolated chlorine in the original compound. Thus compound D is 11,15-dichloro-docosan-1,14-diol. Compounds Fl- and F2-TMIS: Both show Cl3 isotopic clusters at highest m/e 573, consistent with (M-15) of TMS-trichloro-docosan-diol, as well as the same Cl1 cluster at m/e 249 as compound D. Compounds GI- and G2-TMIS: Both have the Cl4 isotope clusters at highest m/e 607 expected for TMS-tetrachloro-docosan-diol as xvell as Cl1 cluster at m/e 249 (GI) and Cl2 cluster at m/e 283 (G2). For compound H-TMIS, the cluster at highest m/e 641 suggests the (imi-15) of TiMS-pentachloro-docosan-

VOOL. 62, 1969 BIOCHEMISTRY: ELOVSON AND VAGELOS 963 diol; but it is not intense enough to permit analysis of the isotope distribution. The spectrum of compound I-TMS (Fig. 3h) shows the highest m/e 675 expected for (M-15) of TMS-hexachloro-docosan-diol; the even-electron isotope cluster at m/e 654 (M-HCl) has the distribution calculated for a pentachloro-compound. The intense Cl2 cluster at m/e 283 suggests that the secondary hydroxyl again is on C-14, with two chlorines distally and four proximally. Discussion.-In stationary 0. danica cells, the total sulfolipid fraction constitutes about 15 per cent of the lipids or about 3 per cent of the dry weight. Figure 1 shows that in cells grown on 1.47 mm Cl-, the majority of these sulfolipids are chlorinated, while growth on 0.47 mm Cl- reduces the extent of chlorination. This is accompanied by a moderate decrease in growth rate.8 Mayers and Haines4 reported that docosan-1, 14-disulfate was recovered in a membrane fraction of 0. danica. Our finding that their detergent properties make the sulfolipids potent enzyme inhibitors also suggests that they must be present in a bound form. Aliphatic chlorine-containing compounds of biological origin are extremely rare and, to our knowledge, have not been described outside the fungi.9 The biosynthesis of the chlorosulfolipids is completely unknown. Miost likely the chlorine is incorporated through electrophilic attack by a chloronium ion, as suggested for the reaction mechanism of chloroperoxidase from Caldariomyces fumago, which has been extensively studied by Hager et al." Where they have been determined, the positions of the secondary hydroxyl and chlorine(s) in the sulfolipids do not suggest a simple pattern, but it is tempting to speculate that the biosynthesis of these compounds is somehow related to that of the unsaturated fatty acids. We are indebted to Dr. David Lipkin for his suggestion of the mercaptide substitution reaction and to H. Holland for help in obtaining the mass spectra. Note added in proof: T. H. Haines (personal communication) has obtained optical rotation data on the 13-chloro-docosan-1,14-diol which indicate that it has the threo-(r)-13-chloro-docosan-1-(r)-14-diol structure (L13, D-14). * Supported by grants from NIH (1-ROl-He 10406), NSF (GB-5142X), and HSAA (5 SO4FR 06115). t Abbreviations used: TMS, trimethylsilyl; HMDS, hexamethyldisilazane; TMSCl, trimethylsilylehloride; BSA, bis(trimethylsilyl) acetamide; GLC, gas-liquid chromatography; TLC, thin-layer chromatography, PPO, 2,5-diphenyloxazole. 1 Folch, J., M. Lees, and G. H. Sloane Stanley, J. Biol. Chem., 226, 497 (1957). 2 Srere, P. A., Biochim. Biophys. Acta, 106, 445 (1965). 3Taketa, K., and B. M. Pogell, J. Biol. Chem., 241, 720 (1966). 4Mayers, G. L., and T. H. Haines, Biochemistry, 6, 1665 (1967). 5 Aaronson, S., and H. Baker, J. Protozool., 6, 282 (1959). 6 Hakomori, S., J. Biochem., 55, 205 (1964). 7Diekman, J., J. B. Thomson, and C. Djerassi, J. Org. Chem., 32, 3904 (1967). 8 Elovson, J. unpublished observation. 9 Miller, M. W., The Pfiizer Handbook of Microbial Metabolites (New York: McGraw-Hill, 1961). lo Morris, D R., and L. P. Hager, J. Biol. Chem., 241, 1763 (1966).