Lipids of Streptomyces sioyaensis V: On the 2-hydroxy-13-methyl-tetradecanoic acid from phosphatidylethanolamine

Chem. Phys. Lipids 3 (1969) 29-38 © North-Holland Publ. Co., Amsterdam

L I P I D S O F S T R E P T O M Y C E S SIO Y A E N S I S V: ON THE 2-HYDROXY-13-METHYL-TETRADECANOIC

ACID

FROM PHOSPHATIDYLETHANOLAMINE JUN'ICHI KAWANAMI, AKIRA KIMURA, YUZO NAKAGAWA and HIDEO OTSUKA Shionogi Research Laboratory, Shionogi and Co., Ltd. Fukushima-ku, Osaka, Japan Received 31 May 1968 1. Phosphatidylethanolamine from St. sioyaensis afforded a double spot on a thin-layer chromatogram, typical of most glycosphingolipids from animal tissues. They were phosphatidylethanolamines one of which had only non-hydroxylated fatty acids and the other hydroxy fatty acids in addition to non-hydroxylated fatty acids, respectively. 2. The distribution of the fatty acids was studied by hydrolysis with snake venom phospholipase A (E.C.3.1.1.4.). Hydroxy fatty acids were located in the .8-position of the glycerol moiety, differing from the results for Brucella abortus phospholipids in which location in the a-position has been reported. 3. The main hydroxy fatty acid was purified by preparative gas-liquid chromatography. The structure of the hydroxy fatty acid was analyzed by oxidation with lead tetraacetate, proton magnetic resonance, and mass spectrometry, etc. From these results, it was assumed that the main acid was 2-hydroxy-I 3-methyltetradecanoic acid. In an earlier p a p e r 1, z), it was r e p o r t e d t h a t the p h o s p h a t i d y l e t h a n o l a m i n e o f Streptomyces sioyaensis was c o m p o s e d o f two g r o u p s which afforded a d o u b l e s p o t on a thin-layer c h r o m a t o g r a m as in the glycolipids from a n i m a l tissues. It was previously assumed that the u p p e r spot has shorter chain fatty acids and the lower spot longer ones 2). This p a p e r describes a m o r e detailed study on the fatty acid m o i e t y o f the p h o s p h a t i d y l e t h a n o l a m i n e from St. sioyaensis. The cephalin fraction was s e p a r a t e d f r o m other lipid classes by D E A E - c e l l u l o s e or silicic acid c o l u m n c h r o m a t o g r a p h y 1,z). The cephalin fraction was further s e p a r a t e d into each p h o s p h a t i d y l e t h a n o l a m i n e by p r e p a r a t i v e thin-layer c h r o m a t o g r a p h y . Each p h o s p h a t i d y l e t h a n o l a m i n e was h y d r o l y z e d separately with 5% m e t h a n o l i c h y d r o c h l o r i c acid to give methyl esters o f the c o m p o n e n t fatty acids. Each fatty acid methyl ester thus o b t a i n e d was analyzed by thin-layer c h r o m a t o g r a p h y (fig. 1) respectively, using d i c h l o r o m e t h a n e as a developer 3) a n d b y gas-liquid c h r o m a t o g r a p h y (fig. 2), in which the lower p h o s p h a t i d y l e t h a n o l a m i n e c o n t a i n e d acids giving rise to shifts in the r e t e n t i o n times after tri-

30

J. K A W A N A M I ET AL.

methylsilylation4), indicating that they were hydroxy fatty acids. Accordingly, it can be stated that the less polar (upper) phosphatidylethanolamine has only non-hydroxylated fatty acids, while the more polar (lower) one has monohydroxy fatty acids in addition to a series of non-hydroxylated fatty acids. Such a finding has often been observed in the glycosphingolipids from animal organs4), but no information was available for bacterial phospholipids. On the other hand, it was found that the hydroxy fatty acids in the more polar phosphatidylethanolamine were located in the fl-position of the

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Fig. 1. Thin-layer chromatogram of methyl ester of fatty acids. The plate was developed in dichloromethane, sprayed with 50~ aq. sulfuric acid and charred. 1. The methyl ester of fatty acids from the upper phosphatidylethanolamine; 2. That from the lower phosphatidylethanolamine; 3. Methyl a-hydroxy stearate.

2-HYDROXY-13-METHYL-TETRADECANOIC ACID

31

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Gas-liquid chromatogram of fatty acid methyl esters from the phosphatidyl-

ethanolamine. Chromatography was run on a 3 m. column of 15 % DEGS on chromosorb W at 190°C. A, those from the upper phosphatidylethanolamine. B, those from the lower phosphatidylethanolamine. glycerol moiety by a study of hydrolysis 2) with snake venom phospholipiase A (E.C.3.1.1.4.). Furthermore, in order to elucidate the structure of the hydroxy fatty acids, they were purified as follows: After the removal of siolipin A 6) from the cephalin fraction, a mixture of phosphatidylethanolamine were subjected to hydrolysis with methanolic hydrochloric acid. The methyl esters of the hydroxy fatty acids were separated from the methyl esters of the non-hydroxylated fatty acids by silicic acid column chromatographyT). It was revealed by gas-liquid chromatography that the methyl esters of the hydroxy fatty acids were composed of a main acid and two minor ones (fig. 3A). As shown in fig. 4, the retention times of these methyl esters are on a straight line drawn through the retention times of the methyl esters of saturated straight chain c~-hydroxy fatty acids. This fraction was further purified by preparative gasliquid chromatography to give one peak (fig. 3B). The methyl ester of the hydroxy acid thus purified was analyzed by a variety of techniques. (1) As shown in fig. 5, its infra-red spectrum indicated this methyl ester was a methyl ester of a hydroxy fatty acid. (2) Gas-liquid chromatographic analysis indicated that the methyl ester had a retention time corresponding to that of the methyl ester of c~-hydroxy pentadecanoic acid. (3) As seen in fig. 6, this methyl ester corresponded to the methyl 2-hydroxy stearate but not the

J. KAWANAMIET AL.

32

methyl 10- and 12-hydroxy stearate on the basis of its mobility on thin-layer chromatography. (4) Although the reaction of this acid with copper nitrate 8) afforded a white precipitate, this reaction was not specific for ct-hydroxy fatty acids in the control experiment. (5) Treatment of this hydroxy acid with lead tetraacetate gave a peak corresponding to that of a branched (iso or anteiso) tetradecanal by gas-liquid chromatography in which the retention time of this aldehyde was on a straight line almost parallel but lower than A

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Fig. 3. Gas-liquid c h r o m a t o g r a m s o f methyl esters of the h y d r o x y fatty acids f r o m the p h o s p h a t i d y l e t h a n o l a m i n e . T h e preparative gas c h r o m a t o g r a p h y was run on a 10 ft × 3/8 in o.d. c o l u m n o f 2 5 ~ D E G S o n c h r o m o s o r b W at 190°C. A, before purification: B, after purification by m e a n s of a preparative G.L.C.

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Fig. 4. Relation between c a r b o n c h a i n length a n d retention times o f methyl esters o f h y d r o x y fatty acids. T h e definition of letters is in the fig. 3. Methyl esters o f 10- a n d 12- h y d r o x y fatty acids. [] Methyl esters of the h y d r o x y fatty acids f r o m the p h o s p h a t i d y l e t h a n o l a m i n e . - - - × - - Methyl esters of n o r m a l a - h y d r o x y fatty acids.

2-HYDROXY-13-METHYL-TETRADECANOIC ACID

33

the line drawn through the retention times of the saturated straight chain aldehyde. (6) A mass spectrum of the methyl ester of this hydroxy fatty acid is shown in fig. 7, together with that of the ester treated with deuterium oxide. These spectra are consistent with that of a methyl ~-hydroxy pentadecanoate as follows: The molecular ion peak at m/e 272 corresponds to C16H3203 which correlates with the result by elementary analysis. This "

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I n f r a - r e d spectrum o f the purified m e t h y l ester of the h y d r o x y f a t t y acid f r o m the phosphatidy]ethanolaminc.

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Fig. 6. Thin-layer chromatogram of methyl esters of hydroxy fatty acids. The plate was developed in dichloromethane, sprayed with 5 0 ~ aq. sulfuric acid and charred. 1. Methyl a-hydroxy stearate. 2. Methyl 10-hydroxy stearate. 3. Methyl 12-hydroxy stearate. 4. Methyl ester of the hydroxy fatty acid from the phosphatidylethanolamine.

34

J. K A W A N A M I

ET AL.

molecular ion peak was somewhat larger than those of the methyl esters of normal fatty acids and of such hydroxy fatty acids as methyl 10- and 12hydroxy stearate. Relatively intense peaks were observed at m/e 90 (89 + 1) and 213 ( M - 59), characteristic for the methyl esters of ~-hydroxy acids. 9,10) 45 --I

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m/e Fig. 7. Mass spectra of methyl ester of the hydroxy fatty acid obtained from the phosphatidylethanolamine. A, Methyl ester of the hydroxy fatty acid. B, Methyl ester of the deuteroxy fatty acid. In addition, relatively intense peaks were also observed at m/e 145 and 159, appearing to give rise in an analogous way through simple 6,7 and 7,8 cleavage, respectively. Other peaks were very similar to those from the isostearoyl alcohol. 10) As shown in fig. 7B, the spectrum of the deuterated compound showed peaks due to deuterium exchange of the active hydrogen. However, mass spectrometry could not reveal the position of the methyl branching in the hydroxy acid, while the methyl esters of non-hydroxylated iso fatty acids gave a peak at m/e (M-65) due to the loss of (CH3OH + CH3 + H 2 0 ). Hence, a study by proton magnetic resonance spectrometry was undertaken. As seen in fig. 8, it showed the presence of a doublet signal ( J = 7 cps, 6H) at 9.14v due to an isopropyl group.

2-HYDROXY-13-METHYL-TETRADECANOICACID

35

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Proton magnetic spectrum of methyl ester of the hydroxy fatty acid obtained from the phosphatidy]ethanolamine.

From these results, it is most probable that this hydroxy fatty acid is 2hydroxy- 13-methyl-tetradecanoic acid.

Experimental part Materials and methods

Reference hydroxy fatty acids e-hydroxy stearic and e-hydroxy-hexacosanoic acid, and 12-hydroxy stearic acid were purchased from CALBIOCHEM. (Los Angeles, U.S.A.) and purified by column chromatography. Pure 10-hydroxy stearic acid 11) was a kind gift of Dr. K. Saito (Kansai Medical School, Osaka).

Phosphatidylethanolamine This lipid had been isolated from St. sioyaensis as described previously 1, 2). The phosphatidylethanolamine moved as a double spot on thin-layer chromatograms, from which the upper and the lower spot were scraped off separately.

Hydrolysis by phospholipiase A About 110 mg of the phosphatidylethanolamine were dissolved in 10 ml of freshly distilled ether. Twenty mg of snake venom (Habu, Trimeresurus flavoviridis, Hallowell) in 10 ml of 0.1 M borate buffer (pH 7.0) and 0.25 ml of 0.0025 M calcium acetate were added to this ethereal solution. This mixture was shaken for 5 hr, with the aid of a shaker (TAIYO INCUBATOR K-If).

36

J. K A W A N A M I ET AL.

Thin-layer chromatography coated with silica gel G was used to establish if degradation was complete. The resultant lyso-phosphatidylethanolamine and fatty acids were separated by thin-layer chromatography on silica plates (20 × 20 cm), or by silicic acid column chromatography, using chloroformmethanol-water (70:20:5, by vol) as a developer.

Hydroxy fatty acids It was revealed by thin-layer chromatography that the fatty acids released by the hydrolysis of the lower phosphatidylethanolamine with snake venom phospholipiase A were composed of both non-hydroxylated and hydroxylated fatty acids. Thin-layer chromatography was performed with dichloromethane as a developer. 3) The bands corresponding to the authentic methyl c~-hydroxy stearate (CALBIOCH~M) were visualized after being sprayed with water, scraped off from the chromatoplate and extracted immediately with chloroform. The methyl esters of the hydroxy fatty acids obtained were analyzed by gas-liquid chromatography and were found to consist of one main peak and two minor peaks. Accordingly, this mixture was further purified by preparative gas-liquid chromatography (an Aerograph Autoprep. A-700) to give one major peak. The methyl ester of the hydroxy fatty acid thus purified was analyzed by thin-layer chromatography, infra-red spectroscopy, mass spectroscopy and so on. [c~]22-3.2_+0.5 (C, 0.805 in chloroform). Anal. Found. C, 70.50; H, 11.74. Calcd. for C16H3zO3, C, 70.59; H, 11.76~.

Analytical methods Optical rotation of the methyl ester of the hydroxy acid was measured in chloroform with a Pcrkin-Elmer Polarimeter, type 141. An infra-red spectrum was taken in CCI 4 with a Nihon Bunko DS-201 B Spectrophotometer. Proton magnetic resonance spectra were measured in carbon tetrachloride, containing tetramethylsilane as an internal standard using a Varian Model A-60 Spectrometer.

Mass spectroscopy (Y.N.) Mass spectra were obtained with a Hitachi RMU 6E single focus mass spectrometer with an energy of 70 eV and 80/tamp, using a direct inlet system at 250°C. The sample treated with deuterium oxide was introduced in a slurry.

Gas-liquid chromatography The methyl esters of the released fatty acids were prepared by treatment with diazomethane in ether. The methyl esters of the fatty acids from lysophosphatidylethanolamine were prepared by transesterification with hydro-

2-HYDROXY-13-METHYL-TETRADECANOIC ACID

37

chloric acid in methanol. The methyl esters were analyzed by means of a Shimadzu gas chromatograph, model 1B, equipped with a flame ionization detection system and 15~ polydiethyleneglycol succinate column as previously described 2, 4). The fatty acids were identified by comparison with the retention times of standards. Preparative gas-liquid chromatography was accomplished by an Aerograph Autoprep A-700, using 25~ polydiethyleneglycol succinate on a chromosorb W column, at 190°C, flow rate 100 ml He/min.

Oxidation with lead tetraacetate Treatment of the hydroxy acid (10 rag) with lead tetraacetate (16.3 mg) in glacial acetic acid (2 ml) at 50°C for 5 hr resulted in the formation of an aldehyde and a carboxylic acid. The aldehyde was isolated from the neutral fraction, while the carboxylic acid was extracted from the basic fraction after acidification with hydrochloric acid, respectively. The aldehyde and the methyl ester of the carboxylic acid were subjected to gas-liquid chromatography. Results and discussion

Composition and distribution of the fatty acids of the phosphatidylethanolamine The overall fatty acid composition of the phosphatidylethanolamine from

St. sioyaensis was already reported2). As seen in fig. 1, the upper phosphatidylethanolamine contained only non-hydroxylated fatty acids and the lower one contained hydroxy fatty acids in addition to non-hydroxylated fatty acids. On the other hand, hydrolysis with snake venom phospholipase A, an enzyme which releases specifically the fatty acids from the 2-ester position12,13), indicated that the hydroxy fatty acids occupied exclusively the 2-position, this being in contrast to the findings on the phospholipid fatty acids from Brucella abortus14).

Structure of the main hydroxy fatty acid As seen in fig. 3, the isolated hydroxy fatty acid was a main constituent among these hydroxy acids. It is clear that the main acid is a hydroxy fatty acid on the basis of elementary analysis, gas-liquid chromatographic study, thin-layer chromatography and the infrared spectrum. Mass spectrometry by deuterium exchange of the active hydrogen indicated the hydroxy acid to be a mono-hydroxy fatty acid. Further, the localization of the hydroxy group on the skeleton of the fatty acid was determined by mass spectroscopy and by the oxidation of this acid with lead tetraacetate; that is, the oxidation with lead tetraacetate gave a tetradecanal, while as seen in fig. 6, its mass

38

J. K A W A N A M I

ET A L .

s p e c t r u m afforded ion peaks at m/e 90, 145, 213(M-59), 272 (molecular ion) characteristic for a methyl ester o f e - h y d r o x y fatty acids. However, with the results o f the a b o v e experiments, a methyl b r a n c h i n g on the fatty acid skeleton c o u l d n o t be confirmed clearly. Hence, p r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y was u n d e r t a k e n in o r d e r to resolve this question. As seen in fig. 8, a d o u b l e t at 9.14z was observed for the t e r m i n a l methyl p r o t o n s (six protons), indicating the h y d r o x y acid to have an i s o - p r o p y l group. Therefore, the h y d r o x y fatty acid o b t a i n e d here m a y be defined as 2-hydroxy-13m e t h y l - t e t r a d e c a n o i c acid. The occurrence o f e- and fl-hydroxy fatty acids have been r e p o r t e d in several bacteria, including species o f Serratia15), E. Colil6), Pseudomonas 17,18), Brucella14) a n d Mycobacteriumt9). H o w e v e r , no i n f o r m a t i o n was available on the fl-localization a n d the b r a n c h e d chain h y d r o x y fatty acids. It m a y be assumed that the occurrence o f this acid in St. sioyaensis shows t h a t such b r a n c h e d chain fatty acids as iso and anteiso acids also m i g h t be d e g r a d a t e d by e - o x i d a t i o n as in a n i m a l b r a i n fatty acids 20) and the localization o f this acid at the fl-position a p p e a r s to indicate t h a t this acid actively might t u r n over as in highly u n s a t u r a t e d fatty acids at the fl-position o f glycerophosphatides.

Acknowledgement T h e a u t h o r s wish to t h a n k Dr. K. Saito ( K a n s a i M e d i c a l School) for his generous gift o f 10-hydroxy stearic acid.

References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20)

A. Kimura, J. Kawanami and H. Otsuka, J. Biochem. (Tokyo) 62 (1967) 384 A. Kimura, J. Kawanami and H. Otsuka, Agr. Biol. Chem. (Tokyo) 31 (1967) 1434 E. Martensson, Biochim. Biophys. Acta 116 (1966) 296, 309 J. Kawanami, J. Biochem. (Tokyo) 62 (1967) 105 H. Okuyama and S. Nojima, J. Biochem. (Tokyo) 57 (1965) 529 J. Kawanami, A. Kimura and H. Otsuka, Biochim. Biophys. Acta 152 (1968) 808 Y. Kishimoto and N. S. Radin, J. Lipid Res. 4 (1963) 130 A. H. Milburn and E. V. Truter, J. Appl. Chem. 12 (1962) 156 R. Ryhage and E. Stenhagen, J. Lipid. Res. 1 (1960) 360; Arkiv. Kemi 15 (1960)545 D. Chapman, The Structure of Lipids, Methuen and Co. Ltd., London (1965) p. 150 K. Saito, J. Biochem. (Tokyo) 59 (1966) 487 G. H. de Haas and U L. M. van Deenen, Biochim. Biophys. Acta 48 (1961) 215 N. H. Tattrie, J. Lipid Res. 1 (1959) 60 O. W. Thiele and D. Busse, Experientia 24 (1968) 112 N. J. Cartwright, Biochem. J. 67 (1957) 663 J. H. Law, Bacteriol. Proc. (1961) 129 S. Bergstrom, H. Theorell and H. Davide, Arch. Biochem. 10 (1946) 165 F. G. Jarvis and M. J. Johnson, J. Am. Chem. Soc. 71 (1949) 4124 J. Asselinean, private communication (Ann. Inst. Pasteur 114 (1968) 305) G. M. Levis and J. F. Mead, J. Biol. Chem. 239 (1964) 77