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Analysis of lipopolysaccharide (lipid A) fatty acids
Journal ofMicrobiologicalMethods
I1 (1990) 195-211
195
Elsevier MIMET 00373
Analysis of lipopolysaccharide (lipid A) fatty acids Horst-Werner Wollenweber and Ernst T. Rietschel Forschungsinstitut Borstel, lnstitut fiir Experimentelle Biologic und Medizin, Borstel, FRG (Received 6 October 1989; revision received i0 March 1990; accepted 23 March 1990)
1 Introduction
Lipid A represents the common, covalently bound lipid component of bacterial lipopolysaccharides (LPS, endotoxin). Lipid A preparations derived from LPS of most bacterial groups possess a comparable chemical architecture. In general [for exceptions, compare 76, 77], lipid A consist of a Bl,6-1inked D-glucosamine disaccharide, which carries (in some cases, substituted) phosphate-groups in position 1 and 4' (lipid A backbone) and long-chain fatty acids in ester and amide bonds [1, 2]. The chemical structure of Escherichia coli and Salmonella lipid A has been elucidated in detail [1, 4, 7, 8]. E. coli-type lipid A (Fig. 1) has been chemically synthesized and bacterial and synthetic E. coli lipid A exhibited identical potent biological activity [3], proving the previous hypothesis that lipid A represents the endotoxic principle of LPS to be correct. More recently, lipid A partial structures have been synthesized the biological analysis of which points to fatty acids as being essential for the mediation of endotoxicity [4]. Thus, it was recognized that the quantity, nature, location and type of linkage of fatty acids determine the degree of biological activity of LPS or lipid A. To elucidate the significance of fatty acids, numerous studies were performed on the fatty acid composition of LPS and lipid A, derived from various genera of Gram-negative bacteria [for summary, see 1, 2, 4, 5]. Depending on the source of lipid A, various types of ester-bound fatty acids were identified. Thus, long-chain, even- or odd-numbered, iso- or anteiso-branched (S)-2-hydroxy and (R)-3-hydroxy fatty acids have been found. In some bacterial groups, the 3-hydroxyl group of ester-bound 3-hydroxy fatty acids is acylated by other fatty acids whereas in other species it is free. Cyclopropane and unsaturated fatty acids have only occasionally been detected in lipid A [compare 6]. In contrast to the broad spectrum of fatty acids involved in ester bonds, as a rule, only one type of fatty acid is amide-linked, viz., (R)-3-hydroxy fatty acids. These 3-hydroxy acids are even- or odd-numbered (iso-branched) and possess, in general, 10-22 C atoms. The 3-hydroxyl group of at least one N-acyl residue has been shown Correspondence to: E.T. Rietschel, Forschungsinstitut Borstel, Parkallee 1-42, 2061 Borstel, FRG.
0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
196 OH
o
o\,_0" \ - \ / .o" \ N. -
0
~ -~\ ,_0 ® \ ~ % / "oH H
H
Fig. 1. Chemicalstructureof E. coli lipidA (4): Hydroxylgroup in position6' representsattachmentsite of polysaccharideportion in LPS. Numbers in circlesindicatenumber of C atoms in acyl chains.
to be usually substituted by other fatty acids [1, 2, 7, 86]. Some bacterial strains contain, besides 3-hydroxy fatty acids, also amide-linked 3-oxo fatty acids which contain a keto-group instead of a hydroxyl function at C atom 3 [8, 76]. Recently, a hydroxy fatty acid with 28 C atoms (27-hydroxyoctacosanoic acid) has been identified to be present in amide or ester linkage in certain lipid A preparations [77, 79]. As a rule, (R)-3-hydroxy fatty acids (ester- and amide-bound) predominate within the fatty acid moiety of lipid A. Since they are generally missing in other cell-wall lipids of Gram-negative bacteria [for exceptions, compare 80, 81], (R)-3-hydroxy fatty acids are peculiar to lipid A and represent characteristic lipid A markers [1, 2, 4, 5, 10-12].
2 Analysis of Fatty Acids For the quantitative and qualitative determination of fatty acids, a number of methods are available [summarized in 13]. These include for example titrimetric [14, 15], colorimetric [16-21], radiochemical [22] and chromatographic [23-25] procedures as well as negative-ion fast-atom bombardment-mass spectrometry [9]. Among these, gas-liquid chromatography (GLC), often combined with mass spectrometry (MS), has proven to be a most powerful tool for the identification and estimation of fatty acids. In the following, therefore, procedures will be described which allow the GLC/MS analysis of fatty acids derived from LPS. All procedures are based on the Salmonella minnesota Re mutant strain R595 (mRe). As the LPS of this deep rough mutant contains only Kdo (3-deoxy-D-manno-2-octulosonic acid [5]) and lipid A, quantities should be appropriately adjusted upwards for other lipopolysaccharides to give approximately the same fatty acid concentration. Quantitative analyses are carried out in the presence of two internal standards. Nonhydroxylated fatty acids are quantitated relative to heptadecanoic acid methyl ester whereas hydroxylated fatty acids are determined relative to an added hydroxy fatty acid ester (in the case of LPS from the mRe mutant, 3-hydroxydodecanoic acid may be used
197 [7]). The use of the second standard fatty acid is not obligatory if hydroxyl groups of hydroxylated fatty acids are derivatized prior to GLC analysis [see Section 7.2]. In the present paper, methods for the analysis of fatty acids liberated from LPS (or lipid A) are described. The application of soft-ionization techniques (like fast-atom bombardment and laser-desorption MS) to determine the location of acyl groups at the lipid A backbone are described in [26, 43]. 3 Liberation of Fatty Acids from LPS
3.1 Total fatty acids 3.1.1 Release of fatty acids in the free form 3.1.L1 General Release of free fatty acids from LPS can be achieved by either acidic or alkaline hydrolysis. Acid-catalyzed hydrolysis liberates both ester- and amide-bound fatty acids. Application of acid hydrolysis to structural studies of LPS, however, is limited by the fact that 3-hydroxy fatty acids are partially dehydrated and that (less volatile) artefacts, such as polymerized 3-hydroxy fatty acids (estolides) and other 3-acyloxyacyl residues, are formed [27, 28]. Strong alkaline hydrolysis (KOH, NaOH) as well as hydrazinolysis [29, yielding hydrazides] and hydroxylaminolysis [30, yielding hydroxamates] also liberate both ester- and amide-bound fatty acids. The methods suffer, however, from the fact that substantial amounts ct,~-unsaturated (with smaller amounts of ~,~-unsaturated) fatty acids may be formed [31] by an alkali-catalyzed ~-elimination of 3-acyloxyacyl residues present in LPS [7, 32, 33]. Furthermore, amide-bound ct,~unsaturated acids are not easily liberated by strong alkaline hydrolysis. Thus, for the liberation of fatty acids from LPS a two-step procedure has been proposed which consists of an initial acidic and a second alkaline hydrolysis step in water [28]. The alkaline hydrolysis splits artefacts, such as estolides and formed 3-acyloxyacyl residues (see above). A disadvantage of this method is the need of carbomethylation of free fatty acids prior to GLC/MS analysis (see Section 5.1). Therefore here, for the release of total fatty acids from LPS an acid hydrolysis step followed by an acid-catalyzed (trans)esterification reaction is recommended which converts liberated free fatty acids and artefacts produced by acid, i.e., estolides and 3-acyloxyacyl residues, directly to fatty acid methyl esters (see Section 3.1.2.2). 3.1.2 Release o f total fatty acids as methyl esters (acid-catalyzed transesterification) 3.L2.1 General Fatty acid methyl esters may be obtained directly from LPS by methanolysis in the presence of either Lewis (BCI3, BF3) or Br6nsted (HCI, H2SO4) acids. With BCI3, amide-bound fatty acids are only partially liberated. With BF3-methanol (14% by weight), complete release of ester- and amide-linked fatty acids from LPS is achieved after 6 h at 100°C [34-36]. 3-Methoxy fatty acids may be formed as artefacts during BF3-catalyzed methanolysis. Furthermore, nonpolar
198 WCOT glass capillary columns usually used for GLC of fatty acid esters are strongly activated by trace amounts of boric acid derivatives. Such columns are no longer useful for the GLC analysis of underivatized hydroxy fatty acid methyl esters. Acid (HCl)-catalyzed methanolysis alone does not liberate the amide-linked fatty acids quantitatively. This procedure is only recommended for the detection of amidelinked 3-oxo fatty acids which are lost at harsh aqueous acidic conditions, presumably via a decarboxylation reaction. The presence of 3-oxo fatty acids is, therefore, recognized by treatment of LPS with methanolic HC1 [38] omitting the initial HCI hydrolysis step. Treatment of LPS with HCI, followed by methanolic HC1, liberates O- and N-acyl residues quantitatively and artefact formation is minimal. No further esterification step is necessary and, therefore, this reaction is recommended for the liberation of ester- and amide-bound (total) fatty acids from LPS. 3.1.2.2 Procedure. The mRe LPS (2 mg) and standard heptadecanoic acid (200/~g) and 3-hydroxydodecanoic acid (600/~g) are heated with HCI (4 M, 1 ml) at 100 °C for 4 h in a Teflon-lined screw cap tube (5 ml size). After cooling, the sample is transferred with chloroform (--- 10 ml) to a separatory funnel and water (= 10 ml) is added. Fatty acids are extracted into chloroform (3 × 10 ml), the combined organic phases dried over anhydrous Na2SO4, concentrated (rotary evaporator) and transferred with chloroform to a glass tube (5 ml size) equipped with a tightly fitting Teflon-lined screw cap. To the N2-dried sample HC1 in methanol (-- 2 M a, 1 ml) is added and (trans)esterification allowed to take place for 6 h at 85 °C. When cool, the mixture is concentrated to about half its initial volume by a stream of N z (to partly remove HCI and methanol), an aqueous solution of half-saturated NaCI is added (1 vol) and the fatty acid methyl esters extracted by 2 x 3 vols. of distilled petroleum ether (40-60 °C)/ethylacetate (1:1, v/v). The combined organic phases are, at room temperature, carefully concentrated by a stream of N2. Concentration to complete dryness should be avoided because of the volatility of short-chain fatty acid methyl esters (e.g., dodecanoic acid methyl ester). Hydroxy fatty acid methyl esters present in the mixture may then be derivatized by trifluoroacetylation (see Section 7.2). Otherwise, the residue is redissolved in distilled chloroform (50#1) and subjected to GLC/MS analysis (see Section 8.1). 3.2 Ester-bound fatty acids 3.2.1 General Ester-bound fatty acids may be selectively liberated from LPS by mild hydrazinolysis [39], controlled hydroxylaminolysis[32, 40], treatment with methanolic NaOH [41] and NaOCH 3 [32, 42] or by 'on column' transesterification with m-(trifluoromethylphenyl)-trimetyl ammonium hydroxide [37]. Of these methods, transesterification with NaOCH 3 is preferred since it directly yields fatty acid methyl esters. If a 3-acyloxyacyl residue is present in LPS in ester linkage, NaOCH 3 will induce a ~elimination reaction resulting in the liberation of a free fatty acid (the substituent at a Generatedby carefullyaddingdrop-wiseacetylchloride(1.5 ml) to dry methanol(10 ml) at 0°C.
199
0 C=O
H3CO"c#O
i.2 HC-O-C i H2 R I CH3
H3CO" C~0
0c.,
0c.3 CH
ICH2 R CH3
. C I~ R
L Fig. 2.
H3CO'c~O
!H 2 CH3 CH2N2
!., HC --OCH3
cH~ t CH3
H3CO'~c~O P I R
Transesterification and/3-elimination reaction involving a 3-acyloxyacyl group present in lipid A in ester linkage (2).
the 3-hydroxyl group of the 3-hydroxy fatty acid) and in the formation of an c¢,~unsaturated fatty acid methyl ester with a chain length identical to the 3-hydroxy fatty acid from which it derives (Fig. 2). The liberated unsaturated fatty acid ester, containing a conjugated double-bond (between atoms 2 and 3), is subject to nucleophilic attack by H3CO- ions, resulting in the nearly quantitative formation of the corresponding 3-methoxy fatty acid methyl ester (Fig. 2). The nature of the eliminated substituent can be determined by GLC after additional carbomethylation of the alkaline methanolysate [32, 33]. It should be noted that the eliminated free fatty acids may also be detected (without further derivatization) on GLC analysis if the described glass capillary columns are used (see Section 8.1.1). 3.2.2 Procedure The mRe LPS is freed from water by keeping it under vacuum at 30°C for 12 h. Dry LPS (10 mg) is placed in a 10-ml dry glass tube equipped with a Teflon-lined screw cap and a small Teflon-coated magnetic bar. NaOCH3 in absolute methanola (= 0.25 N b, 5 ml), heptadecanoic acid methyl ester (1 mg) and 3-hydroxydodecanoic acid methyl ester (1 mg) are added and the suspension kept under magnetic stirring at 37 °C for = 15 h. After removal of the magnetic bar, the tube is centrifuged (10 min, 2000×g) and the supernatant decanted into a separatory funnel (I00 ml size). [The sediment (de-O-acylated LPS, LPS-OH) is washed with cold methanol (1 ml) and kept for the analysis of amide-bound fatty acids (see Section 3.3.2).1 The combined supernatants are acidified (pH 1-3) with HCI (0.25 N, = 10 ml), a solution of half-saturated NaCI (2 ml) is added and the mixture extracted with distilled chloroform (3 x 20 ml). The combined chloroform extracts are well-dried over anhydrous Na2SO 4, passed through a paper filter into a round-bottom flask and concentrated (rotary evaporator, water bath < 30 °C). The fatty acid methyl esters are dissolved in distilled diethyl ether (1 ml), transferred to a small vial with internal cone shape (reactivial with Mininert Valve; Pierce Chemical) and ether is removed by a gentle stream of N 2. The residue is redissolved in chloroform (200/zl) and subjected to a The use of absolute methanol is essential since 3-hydroxy fatty acid methyl esters are highly susceptible to alkaline saponification. b 575 mg Na (shortly washed with methanol and ether) dissolved in 100 ml of absolute methanol (hood!),
200 GLC/MS analysis (see Section 8.1). To elucidate the nature of/3-eliminated fatty acids, the methanolysate is additionally carbomethylated with diazomethane (see Section 5.2.2) and again analysed by GLC/MS. 3.3 A m i d e - b o u n d f a t t y acids 3.3.1 General
Treatment of LPS with methanolic NaOCH 3 yields a sediment which represents de-O-acylated LPS (LPS-OH) containing amide-bound fatty acids. These fatty acids can be liberated from LPS-OH by acid hydrolysis. 3-Acyloxyacyl residues present in LPS in amide linkage undergo a/3-elimination reaction caused by NaOCH 3 resulting in the partial (in Salmonella, -~25070) liberation of the free fatty acid previously linked to the 3-hydroxyl group of the amide-linked 3-hydroxy fatty acid. Concomitantly, the corresponding, still amide-bound, a,~unsaturated fatty acid is formed which, however, is not attacked by H3CO- ions (but compare Section 3.2.1). This amide-linked a,~unsaturated fatty acid is not quantitatively liberated by conventional hydrolysis procedures and for its estimation indirect methods, such as oxidation with MnO4-/IO 4- or reduction with deuterium, have been described [7]. Amide-bound 3-hydroxy fatty acids, still linked to the lipid A glucosamine residues, have also been directly [45, 82] or after controlled degradation of glucosamine [46] analysed by GLC/MS. 3.3.2 Procedure
The sediment (LPS-OH) obtained after treatment of LPS with methanolic NaOCH 3 (Section 3.2.2) is, is for purification, dissolved (in the original glass tube) by ultrasonication in distilled water (~-0.5 ml) and acidified with 2 N acetic acid (pH 2-4). To this solution, cold distilled ethanol ( ~ 8 ml) is added and, after shaking, LPS-OH is allowed to precipitate at 4 °C for 1 h. If precipitation does not occur, 0.1-0.5 ml petroleum ether (40-60°C) is added. The tube is centrifuged (2000xg, 3 min), the supernatant removed by suction, the precipitated LPS-OH washed three times with cold distilled acetone, redissolved in distilled water (2 ml, ultrasonication, warming) and lyophilized (yield ~ 5 mg). For liberation and analysis of amide-bound fatty acids, LPS-OH (2 mg) is treated in the same way as LPS (Section 3.1.2.2). 4 Liberation of 3-Acyioxyacyl Residues from LPS 4.1 General
Many LPS, including that of mRe bacteria, contain ester- and amide-bound 3-acyloxyacyl-residues [1, 2, 4, 7, 32, 33, 78]. The presence of an ester-bound 3-acyloxyacyl group is indicated by the formation of 3-methoxy fatty acid methyl ester on treatment of LPS with NaOCH 3 (see Section 3.2.2). Ester-bound 3-acyloxyacyl groups can also be liberated intact from LPS by short alkaline hydrolysis (0.25 N NaOH, 4 min, 56 °C [32]) and thus be analysed directly by GLC/MS (see Section 8.2). As shown in Fig. 3, amide-bound 3-acyloxyacyl-residues are liberated from LPS [2,
201 O
O
NH ~-0 CH2 O I I1 HC-O-C-R I ( CH2)n [ 3 CH
INI CH3J/Ag" ~
C-OCH 3 CH2 0 I II HC-O-C- R I (CH2)n ~H 3
0% rnRd2ooCaCid ~
/OCH3
CI CH2 0 I I HC- O-C-R I (CH2)/7 I 3 CH
C=0
NH3 +
Fig. 3. Steps involvedin selectiveliberationof 3-acyloxyacylgroup presentin lipid A in amidelinkage (2, 7).
7] in the form of their methyl esters after conversion of the amide bond (by methyliodide in the presence of Ag salts) to an acid-labile imidate (double bond between C and N). Because of the better solubility in organic solvents, this reaction is carried out with free lipid A which is obtained as a precipitate after mild-acid hydrolysis of LPS (1070 acetic acid, 1 h, 100°C; washing with 0.1 N HCI, 5 ml [5]). Due to trace amounts of water present, also ester-bound 3-acyloxyacyl groups are liberated to a small extent. To distinguish between ester- and amide-bound 3-acyloxyacyl residues, therefore, the procedure is carried out once in the presence and once in the absence of the methylating reagent (methyliodide).
4.2 Procedure [7] Two samples, mixtures of free lipid A (2 mg), Ag20 (7 mg [prepared according to 44]) and silver-trifluoromethanesulfonate (15 mg; Merck, Darmstadt, FRG) are kept for 12 h in an evaporated desiccator over P205 in the dark. To each sample, water-free petroleum ether (2 ml, 40-60 °C) is added, and the vials, closed with Teflon-lined screw caps, are flushed with a gentle stream of dry N 2 by syringes and sonicated (15 min, 20 °C, dark). Distilled CH3I (10/zl) is added to one sample and methylation allowed to take place under sonication (2 h, 30-40 °C, dark). From the other sample, CHaI is omitted but otherwise it is subjected to the same procedure as the methylated sample. The reaction mixture is transferred to a centrifuge tube containing an aqueous solution of NaES203 (0.5 M, 2 ml). 3-(Tetradecanoyloxy)tetradecanoic acid ethyl ester (prepared according to [47] and esterified with diazoethane, see Section 5.2.1) is added as internal standard, the reaction mixture acidified with HaPO 4 (10070) to pH ~ 2 and shaken (15 min, 20 °C), or, more efficiently, ultrasonicated (30 min, 25-30 °C). After centrifugation (2000 x g, 10 min), the reaction mixture is extracted with petroleum ether/ethylacetate (1:1, v/v, 3 x 2 ml) and the respective combined organic phases are washed with distilled water (2 x 5 ml), dried over Na2SO 4, concentrated to ~ 1 ml and purified by application to a small column (20× 3 mm) packed with silica gel 60 (100-240 mesh) which is eluted with petroleum ether/ethylacetate (1:1, v/v, 10 ml). The eluate is concentrated, carbomethylated with diazomethane (see Section 5.2.2) and analysed by GLC/MS (see Section 8.2).
202 5 Carbomethylation of Free Fatty Acids (Methyl Ester Formation) 5.1 General Fatty acids are usually analysed by GLC in the form of their volatile methyl esters and several convenient methods for methyl ester formation are available. These methods include reaction of fatty acids with methanol in the presence of an acidic catalyst (HCI, H2SO4, BC13, BF3), treatment with N,N'-dicyclohexyl carbodiimide and methanol [48] or diazomethane [49, 50, compare also 51]. Detailed procedures for esterification of fatty acids with HCl-methanol [52, 53] and BF3-methanol [32, 34, 54-56] have been described. Here, the preparation of fatty acid and 3-acyloxyacylmethyl esters by diazomethane will be discussed. This procedure leads to rapid and complete esterification. It has been noted, however, that ct,~-unsaturated fatty acids will react with diazomethane to form pyrazoline derivatives [57] which are not detected under the conditions used for GIAZ/MS. Therefore, if ot,~-unsaturated fatty acids are to be expected, the LPS hydrolysate should (for comparison) also be esterified by other methods (e.g., MeOH/HCI). 3-Hydroxy fatty acids are partially converted by diazomethane to the 3-methoxy fatty acid derivative. This artefact is, however, formed to only a small extent (--1%). Larger amounts of diazomethane are prepared from p-toluenesulfonyl-N,Nmethylnitrosamid (Diazald; Aldrich, Milwaukee, Wisconsin) by distillation with diethyl ether in glassware with clear-seal joints (Diazald Kit; Aldrich) [compare 58] to prevent contamination and explosion. (The distillation should be performed under a hood and behind a safety shield.) The etheral solution can be stored at -30°C for some weeks. Smaller amounts of diazomethane are easily prepared with the help of a diazomethane generator a or by extraction into diethyl ether from N,N-nitrosomethyl urea b which should, however, be handled with caution. Diazoethane is prepared in a similar way (using N,N-nitrosoethyl urea; Serva, Heidelberg, FRG). 5.2 Procedures 5.2.1 Preparation of diazomethane (or diazoethane) in ether b Diethylether (10 ml) and 40% KOH (2 ml) in a separatory funnel (50 ml size) are cooled to 0 °C (ice bath). N,N-nitrosomethyl urea (or N,N-nitrosoethyl urea, 1 g; Serva) is gradually added under gentle stirring. After 5 min, the alkaline water phase is removed, the yellow ether phase transferred to a precooled Erlenmeyer flask (50 ml size) and kept for 30 min over KOH pellets at 0 °C. This diazomethane solution can, in general, be used without further purification, 5.2.2 Carbomethylation (methyl ester formation) To the solution of free fatty acids (1 mg) in ether (1 ml), first distilled methanol a Wheaton, 1000 N Tenth Street, MillviUe, New Jersey 08332. b N,N-nitrosomethyl urea is a carcinogen. Gloves should be used and the procedure should be performed under a hood.
203 (0.1 ml, Whirlmix) is added as a catalyst at room temperature. Diazomethane in ether (Section 5.2.1) is then added in portions (0.1 ml) until the yellow colour persists. After 10 min, solvents and excess diazomethane are removed (under a hood) by a gentle stream of N 2. The fatty acid methyl esters are redissolved in distilled chloroform and subjected to GLC analysis.
6. Stereochemicai Analysis of Hydroxy Fatty Acids 6.1 General The stereochemistry of 2- and 3-hydroxy fatty acids present in LPS has been studied by optical rotation measurements [5, 11, 32, 35]. This method, however, requires large amounts of material and the possible presence of both optical antipodes makes optical rotation measurements difficult. Therefore, based on results of Karlsson and Pascher [59], a GLC procedure was developed for the analysis of diastereomeric derivatives of bacterial hydroxy fatty acids [11, 60]. By this method, both 2- and 3-hydroxy fatty acids are separated according to chain length and configuration. 6.2 Procedure The mRe LPS (10 mg) is hydrolyzed in a sealed tube with strong alkali (4 N KOH, 5 h, 100°C). The liberated (hydroxy) fatty acids are, after acidification (pH ~ 1) extracted with distilled chloroform (see Section 3.2.2), after drying (NaESO4) and concentration transferred with water-free diethyi ether (0.5 ml) to a dry a glass tube (10 ml size, with a glass stopper), kept at --- -15 °C (ice/NaCl) and mixed with cool (-15 °C) BF3-ethyl etherate (10-20 ~l; Fluka, Buchs). To this solution, precooled diazomethane in ether (see Section 5.2.1) is gradually added (1-ml portions) until the yellow colour persists for = 30 s [61, 62] (~-8 ml diazomethane solution). After 10 rain, the reaction mixture is filtered through paper into a separatory funnel and washed once with saturated NaHCO 3 and twice with distilled water. The ether phase, after drying over NaESO4, is filtered into a round-bottom flask (25-ml size), partially evaporated (rotary evaporator), transferred to a 15-ml glass tube and taken to dryness with a stream of N2b. KOH (1 N, 1 ml) is added and the methoxy fatty acid methyl esters saponified at 70 °C for 1-2 h. The free methoxy fatty acids are extracted with chloroform and applied to chloroform-washed silicic acid thin-layer plates (Kieselgel H; Merck). Chromatography is performed with petroleum ether-diethyl ether-acetic acid (35 : 15 : 1, v/v/v) and fatty acids are visualized by iodine vapour. The area corresponding to methoxy fatty acids (Rf=0.5) is eluted from the plate with distilled chloroform, the extract transferred to a serum vial (5 ml size) and taken to dryness by N 2. The vial is closed with a Teflon-lined Bordelcap (Machery and Nagel) and flushed by two cannulae with N 2. The methoxy fatty acids are dissolved in dry chloroform (1 ml, passed into the vial through a cannula) and, after addition of thionyl chloride (0.25 ml), the vial is kept at 60 °C for 30 min. Chloroform and excess SOCl 2 are flushed from the vial with N2, the methoxy fatty acid chlorides dissolved in dry chlo-
a The useof water-freesolventsand dry glasswareis essentialfor obtaininggoodyields(> 90%) of methoxy fatty acid derivatives. b Since trance amounts of boric acid derivatives activate glass-capillary columns, the formation of 3-methoxy fatty acid methylesters should be checkedby using packed (and not capillary)columns.
204 roform (1 ml) and L-phenylethylamine (0.25 ml, Merck) is added and the mixture kept at room temperature for 30 min. The vial is opened, the chloroform phase washed (separatory funnel) once with HCI (0.01 N) and twice with distilled water, dried over Na2SO4, filtered and taken to dryness (rotary evaporator). The methoxy fatty acid-zphenylethylamides are redissolved in distilled chloroform (50 #l) and analysed by GLC/MS (see Section 8.3). 7 Other Modifications of Hydroxy Fatty Acids 7.1 General
A series of modifications of the hydroxyl group of 2- and 3-hydroxy fatty acids of LPS have been described. These modifications may be useful for the identification of hydroxy fatty acids by GLC, especially if MS is not available. The hydroxyl group of both 2- and 3-hydroxy fatty acid methyl esters may be acetylated [11, 28], trifluoroacetylated [38, 63, 64, 83, 84, see below], trimethylsilylated [32, 65], tbutyldimethylsilylated [2, 66] and O-methylated with BF3-etherate/diazomethane (see Section 6.2) or with CH3I/Ag20 [59]. Reactions specific for 3-hydroxy fatty acids include reduction of the methyl ester and conversion into acetonides [67], dehydration of the free 3-hydroxy fatty acid by either treatment with acetic acid anhydride [60], PBr3 and potassium t-butylate [68, 69] or concentrated H2SO4 [16]. The resulting ot,flunsaturated fatty acids can be converted to the 3-picolinyl ester [85] or to the corresponding saturated fatty acids either by hydrogenation with Pd on charcoal (10%) as a catalyst [7, 32] or by nascent H2 [69]. Furthermore, they may be oxidized with KMnO4 to a saturated fatty acid, possessing two C atoms less than the original a,flunsaturated and 3-hydroxy fatty acid, respectively [7, 32]. For the quantitative estimation of hydroxylated fatty acids (notably, 3-hydroxy fatty acids), the use of a hydroxylated internal standard is essential (compare Sections 2 and 3.1.2.2). The addition of a hydroxylated standard is, however, not obligatory if the hydroxyl groups of the hydroxy fatty acids to be analysed are protected. As protecting groups, trifluoroacetyl residues have been found to be useful. The derivatization procedure leads to rapid and complete acylation and the derivatives obtained are stable and exhibit excellent chromatographic behaviour. Therefore, for the derivatization of hydroxyl groups present in hydroxy fatty acid methyl esters, trifluoroacetylation is recommended (GLC profiles and retention times of trifluoroacetylated 3-hydroxy fatty acid methyl esters are given in [83, 84]). 7.2 Procedure [38]
To a mixture of fatty acid methyl esters containing hydroxy fatty acids (in a small glass tube, 5 ml size, with a Teflon-lined screw cap) trifluoroacetic anhydride in acetonitrile (50/zl, 1 : 1, v/v) is added. The solution is heated (boiling) for 2 min and after 10 min at room temperature diluted with acetonitrile (100/A). The mixture is (without further manipulations) directly analysed by GLC.
205
8 GLC/MS Analysis of Fatty Acid Derivatives 8.1 Fatty acid methyl esters 8.L1 GLC The identity of long-chain fatty acids a may be tentatively determined on the basis of relative retention times obtained with different liquid phases and comparison with authentic reference substances. In Table 1, the relative retention times (tR of tetradecanoic acid methyl ester = 1.00) derived from S. minnesota Re LPS as obtained. on three liquid phases (packed columns) with different polarities are given. The stationary phases used (operating temperature given in the table) were: (i) SE-30 (10%0 on Gas Chrom Q, 100-120 mesh), (ii) Castorwax (2.5%o on Chromosorb W, 80-100 mesh) and (iii) EGSS-X (15% on Gas Chrom P, 100-120 mesh). In all cases, glass columns (0.3 × 200 cm) were used with a N2 flow of 30 ml. min -1. In general, for qualitative and quantitative GLC/MS analyses of fatty acid methyl esters capillary columns (glass and fused silica) are preferable (for exceptions, see Section 6.2) and in Table 2 relative retention times of bacterial fatty acids as obtained on two types of nonpolar phases (OV-101; Chrompack, Berlin, FRG; SE-54; Weeke, Duisburg, FRG) are listed. Glass capillary columns (WCOT 25 m, i.d. 0.25 mm, split-mode injection type, split ratio 1:40) were used, connected to a gas chromatograph (Varian 3700) equipped with a flame-ionization detector and combined with an automatic integrator (Hewlett-Packard 3380A). H 2 ( ~- 2 ml. rain -1) served as carrier gas. To obtain
TABLE 1 NATURE AND RELATIVE RETENTION TIMES OF FATTY ACIDS PRESENT IN HYDROLYSATES OF S. MINNESOTA Re LPS AS ANALYSED ON PACKED COLUMNS (tR of 14:0 = 1.00) Fatty acid methyl ester
Relative retention times (tR) SE-30 (10%) (170°C)
Castorwax (3%) (175 °C)
EGSS-X (15a/0) (140°C)
12:0 14:0 16:0 2-OH-14:0 3-OH-14:0
0.46 1.00 2.23 1.58 1.74
0.40 1.00 2.45 2.42 3.16
0.51 1.00 1.98 5.00 7.90
A3-14:la A2-14:1a 3-OCH3-14:0 a
1.00 1.20 1.52
1.00 1.46 1.82
1.36 1.70 2.42
17:0b 3-OH-12:0 b
3.33 0.80
3.81 1.26
2.80 4.00
a Artefact formed from 3-OH-14:0. b Internal standard.
a For GLC analysis of acetyl groups in LPS, compare [70].
206 TABLE 2 N A T U R E A N D R E L A T I V E R E T E N T I O N TIMES OF F A T T Y ACIDS P R E S E N T IN H Y D R O L Y SATES OF S. M I N N E S O T A Re LPS AS A N A L Y S E D ON GLASS C A P I L L A R Y C O L U M N S (IR of 14:0 = 1.00) Fatty acid methyl ester
Relative retention times (tR) OV-101 (150°C)
SE-54 (140°C)
0.39 1.00 2.50 1.66 1.89
0.36 1.00 2.70 1.76 2.04
A3-14:1 a A2-14:1 a 3-OCH3-14:0 a
0.96 1.22 1.70
0.95 1.27 1.82
17:0 b 3-OH-12:0 b
3.95 0.63
4.39 0.62
12:0 14:0 16:0 2-OH-14:0 3-OH-14:0
a Artefact formed from 3-OH-14:0. b Internal standards.
reproducible results, it is important that the proper analysis conditions (such as amount of sample and volume, geometry of injection port, injection technique [71]) are determined with the aid of a standard mixture of representative fatty acid methyl esters. 8.1.2 MS Electron-impact (EI) MS of synthetic and bacterial 2-hydroxy, 3-hydroxy, 3-oxo and nonhydroxylated fatty acids has been described in detail [7, 8, 11, 28, 35, 36, 45, 46, 63-65, 69, 72-74]. The EI fragmentation pattern of 2-hydroxy fatty acid methyl esters is characterized by peaks at m/z=M, M-32, M-59 and 90 (CH2OHCOOCH3). The base peak is found at m/z= 83. Fragments characteristic for 3-hydroxy fatty acid methyl esters include m/z=M-18, M-50, 74 and 103 (CHOHCH2COOCH3), with the base peak at m/z=43. Spectra of 3-methoxy fatty acid methyl esters exhibit characteristic fragments at m/z--M-15, M-30 and 117 (CHOCH3CH2COOCH 3, base peak at m/z = 75). 3-Oxo fatty acid methyl esters are characterized by peaks at m/z--M and M-73, the base peak being at m / z = l l 6 (CH2COHCHECOOCH3 [74]). 8.2 3-Acyloxyacyl methyl esters 8.2.1 GLC 3-Acyloxyacyl methyl esters, the preparation of which has been described [7, 28, 47], are analysed on capillary columns and Table 3 summarizes relative retention times (t R of 3-(tetradecanoyloxy)tetradecanoic acid = 1.00). Isomers, such as 3-(tetradecanoyloxy)dodecanoic acid methyl ester [3-O(14:0)12:0] and 3-(dodecanoyloxy)
207 TABLE 3 NATURE AND RELATIVE RETENTION TIMES OF SYNTHETIC (S) OR BACTERIAL (B) 3ACYLOXYACYL RESIDUES (tR of 3-0 (14:0)- 14:0 = 1.00) 3-Acyloxyacyl methyl ester
Origin
Relative retention times OV-101 (230 °C)
SE-54 (230 °C)
3-O(12:0)-12:0 3-O(2-OCH3-12:0)- 12:0 3-O(2-OH-12:0)-12:0 3-O(3-OH-12:0)-12:0 3-O(12:0)-14:0 3-O(14:0)-14:0 3-O(A2-14:1)-14:0 3-O(2-OCH 3-14:0)-14:0 3-O(2-OH-14:0)-14:0 3-O(3-OH-14:0)-14:0 3-O(16:0)-14:0
S S S S B B S B B S B
0.33 0.41 0.43 ND b 0.58 1.00 1.19 1.25 1.28 1.55 1.77
0.31 ND 0.44 0.48 0.56 1.00 1.20 1.25 1.30 1.56 1.76
3-O(14:0)-14:0 a
S
1.19
1.17
a Ethyl ester, internal standard. b ND, not determined.
tetradecanoic acid methyl ester [3-O(12:0)14:0], exhibit identical polarity and, thus, identical retention times. Such isomers can, however, be unequivocally identified by their specific fragmentation pattern as obtained by MS (see Section 8.2.2).
8.2.2 MS As an example, the MS fragmentation pattern (EI mode) of 3-(dodecanoyloxy)tetradecanoic acid methyl ester shows characteristic fragments at m/z=440 (M+), 409 (M-31), 313 (M-127), 300 (M-140), 257 (M-183), 241/240 (M-199/200), 209/208 (241/240-32) and a prominent peak at 183 (M-257, CH3(CH2)10CO ). The latter fragment mainly derives from the dodecanoyl substituent. Thus, in the spectrum of 3-(tetradecanoyloxy)tetradecanoic acid methyl ester the corresponding peak appears at m/z=211. A number of mass spectra from 3-acyloxyacyl ester groups have been published [6, 7, 28, 75]. Structures, like 3-(2-methoxytetradecanoyl)oxytetradecanoic acid methyl ester, exhibit a similar fragmentation pattern as 2-methoxytetradecanoic acid methyl ester with a prominent peak at m/z =213 (M-59 [7]). The two compounds are, however, easily distinguished by their GLC retention times. 8.3 (R,S)-2- and 3-methoxy fatty acid-L-phenylethylamides [11] &3.1 GLC GLC analysis of (R)- or (S)-2- and 3-methoxy fatty acid-L-phenylethylamides is carried out on packed columns (see Section 6.2) using OV-I (3°/o on Gas Chrom Q, 100-120 mesh) with N2 (30 ml. min: ~) as carrier gas (glass column, 0.3 x 200 cm). Ta-
208 ble 4 gives relative retention times of various fatty acid-I.-phenylethylamides (t R o f tetradecanoic acid-L-phenylethylamide = 1.00) on OV-1 (3°70) at 205 °C. As is obvious from Table 4, the (R)-isomers o f 2- and 3-methoxy fatty acid-L-phenylethylamides elute from the column before the corresponding (S)-isomers.
8.3.2 M S EI-mass spectra o f racemic 3-methoxytetradecanoic acid-L-phenylethylamides ( M = 361) showed intensive fragments at m/z--120 (M-241) and 105 (M-241-15), both deriving from the phenylethylamide portion o f the molecule. In addition (inter alia), characteristic peaks were seen at m / z = 361 (M) and 206 (120+ 86). On MS o f racemic 2-methoxytetradecanoic acid-L-phenylethylamides (M = 361), a prominent fragment at m / z = 120 (M-241) and a peak at m / z = 105 (M-241-15) were seen. Furthermore, typical fragments were observed at m/z--361 (M), 213 (M-120-28) and 193 (120+72+1). TABLE 4 RELATIVE RETENTION TIMES OF L-PHENYLETHYLAMIDESOF STANDARD (METHOXY) FATTY ACIDS AS ANALYSED ON PACKED COLUMNS (tR OF TETRADECANOIC ACID LPHENYLETHYLAMIDE = 1.00, = 6 min) Fatty acid-Lphenylethylamide
Relative retention time (tR) OV-I (205°C)
(R)-3-OCH3-10:0 (S)-3-OCH3-10:0
0.32 0.34
(R)-3-OCH3-12:0 (S)-3-OCH3-12:0
0.63 0.67
(R)-3-OCH3-14:0 (S)-3-OCH3-14:0
1.23 1.32
(R)-3-OCH3-16:0 (S)-3-OCH3-16:0
2.41 2.61
(R)-3-OCH3-18:0 (S)-3-OCH3-18:0
4.70 5.10
(R)-3-OCH3-9-CH3-10:0 (S)-3-OCH3-9-CH3-10:0
0.41 0.43
(R)-3-OCH3-11-CH3-12:0 (S)-3-OCH3-11-CH3-12:0
0.81 0.85
(R)-2-OCH3-12:0 (S)-2-OCH3-12:0
0.51 0.59
(R)-2-OCH3-14:0 (S)-2-OCH3-14:0
1.01 1.15
(R)-2-OCH3-16:0 (S)-2-OCH3-16:0
1.98 2.25
10:0 12:0 14:0
0.25 0.50 1.00
209
Acknowledgements We thank K. Bryn, G. Rosenfelder and O. Liideritz for critically reading this manuscript, I. Bendt for excellent secretarial assistance and the Fonds der Chemie, Frankfurt, FRG, for financial support.
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I1 (1990) 195-211
195
Elsevier MIMET 00373
Analysis of lipopolysaccharide (lipid A) fatty acids Horst-Werner Wollenweber and Ernst T. Rietschel Forschungsinstitut Borstel, lnstitut fiir Experimentelle Biologic und Medizin, Borstel, FRG (Received 6 October 1989; revision received i0 March 1990; accepted 23 March 1990)
1 Introduction
Lipid A represents the common, covalently bound lipid component of bacterial lipopolysaccharides (LPS, endotoxin). Lipid A preparations derived from LPS of most bacterial groups possess a comparable chemical architecture. In general [for exceptions, compare 76, 77], lipid A consist of a Bl,6-1inked D-glucosamine disaccharide, which carries (in some cases, substituted) phosphate-groups in position 1 and 4' (lipid A backbone) and long-chain fatty acids in ester and amide bonds [1, 2]. The chemical structure of Escherichia coli and Salmonella lipid A has been elucidated in detail [1, 4, 7, 8]. E. coli-type lipid A (Fig. 1) has been chemically synthesized and bacterial and synthetic E. coli lipid A exhibited identical potent biological activity [3], proving the previous hypothesis that lipid A represents the endotoxic principle of LPS to be correct. More recently, lipid A partial structures have been synthesized the biological analysis of which points to fatty acids as being essential for the mediation of endotoxicity [4]. Thus, it was recognized that the quantity, nature, location and type of linkage of fatty acids determine the degree of biological activity of LPS or lipid A. To elucidate the significance of fatty acids, numerous studies were performed on the fatty acid composition of LPS and lipid A, derived from various genera of Gram-negative bacteria [for summary, see 1, 2, 4, 5]. Depending on the source of lipid A, various types of ester-bound fatty acids were identified. Thus, long-chain, even- or odd-numbered, iso- or anteiso-branched (S)-2-hydroxy and (R)-3-hydroxy fatty acids have been found. In some bacterial groups, the 3-hydroxyl group of ester-bound 3-hydroxy fatty acids is acylated by other fatty acids whereas in other species it is free. Cyclopropane and unsaturated fatty acids have only occasionally been detected in lipid A [compare 6]. In contrast to the broad spectrum of fatty acids involved in ester bonds, as a rule, only one type of fatty acid is amide-linked, viz., (R)-3-hydroxy fatty acids. These 3-hydroxy acids are even- or odd-numbered (iso-branched) and possess, in general, 10-22 C atoms. The 3-hydroxyl group of at least one N-acyl residue has been shown Correspondence to: E.T. Rietschel, Forschungsinstitut Borstel, Parkallee 1-42, 2061 Borstel, FRG.
0167-7012/90/$ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
196 OH
o
o\,_0" \ - \ / .o" \ N. -
0
~ -~\ ,_0 ® \ ~ % / "oH H
H
Fig. 1. Chemicalstructureof E. coli lipidA (4): Hydroxylgroup in position6' representsattachmentsite of polysaccharideportion in LPS. Numbers in circlesindicatenumber of C atoms in acyl chains.
to be usually substituted by other fatty acids [1, 2, 7, 86]. Some bacterial strains contain, besides 3-hydroxy fatty acids, also amide-linked 3-oxo fatty acids which contain a keto-group instead of a hydroxyl function at C atom 3 [8, 76]. Recently, a hydroxy fatty acid with 28 C atoms (27-hydroxyoctacosanoic acid) has been identified to be present in amide or ester linkage in certain lipid A preparations [77, 79]. As a rule, (R)-3-hydroxy fatty acids (ester- and amide-bound) predominate within the fatty acid moiety of lipid A. Since they are generally missing in other cell-wall lipids of Gram-negative bacteria [for exceptions, compare 80, 81], (R)-3-hydroxy fatty acids are peculiar to lipid A and represent characteristic lipid A markers [1, 2, 4, 5, 10-12].
2 Analysis of Fatty Acids For the quantitative and qualitative determination of fatty acids, a number of methods are available [summarized in 13]. These include for example titrimetric [14, 15], colorimetric [16-21], radiochemical [22] and chromatographic [23-25] procedures as well as negative-ion fast-atom bombardment-mass spectrometry [9]. Among these, gas-liquid chromatography (GLC), often combined with mass spectrometry (MS), has proven to be a most powerful tool for the identification and estimation of fatty acids. In the following, therefore, procedures will be described which allow the GLC/MS analysis of fatty acids derived from LPS. All procedures are based on the Salmonella minnesota Re mutant strain R595 (mRe). As the LPS of this deep rough mutant contains only Kdo (3-deoxy-D-manno-2-octulosonic acid [5]) and lipid A, quantities should be appropriately adjusted upwards for other lipopolysaccharides to give approximately the same fatty acid concentration. Quantitative analyses are carried out in the presence of two internal standards. Nonhydroxylated fatty acids are quantitated relative to heptadecanoic acid methyl ester whereas hydroxylated fatty acids are determined relative to an added hydroxy fatty acid ester (in the case of LPS from the mRe mutant, 3-hydroxydodecanoic acid may be used
197 [7]). The use of the second standard fatty acid is not obligatory if hydroxyl groups of hydroxylated fatty acids are derivatized prior to GLC analysis [see Section 7.2]. In the present paper, methods for the analysis of fatty acids liberated from LPS (or lipid A) are described. The application of soft-ionization techniques (like fast-atom bombardment and laser-desorption MS) to determine the location of acyl groups at the lipid A backbone are described in [26, 43]. 3 Liberation of Fatty Acids from LPS
3.1 Total fatty acids 3.1.1 Release of fatty acids in the free form 3.1.L1 General Release of free fatty acids from LPS can be achieved by either acidic or alkaline hydrolysis. Acid-catalyzed hydrolysis liberates both ester- and amide-bound fatty acids. Application of acid hydrolysis to structural studies of LPS, however, is limited by the fact that 3-hydroxy fatty acids are partially dehydrated and that (less volatile) artefacts, such as polymerized 3-hydroxy fatty acids (estolides) and other 3-acyloxyacyl residues, are formed [27, 28]. Strong alkaline hydrolysis (KOH, NaOH) as well as hydrazinolysis [29, yielding hydrazides] and hydroxylaminolysis [30, yielding hydroxamates] also liberate both ester- and amide-bound fatty acids. The methods suffer, however, from the fact that substantial amounts ct,~-unsaturated (with smaller amounts of ~,~-unsaturated) fatty acids may be formed [31] by an alkali-catalyzed ~-elimination of 3-acyloxyacyl residues present in LPS [7, 32, 33]. Furthermore, amide-bound ct,~unsaturated acids are not easily liberated by strong alkaline hydrolysis. Thus, for the liberation of fatty acids from LPS a two-step procedure has been proposed which consists of an initial acidic and a second alkaline hydrolysis step in water [28]. The alkaline hydrolysis splits artefacts, such as estolides and formed 3-acyloxyacyl residues (see above). A disadvantage of this method is the need of carbomethylation of free fatty acids prior to GLC/MS analysis (see Section 5.1). Therefore here, for the release of total fatty acids from LPS an acid hydrolysis step followed by an acid-catalyzed (trans)esterification reaction is recommended which converts liberated free fatty acids and artefacts produced by acid, i.e., estolides and 3-acyloxyacyl residues, directly to fatty acid methyl esters (see Section 3.1.2.2). 3.1.2 Release o f total fatty acids as methyl esters (acid-catalyzed transesterification) 3.L2.1 General Fatty acid methyl esters may be obtained directly from LPS by methanolysis in the presence of either Lewis (BCI3, BF3) or Br6nsted (HCI, H2SO4) acids. With BCI3, amide-bound fatty acids are only partially liberated. With BF3-methanol (14% by weight), complete release of ester- and amide-linked fatty acids from LPS is achieved after 6 h at 100°C [34-36]. 3-Methoxy fatty acids may be formed as artefacts during BF3-catalyzed methanolysis. Furthermore, nonpolar
198 WCOT glass capillary columns usually used for GLC of fatty acid esters are strongly activated by trace amounts of boric acid derivatives. Such columns are no longer useful for the GLC analysis of underivatized hydroxy fatty acid methyl esters. Acid (HCl)-catalyzed methanolysis alone does not liberate the amide-linked fatty acids quantitatively. This procedure is only recommended for the detection of amidelinked 3-oxo fatty acids which are lost at harsh aqueous acidic conditions, presumably via a decarboxylation reaction. The presence of 3-oxo fatty acids is, therefore, recognized by treatment of LPS with methanolic HC1 [38] omitting the initial HCI hydrolysis step. Treatment of LPS with HCI, followed by methanolic HC1, liberates O- and N-acyl residues quantitatively and artefact formation is minimal. No further esterification step is necessary and, therefore, this reaction is recommended for the liberation of ester- and amide-bound (total) fatty acids from LPS. 3.1.2.2 Procedure. The mRe LPS (2 mg) and standard heptadecanoic acid (200/~g) and 3-hydroxydodecanoic acid (600/~g) are heated with HCI (4 M, 1 ml) at 100 °C for 4 h in a Teflon-lined screw cap tube (5 ml size). After cooling, the sample is transferred with chloroform (--- 10 ml) to a separatory funnel and water (= 10 ml) is added. Fatty acids are extracted into chloroform (3 × 10 ml), the combined organic phases dried over anhydrous Na2SO4, concentrated (rotary evaporator) and transferred with chloroform to a glass tube (5 ml size) equipped with a tightly fitting Teflon-lined screw cap. To the N2-dried sample HC1 in methanol (-- 2 M a, 1 ml) is added and (trans)esterification allowed to take place for 6 h at 85 °C. When cool, the mixture is concentrated to about half its initial volume by a stream of N z (to partly remove HCI and methanol), an aqueous solution of half-saturated NaCI is added (1 vol) and the fatty acid methyl esters extracted by 2 x 3 vols. of distilled petroleum ether (40-60 °C)/ethylacetate (1:1, v/v). The combined organic phases are, at room temperature, carefully concentrated by a stream of N2. Concentration to complete dryness should be avoided because of the volatility of short-chain fatty acid methyl esters (e.g., dodecanoic acid methyl ester). Hydroxy fatty acid methyl esters present in the mixture may then be derivatized by trifluoroacetylation (see Section 7.2). Otherwise, the residue is redissolved in distilled chloroform (50#1) and subjected to GLC/MS analysis (see Section 8.1). 3.2 Ester-bound fatty acids 3.2.1 General Ester-bound fatty acids may be selectively liberated from LPS by mild hydrazinolysis [39], controlled hydroxylaminolysis[32, 40], treatment with methanolic NaOH [41] and NaOCH 3 [32, 42] or by 'on column' transesterification with m-(trifluoromethylphenyl)-trimetyl ammonium hydroxide [37]. Of these methods, transesterification with NaOCH 3 is preferred since it directly yields fatty acid methyl esters. If a 3-acyloxyacyl residue is present in LPS in ester linkage, NaOCH 3 will induce a ~elimination reaction resulting in the liberation of a free fatty acid (the substituent at a Generatedby carefullyaddingdrop-wiseacetylchloride(1.5 ml) to dry methanol(10 ml) at 0°C.
199
0 C=O
H3CO"c#O
i.2 HC-O-C i H2 R I CH3
H3CO" C~0
0c.,
0c.3 CH
ICH2 R CH3
. C I~ R
L Fig. 2.
H3CO'c~O
!H 2 CH3 CH2N2
!., HC --OCH3
cH~ t CH3
H3CO'~c~O P I R
Transesterification and/3-elimination reaction involving a 3-acyloxyacyl group present in lipid A in ester linkage (2).
the 3-hydroxyl group of the 3-hydroxy fatty acid) and in the formation of an c¢,~unsaturated fatty acid methyl ester with a chain length identical to the 3-hydroxy fatty acid from which it derives (Fig. 2). The liberated unsaturated fatty acid ester, containing a conjugated double-bond (between atoms 2 and 3), is subject to nucleophilic attack by H3CO- ions, resulting in the nearly quantitative formation of the corresponding 3-methoxy fatty acid methyl ester (Fig. 2). The nature of the eliminated substituent can be determined by GLC after additional carbomethylation of the alkaline methanolysate [32, 33]. It should be noted that the eliminated free fatty acids may also be detected (without further derivatization) on GLC analysis if the described glass capillary columns are used (see Section 8.1.1). 3.2.2 Procedure The mRe LPS is freed from water by keeping it under vacuum at 30°C for 12 h. Dry LPS (10 mg) is placed in a 10-ml dry glass tube equipped with a Teflon-lined screw cap and a small Teflon-coated magnetic bar. NaOCH3 in absolute methanola (= 0.25 N b, 5 ml), heptadecanoic acid methyl ester (1 mg) and 3-hydroxydodecanoic acid methyl ester (1 mg) are added and the suspension kept under magnetic stirring at 37 °C for = 15 h. After removal of the magnetic bar, the tube is centrifuged (10 min, 2000×g) and the supernatant decanted into a separatory funnel (I00 ml size). [The sediment (de-O-acylated LPS, LPS-OH) is washed with cold methanol (1 ml) and kept for the analysis of amide-bound fatty acids (see Section 3.3.2).1 The combined supernatants are acidified (pH 1-3) with HCI (0.25 N, = 10 ml), a solution of half-saturated NaCI (2 ml) is added and the mixture extracted with distilled chloroform (3 x 20 ml). The combined chloroform extracts are well-dried over anhydrous Na2SO 4, passed through a paper filter into a round-bottom flask and concentrated (rotary evaporator, water bath < 30 °C). The fatty acid methyl esters are dissolved in distilled diethyl ether (1 ml), transferred to a small vial with internal cone shape (reactivial with Mininert Valve; Pierce Chemical) and ether is removed by a gentle stream of N 2. The residue is redissolved in chloroform (200/zl) and subjected to a The use of absolute methanol is essential since 3-hydroxy fatty acid methyl esters are highly susceptible to alkaline saponification. b 575 mg Na (shortly washed with methanol and ether) dissolved in 100 ml of absolute methanol (hood!),
200 GLC/MS analysis (see Section 8.1). To elucidate the nature of/3-eliminated fatty acids, the methanolysate is additionally carbomethylated with diazomethane (see Section 5.2.2) and again analysed by GLC/MS. 3.3 A m i d e - b o u n d f a t t y acids 3.3.1 General
Treatment of LPS with methanolic NaOCH 3 yields a sediment which represents de-O-acylated LPS (LPS-OH) containing amide-bound fatty acids. These fatty acids can be liberated from LPS-OH by acid hydrolysis. 3-Acyloxyacyl residues present in LPS in amide linkage undergo a/3-elimination reaction caused by NaOCH 3 resulting in the partial (in Salmonella, -~25070) liberation of the free fatty acid previously linked to the 3-hydroxyl group of the amide-linked 3-hydroxy fatty acid. Concomitantly, the corresponding, still amide-bound, a,~unsaturated fatty acid is formed which, however, is not attacked by H3CO- ions (but compare Section 3.2.1). This amide-linked a,~unsaturated fatty acid is not quantitatively liberated by conventional hydrolysis procedures and for its estimation indirect methods, such as oxidation with MnO4-/IO 4- or reduction with deuterium, have been described [7]. Amide-bound 3-hydroxy fatty acids, still linked to the lipid A glucosamine residues, have also been directly [45, 82] or after controlled degradation of glucosamine [46] analysed by GLC/MS. 3.3.2 Procedure
The sediment (LPS-OH) obtained after treatment of LPS with methanolic NaOCH 3 (Section 3.2.2) is, is for purification, dissolved (in the original glass tube) by ultrasonication in distilled water (~-0.5 ml) and acidified with 2 N acetic acid (pH 2-4). To this solution, cold distilled ethanol ( ~ 8 ml) is added and, after shaking, LPS-OH is allowed to precipitate at 4 °C for 1 h. If precipitation does not occur, 0.1-0.5 ml petroleum ether (40-60°C) is added. The tube is centrifuged (2000xg, 3 min), the supernatant removed by suction, the precipitated LPS-OH washed three times with cold distilled acetone, redissolved in distilled water (2 ml, ultrasonication, warming) and lyophilized (yield ~ 5 mg). For liberation and analysis of amide-bound fatty acids, LPS-OH (2 mg) is treated in the same way as LPS (Section 3.1.2.2). 4 Liberation of 3-Acyioxyacyl Residues from LPS 4.1 General
Many LPS, including that of mRe bacteria, contain ester- and amide-bound 3-acyloxyacyl-residues [1, 2, 4, 7, 32, 33, 78]. The presence of an ester-bound 3-acyloxyacyl group is indicated by the formation of 3-methoxy fatty acid methyl ester on treatment of LPS with NaOCH 3 (see Section 3.2.2). Ester-bound 3-acyloxyacyl groups can also be liberated intact from LPS by short alkaline hydrolysis (0.25 N NaOH, 4 min, 56 °C [32]) and thus be analysed directly by GLC/MS (see Section 8.2). As shown in Fig. 3, amide-bound 3-acyloxyacyl-residues are liberated from LPS [2,
201 O
O
NH ~-0 CH2 O I I1 HC-O-C-R I ( CH2)n [ 3 CH
INI CH3J/Ag" ~
C-OCH 3 CH2 0 I II HC-O-C- R I (CH2)n ~H 3
0% rnRd2ooCaCid ~
/OCH3
CI CH2 0 I I HC- O-C-R I (CH2)/7 I 3 CH
C=0
NH3 +
Fig. 3. Steps involvedin selectiveliberationof 3-acyloxyacylgroup presentin lipid A in amidelinkage (2, 7).
7] in the form of their methyl esters after conversion of the amide bond (by methyliodide in the presence of Ag salts) to an acid-labile imidate (double bond between C and N). Because of the better solubility in organic solvents, this reaction is carried out with free lipid A which is obtained as a precipitate after mild-acid hydrolysis of LPS (1070 acetic acid, 1 h, 100°C; washing with 0.1 N HCI, 5 ml [5]). Due to trace amounts of water present, also ester-bound 3-acyloxyacyl groups are liberated to a small extent. To distinguish between ester- and amide-bound 3-acyloxyacyl residues, therefore, the procedure is carried out once in the presence and once in the absence of the methylating reagent (methyliodide).
4.2 Procedure [7] Two samples, mixtures of free lipid A (2 mg), Ag20 (7 mg [prepared according to 44]) and silver-trifluoromethanesulfonate (15 mg; Merck, Darmstadt, FRG) are kept for 12 h in an evaporated desiccator over P205 in the dark. To each sample, water-free petroleum ether (2 ml, 40-60 °C) is added, and the vials, closed with Teflon-lined screw caps, are flushed with a gentle stream of dry N 2 by syringes and sonicated (15 min, 20 °C, dark). Distilled CH3I (10/zl) is added to one sample and methylation allowed to take place under sonication (2 h, 30-40 °C, dark). From the other sample, CHaI is omitted but otherwise it is subjected to the same procedure as the methylated sample. The reaction mixture is transferred to a centrifuge tube containing an aqueous solution of NaES203 (0.5 M, 2 ml). 3-(Tetradecanoyloxy)tetradecanoic acid ethyl ester (prepared according to [47] and esterified with diazoethane, see Section 5.2.1) is added as internal standard, the reaction mixture acidified with HaPO 4 (10070) to pH ~ 2 and shaken (15 min, 20 °C), or, more efficiently, ultrasonicated (30 min, 25-30 °C). After centrifugation (2000 x g, 10 min), the reaction mixture is extracted with petroleum ether/ethylacetate (1:1, v/v, 3 x 2 ml) and the respective combined organic phases are washed with distilled water (2 x 5 ml), dried over Na2SO 4, concentrated to ~ 1 ml and purified by application to a small column (20× 3 mm) packed with silica gel 60 (100-240 mesh) which is eluted with petroleum ether/ethylacetate (1:1, v/v, 10 ml). The eluate is concentrated, carbomethylated with diazomethane (see Section 5.2.2) and analysed by GLC/MS (see Section 8.2).
202 5 Carbomethylation of Free Fatty Acids (Methyl Ester Formation) 5.1 General Fatty acids are usually analysed by GLC in the form of their volatile methyl esters and several convenient methods for methyl ester formation are available. These methods include reaction of fatty acids with methanol in the presence of an acidic catalyst (HCI, H2SO4, BC13, BF3), treatment with N,N'-dicyclohexyl carbodiimide and methanol [48] or diazomethane [49, 50, compare also 51]. Detailed procedures for esterification of fatty acids with HCl-methanol [52, 53] and BF3-methanol [32, 34, 54-56] have been described. Here, the preparation of fatty acid and 3-acyloxyacylmethyl esters by diazomethane will be discussed. This procedure leads to rapid and complete esterification. It has been noted, however, that ct,~-unsaturated fatty acids will react with diazomethane to form pyrazoline derivatives [57] which are not detected under the conditions used for GIAZ/MS. Therefore, if ot,~-unsaturated fatty acids are to be expected, the LPS hydrolysate should (for comparison) also be esterified by other methods (e.g., MeOH/HCI). 3-Hydroxy fatty acids are partially converted by diazomethane to the 3-methoxy fatty acid derivative. This artefact is, however, formed to only a small extent (--1%). Larger amounts of diazomethane are prepared from p-toluenesulfonyl-N,Nmethylnitrosamid (Diazald; Aldrich, Milwaukee, Wisconsin) by distillation with diethyl ether in glassware with clear-seal joints (Diazald Kit; Aldrich) [compare 58] to prevent contamination and explosion. (The distillation should be performed under a hood and behind a safety shield.) The etheral solution can be stored at -30°C for some weeks. Smaller amounts of diazomethane are easily prepared with the help of a diazomethane generator a or by extraction into diethyl ether from N,N-nitrosomethyl urea b which should, however, be handled with caution. Diazoethane is prepared in a similar way (using N,N-nitrosoethyl urea; Serva, Heidelberg, FRG). 5.2 Procedures 5.2.1 Preparation of diazomethane (or diazoethane) in ether b Diethylether (10 ml) and 40% KOH (2 ml) in a separatory funnel (50 ml size) are cooled to 0 °C (ice bath). N,N-nitrosomethyl urea (or N,N-nitrosoethyl urea, 1 g; Serva) is gradually added under gentle stirring. After 5 min, the alkaline water phase is removed, the yellow ether phase transferred to a precooled Erlenmeyer flask (50 ml size) and kept for 30 min over KOH pellets at 0 °C. This diazomethane solution can, in general, be used without further purification, 5.2.2 Carbomethylation (methyl ester formation) To the solution of free fatty acids (1 mg) in ether (1 ml), first distilled methanol a Wheaton, 1000 N Tenth Street, MillviUe, New Jersey 08332. b N,N-nitrosomethyl urea is a carcinogen. Gloves should be used and the procedure should be performed under a hood.
203 (0.1 ml, Whirlmix) is added as a catalyst at room temperature. Diazomethane in ether (Section 5.2.1) is then added in portions (0.1 ml) until the yellow colour persists. After 10 min, solvents and excess diazomethane are removed (under a hood) by a gentle stream of N 2. The fatty acid methyl esters are redissolved in distilled chloroform and subjected to GLC analysis.
6. Stereochemicai Analysis of Hydroxy Fatty Acids 6.1 General The stereochemistry of 2- and 3-hydroxy fatty acids present in LPS has been studied by optical rotation measurements [5, 11, 32, 35]. This method, however, requires large amounts of material and the possible presence of both optical antipodes makes optical rotation measurements difficult. Therefore, based on results of Karlsson and Pascher [59], a GLC procedure was developed for the analysis of diastereomeric derivatives of bacterial hydroxy fatty acids [11, 60]. By this method, both 2- and 3-hydroxy fatty acids are separated according to chain length and configuration. 6.2 Procedure The mRe LPS (10 mg) is hydrolyzed in a sealed tube with strong alkali (4 N KOH, 5 h, 100°C). The liberated (hydroxy) fatty acids are, after acidification (pH ~ 1) extracted with distilled chloroform (see Section 3.2.2), after drying (NaESO4) and concentration transferred with water-free diethyi ether (0.5 ml) to a dry a glass tube (10 ml size, with a glass stopper), kept at --- -15 °C (ice/NaCl) and mixed with cool (-15 °C) BF3-ethyl etherate (10-20 ~l; Fluka, Buchs). To this solution, precooled diazomethane in ether (see Section 5.2.1) is gradually added (1-ml portions) until the yellow colour persists for = 30 s [61, 62] (~-8 ml diazomethane solution). After 10 rain, the reaction mixture is filtered through paper into a separatory funnel and washed once with saturated NaHCO 3 and twice with distilled water. The ether phase, after drying over NaESO4, is filtered into a round-bottom flask (25-ml size), partially evaporated (rotary evaporator), transferred to a 15-ml glass tube and taken to dryness with a stream of N2b. KOH (1 N, 1 ml) is added and the methoxy fatty acid methyl esters saponified at 70 °C for 1-2 h. The free methoxy fatty acids are extracted with chloroform and applied to chloroform-washed silicic acid thin-layer plates (Kieselgel H; Merck). Chromatography is performed with petroleum ether-diethyl ether-acetic acid (35 : 15 : 1, v/v/v) and fatty acids are visualized by iodine vapour. The area corresponding to methoxy fatty acids (Rf=0.5) is eluted from the plate with distilled chloroform, the extract transferred to a serum vial (5 ml size) and taken to dryness by N 2. The vial is closed with a Teflon-lined Bordelcap (Machery and Nagel) and flushed by two cannulae with N 2. The methoxy fatty acids are dissolved in dry chloroform (1 ml, passed into the vial through a cannula) and, after addition of thionyl chloride (0.25 ml), the vial is kept at 60 °C for 30 min. Chloroform and excess SOCl 2 are flushed from the vial with N2, the methoxy fatty acid chlorides dissolved in dry chlo-
a The useof water-freesolventsand dry glasswareis essentialfor obtaininggoodyields(> 90%) of methoxy fatty acid derivatives. b Since trance amounts of boric acid derivatives activate glass-capillary columns, the formation of 3-methoxy fatty acid methylesters should be checkedby using packed (and not capillary)columns.
204 roform (1 ml) and L-phenylethylamine (0.25 ml, Merck) is added and the mixture kept at room temperature for 30 min. The vial is opened, the chloroform phase washed (separatory funnel) once with HCI (0.01 N) and twice with distilled water, dried over Na2SO4, filtered and taken to dryness (rotary evaporator). The methoxy fatty acid-zphenylethylamides are redissolved in distilled chloroform (50 #l) and analysed by GLC/MS (see Section 8.3). 7 Other Modifications of Hydroxy Fatty Acids 7.1 General
A series of modifications of the hydroxyl group of 2- and 3-hydroxy fatty acids of LPS have been described. These modifications may be useful for the identification of hydroxy fatty acids by GLC, especially if MS is not available. The hydroxyl group of both 2- and 3-hydroxy fatty acid methyl esters may be acetylated [11, 28], trifluoroacetylated [38, 63, 64, 83, 84, see below], trimethylsilylated [32, 65], tbutyldimethylsilylated [2, 66] and O-methylated with BF3-etherate/diazomethane (see Section 6.2) or with CH3I/Ag20 [59]. Reactions specific for 3-hydroxy fatty acids include reduction of the methyl ester and conversion into acetonides [67], dehydration of the free 3-hydroxy fatty acid by either treatment with acetic acid anhydride [60], PBr3 and potassium t-butylate [68, 69] or concentrated H2SO4 [16]. The resulting ot,flunsaturated fatty acids can be converted to the 3-picolinyl ester [85] or to the corresponding saturated fatty acids either by hydrogenation with Pd on charcoal (10%) as a catalyst [7, 32] or by nascent H2 [69]. Furthermore, they may be oxidized with KMnO4 to a saturated fatty acid, possessing two C atoms less than the original a,flunsaturated and 3-hydroxy fatty acid, respectively [7, 32]. For the quantitative estimation of hydroxylated fatty acids (notably, 3-hydroxy fatty acids), the use of a hydroxylated internal standard is essential (compare Sections 2 and 3.1.2.2). The addition of a hydroxylated standard is, however, not obligatory if the hydroxyl groups of the hydroxy fatty acids to be analysed are protected. As protecting groups, trifluoroacetyl residues have been found to be useful. The derivatization procedure leads to rapid and complete acylation and the derivatives obtained are stable and exhibit excellent chromatographic behaviour. Therefore, for the derivatization of hydroxyl groups present in hydroxy fatty acid methyl esters, trifluoroacetylation is recommended (GLC profiles and retention times of trifluoroacetylated 3-hydroxy fatty acid methyl esters are given in [83, 84]). 7.2 Procedure [38]
To a mixture of fatty acid methyl esters containing hydroxy fatty acids (in a small glass tube, 5 ml size, with a Teflon-lined screw cap) trifluoroacetic anhydride in acetonitrile (50/zl, 1 : 1, v/v) is added. The solution is heated (boiling) for 2 min and after 10 min at room temperature diluted with acetonitrile (100/A). The mixture is (without further manipulations) directly analysed by GLC.
205
8 GLC/MS Analysis of Fatty Acid Derivatives 8.1 Fatty acid methyl esters 8.L1 GLC The identity of long-chain fatty acids a may be tentatively determined on the basis of relative retention times obtained with different liquid phases and comparison with authentic reference substances. In Table 1, the relative retention times (tR of tetradecanoic acid methyl ester = 1.00) derived from S. minnesota Re LPS as obtained. on three liquid phases (packed columns) with different polarities are given. The stationary phases used (operating temperature given in the table) were: (i) SE-30 (10%0 on Gas Chrom Q, 100-120 mesh), (ii) Castorwax (2.5%o on Chromosorb W, 80-100 mesh) and (iii) EGSS-X (15% on Gas Chrom P, 100-120 mesh). In all cases, glass columns (0.3 × 200 cm) were used with a N2 flow of 30 ml. min -1. In general, for qualitative and quantitative GLC/MS analyses of fatty acid methyl esters capillary columns (glass and fused silica) are preferable (for exceptions, see Section 6.2) and in Table 2 relative retention times of bacterial fatty acids as obtained on two types of nonpolar phases (OV-101; Chrompack, Berlin, FRG; SE-54; Weeke, Duisburg, FRG) are listed. Glass capillary columns (WCOT 25 m, i.d. 0.25 mm, split-mode injection type, split ratio 1:40) were used, connected to a gas chromatograph (Varian 3700) equipped with a flame-ionization detector and combined with an automatic integrator (Hewlett-Packard 3380A). H 2 ( ~- 2 ml. rain -1) served as carrier gas. To obtain
TABLE 1 NATURE AND RELATIVE RETENTION TIMES OF FATTY ACIDS PRESENT IN HYDROLYSATES OF S. MINNESOTA Re LPS AS ANALYSED ON PACKED COLUMNS (tR of 14:0 = 1.00) Fatty acid methyl ester
Relative retention times (tR) SE-30 (10%) (170°C)
Castorwax (3%) (175 °C)
EGSS-X (15a/0) (140°C)
12:0 14:0 16:0 2-OH-14:0 3-OH-14:0
0.46 1.00 2.23 1.58 1.74
0.40 1.00 2.45 2.42 3.16
0.51 1.00 1.98 5.00 7.90
A3-14:la A2-14:1a 3-OCH3-14:0 a
1.00 1.20 1.52
1.00 1.46 1.82
1.36 1.70 2.42
17:0b 3-OH-12:0 b
3.33 0.80
3.81 1.26
2.80 4.00
a Artefact formed from 3-OH-14:0. b Internal standard.
a For GLC analysis of acetyl groups in LPS, compare [70].
206 TABLE 2 N A T U R E A N D R E L A T I V E R E T E N T I O N TIMES OF F A T T Y ACIDS P R E S E N T IN H Y D R O L Y SATES OF S. M I N N E S O T A Re LPS AS A N A L Y S E D ON GLASS C A P I L L A R Y C O L U M N S (IR of 14:0 = 1.00) Fatty acid methyl ester
Relative retention times (tR) OV-101 (150°C)
SE-54 (140°C)
0.39 1.00 2.50 1.66 1.89
0.36 1.00 2.70 1.76 2.04
A3-14:1 a A2-14:1 a 3-OCH3-14:0 a
0.96 1.22 1.70
0.95 1.27 1.82
17:0 b 3-OH-12:0 b
3.95 0.63
4.39 0.62
12:0 14:0 16:0 2-OH-14:0 3-OH-14:0
a Artefact formed from 3-OH-14:0. b Internal standards.
reproducible results, it is important that the proper analysis conditions (such as amount of sample and volume, geometry of injection port, injection technique [71]) are determined with the aid of a standard mixture of representative fatty acid methyl esters. 8.1.2 MS Electron-impact (EI) MS of synthetic and bacterial 2-hydroxy, 3-hydroxy, 3-oxo and nonhydroxylated fatty acids has been described in detail [7, 8, 11, 28, 35, 36, 45, 46, 63-65, 69, 72-74]. The EI fragmentation pattern of 2-hydroxy fatty acid methyl esters is characterized by peaks at m/z=M, M-32, M-59 and 90 (CH2OHCOOCH3). The base peak is found at m/z= 83. Fragments characteristic for 3-hydroxy fatty acid methyl esters include m/z=M-18, M-50, 74 and 103 (CHOHCH2COOCH3), with the base peak at m/z=43. Spectra of 3-methoxy fatty acid methyl esters exhibit characteristic fragments at m/z--M-15, M-30 and 117 (CHOCH3CH2COOCH 3, base peak at m/z = 75). 3-Oxo fatty acid methyl esters are characterized by peaks at m/z--M and M-73, the base peak being at m / z = l l 6 (CH2COHCHECOOCH3 [74]). 8.2 3-Acyloxyacyl methyl esters 8.2.1 GLC 3-Acyloxyacyl methyl esters, the preparation of which has been described [7, 28, 47], are analysed on capillary columns and Table 3 summarizes relative retention times (t R of 3-(tetradecanoyloxy)tetradecanoic acid = 1.00). Isomers, such as 3-(tetradecanoyloxy)dodecanoic acid methyl ester [3-O(14:0)12:0] and 3-(dodecanoyloxy)
207 TABLE 3 NATURE AND RELATIVE RETENTION TIMES OF SYNTHETIC (S) OR BACTERIAL (B) 3ACYLOXYACYL RESIDUES (tR of 3-0 (14:0)- 14:0 = 1.00) 3-Acyloxyacyl methyl ester
Origin
Relative retention times OV-101 (230 °C)
SE-54 (230 °C)
3-O(12:0)-12:0 3-O(2-OCH3-12:0)- 12:0 3-O(2-OH-12:0)-12:0 3-O(3-OH-12:0)-12:0 3-O(12:0)-14:0 3-O(14:0)-14:0 3-O(A2-14:1)-14:0 3-O(2-OCH 3-14:0)-14:0 3-O(2-OH-14:0)-14:0 3-O(3-OH-14:0)-14:0 3-O(16:0)-14:0
S S S S B B S B B S B
0.33 0.41 0.43 ND b 0.58 1.00 1.19 1.25 1.28 1.55 1.77
0.31 ND 0.44 0.48 0.56 1.00 1.20 1.25 1.30 1.56 1.76
3-O(14:0)-14:0 a
S
1.19
1.17
a Ethyl ester, internal standard. b ND, not determined.
tetradecanoic acid methyl ester [3-O(12:0)14:0], exhibit identical polarity and, thus, identical retention times. Such isomers can, however, be unequivocally identified by their specific fragmentation pattern as obtained by MS (see Section 8.2.2).
8.2.2 MS As an example, the MS fragmentation pattern (EI mode) of 3-(dodecanoyloxy)tetradecanoic acid methyl ester shows characteristic fragments at m/z=440 (M+), 409 (M-31), 313 (M-127), 300 (M-140), 257 (M-183), 241/240 (M-199/200), 209/208 (241/240-32) and a prominent peak at 183 (M-257, CH3(CH2)10CO ). The latter fragment mainly derives from the dodecanoyl substituent. Thus, in the spectrum of 3-(tetradecanoyloxy)tetradecanoic acid methyl ester the corresponding peak appears at m/z=211. A number of mass spectra from 3-acyloxyacyl ester groups have been published [6, 7, 28, 75]. Structures, like 3-(2-methoxytetradecanoyl)oxytetradecanoic acid methyl ester, exhibit a similar fragmentation pattern as 2-methoxytetradecanoic acid methyl ester with a prominent peak at m/z =213 (M-59 [7]). The two compounds are, however, easily distinguished by their GLC retention times. 8.3 (R,S)-2- and 3-methoxy fatty acid-L-phenylethylamides [11] &3.1 GLC GLC analysis of (R)- or (S)-2- and 3-methoxy fatty acid-L-phenylethylamides is carried out on packed columns (see Section 6.2) using OV-I (3°/o on Gas Chrom Q, 100-120 mesh) with N2 (30 ml. min: ~) as carrier gas (glass column, 0.3 x 200 cm). Ta-
208 ble 4 gives relative retention times of various fatty acid-I.-phenylethylamides (t R o f tetradecanoic acid-L-phenylethylamide = 1.00) on OV-1 (3°70) at 205 °C. As is obvious from Table 4, the (R)-isomers o f 2- and 3-methoxy fatty acid-L-phenylethylamides elute from the column before the corresponding (S)-isomers.
8.3.2 M S EI-mass spectra o f racemic 3-methoxytetradecanoic acid-L-phenylethylamides ( M = 361) showed intensive fragments at m/z--120 (M-241) and 105 (M-241-15), both deriving from the phenylethylamide portion o f the molecule. In addition (inter alia), characteristic peaks were seen at m / z = 361 (M) and 206 (120+ 86). On MS o f racemic 2-methoxytetradecanoic acid-L-phenylethylamides (M = 361), a prominent fragment at m / z = 120 (M-241) and a peak at m / z = 105 (M-241-15) were seen. Furthermore, typical fragments were observed at m/z--361 (M), 213 (M-120-28) and 193 (120+72+1). TABLE 4 RELATIVE RETENTION TIMES OF L-PHENYLETHYLAMIDESOF STANDARD (METHOXY) FATTY ACIDS AS ANALYSED ON PACKED COLUMNS (tR OF TETRADECANOIC ACID LPHENYLETHYLAMIDE = 1.00, = 6 min) Fatty acid-Lphenylethylamide
Relative retention time (tR) OV-I (205°C)
(R)-3-OCH3-10:0 (S)-3-OCH3-10:0
0.32 0.34
(R)-3-OCH3-12:0 (S)-3-OCH3-12:0
0.63 0.67
(R)-3-OCH3-14:0 (S)-3-OCH3-14:0
1.23 1.32
(R)-3-OCH3-16:0 (S)-3-OCH3-16:0
2.41 2.61
(R)-3-OCH3-18:0 (S)-3-OCH3-18:0
4.70 5.10
(R)-3-OCH3-9-CH3-10:0 (S)-3-OCH3-9-CH3-10:0
0.41 0.43
(R)-3-OCH3-11-CH3-12:0 (S)-3-OCH3-11-CH3-12:0
0.81 0.85
(R)-2-OCH3-12:0 (S)-2-OCH3-12:0
0.51 0.59
(R)-2-OCH3-14:0 (S)-2-OCH3-14:0
1.01 1.15
(R)-2-OCH3-16:0 (S)-2-OCH3-16:0
1.98 2.25
10:0 12:0 14:0
0.25 0.50 1.00
209
Acknowledgements We thank K. Bryn, G. Rosenfelder and O. Liideritz for critically reading this manuscript, I. Bendt for excellent secretarial assistance and the Fonds der Chemie, Frankfurt, FRG, for financial support.
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