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Structure determination of mycolic acids by using charge remote fragmentation
Chemistry and Physics of Lipids, 51 (1989) 31--38 Elsevier Scientific Publishers Ireland Ltd.
31
Structure determination of mycolic acids by using charge remote fragmentation Arlette Savagnac, H616ne Aurelle, Christiane Casas, Francois Couderc, Pierre Gavard, Danielle Prom6 and Jean-Claude Prom6 Centre de Recherche de Biochimie et G~n&tiqueCeilulaires C.N.R.S., 118 Route de Narbonne 31062 Toulouse Cedex (France)
(Received December 5th, 1988; revised and accepted February 2rid, 1989) The collision-induced remote site fragmentation process of closed-shell ions, such as carboxylate anions, is a very potent analytical tool for the structural determination of fatty acids. This leads to an easy location of branch points, double bonds, cyclopropane rings and other functional groups. Although corynomycolicacid mixtures from Corynebacterium diphtheriae can be directly analyzed by negative-ion fast atom bombardment combined with collisionally activated decomposition spectra, mycolic acid mixtures from mycobacteria need a preliminary chemical cleavage. They are oxidized to /~-keto esters and then submitted to a retro-Clalsen reaction. The resulting fatty acids were then converted into pentafluorobenzyl derivatives and introduced directly into a high pressure ion source working in the negative ion mode. The resulting gas phase carboxylate anions are activated to decompose by collision with helium atoms. When applied to M3-mycolicacids from Mycobacterium failax, this method allows for the characterization of a new tri-unsaturated mycolicacid, which has the middle and the remote double bonds separated by two methylene groups. Keywords: mycolicacids; mycobaeteria; corynebaeteria; mass spectrometry; negative ions; remote site fragmentation.
Introduction Mycolic acids (very long a-alkyl-/I-hydroxy fatty acids) are the most characteristic components in the cell wall lipids o f "acid fast" bacteria such as Mycobacteria and related taxa (Nocardia, Corynebacteria, Rhodococcus) [1-2]. Despite numerous efforts, their structures remain only partially determined. This is due to the difficulty in isolating a pure c o m p o u n d from the complex mixture o f isomers and homologues. Recently, Gross and coworkers have proposed a new mass spectrometric method for the structural determination o f fatty acids, [3--5]. This mass spectrometry process is called "charge remote fragmentation", and it occurs when the internal energy o f "closed-shell" ions (such as carboxylate anions) is raised up by collision with Correspondence to: J.-C. Prom6.
neutral atoms [6--7]. The electric charges of such gas-phase ions remain localized during the fragmentation process, thus avoiding isomerisation of an aliphatic chain before any carboncarbon cleavage takes place. In the original procedure, gas-phase ions are produced by fast atom bombardment. We recently described an improvement to this technique that provides a powerful analytical tool for the study o f complex mixtures [8--10]. In this method gas-phase carboxylate anions are produced by dissociative electron capture o f pentafluorobenzyl esters of fatty acids (PFB derivatives). This modification allows GC separation before ionization and increases the ionization yield to a large extent to provide sensitive and fast determinations. The aim o f this paper is to describe a strategy for structural determination o f mycolic acids, using the charge remote fragmentation process. Two kinds o f mycolic acids will be studied: corynomycolic acids, from C o r y n e b a c t e r i u m
0009-3084/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
32
diphtheriae [11], which are short-chain mycotic acids, and M 3 mycotic acids from Mycobacterium failax, which possess a very long aliphatic chain with three double bonds [12]. In the latter study, the authors were unable to locate precisely the intermediary double bond in all isomers by chemical degradations, because of the complexity of the mycotic acid mixture. Experimental Corynomycolic acids and mycolic acids were extracted and purified from C. diphtheriae and M. fallax as described [11,12]. The mass spectrometer was a reverse geometry ZAB-HS instrument (VG-analytical, Manchester). A high pressure ion source filled with methane was used for electron capture negative ionization. The electron energy was 50 eV. Alternatively, negative FAB-ionization in a triethanolamine matrix was used to produce carboxylate anions from free fatty acids. Helium was introduced into the collision cell located in the second field free area in such a way that a signal arising from a carboxyiate anion was reduced to approximately 30% of its original value. CAD/MIKE spectra were collected by the data system (VG-2035). Scan speed was 800 eV/s.
Oxidation o f mycolic acid methyl esters Crude a-mycotic acids from M. fallax (400 mg) were dissolved in 5 ml anhydrous methylene chloride and added under stirring to a mixture of 400 mg pyridinium chlorochromate, 150 mg calcium carbonate and 10 ml methylene chloride. After 2 h at 40°C, the mixture was extracted with diethyiether, washed and the corresponding /1-ketoesters were purified by siticic acid column chromatography. The yield was 90%. Infrared carbonyl stretching frequencies at 1700 and 1740 cm -1 were observed.
Retro-Claisen reaction assisted by a-trehalose The O.keto~sters (350 mg) were added to a mixture of 180 mg anhydrous potassium carbonate, 600 mg anhydrous a-trehalose in 30 ml
dimethylformamide. The mixture was stirred at 80°C for 6 h under vacuum (125 Torr). After cooling, the mixture was acidified and extracted with chloroform. The mixture of a-trehalose esters was hydrolyzed with 5%0 KOH in methanol/water 1:1 (60°C, 2 h). After acidification and extraction with diethyl ether, the fatty acids were methylated with ethereal diazomethane. Long chain ketones resulting from the degradation of unreacted/~-ketoesters (40%0) were separated from the fatty acid methyl esters (60%) by silicic acid chromatography.
Fractionation o f fatty acid methyl esters by silver nitrate chromatography Chromatography on silver nitrate impregnated silicic acid column allows the separation of fatty acid methyl ester mixtures according both to the number of double bonds and their steric environment. The fatty acid methyl esters resulting from the retro-Claisen reaction (130 rag) were fractionated on 30 g of freshly prepared silver nitrate impregnated siticic acid. Successive elutions with increasing amounts of diethylether in hexane gave four main fractions. The first fraction consisted of a mixture of straight chain C-22 and C-24 fatty acid esters, as identified by GC/MS. The two subsequent fractions contained di-unsaturated long-chain fatty acids as identified by mass spectrometry (direct inlet, electron impact). The fourth fraction contained tri-unsaturated fatty acid methyl esters (direct inlet) corresponding to what was expected from the main chain of the M3-mycotic acids, namely m/z 824 (C56), 810 (C55), 796 (C54), 782 (C53) and 768 (C52) in the approximate ratio 2 : 1 : 7 : 1 : 6 (the carbon number of each fatty acid is indicated between brackets).
Synthesis o f PFB derivatives Less than 1 mg of free fatty acids was mixed with pentafluorobenzyibromide (2 ~l), diisopropylethylamine (2 /A), acetonitrile (50 ~l) and methanol (10 ~). The mixture was stirred at room temperature for 1 h. An aliquot (1 /A) was then directly introduced into the crucible of the direct inlet probe of the mass spectrometer.
33 . ,,.,
255
/
253 \
!227
283 281 [[ ,t[J..t I1' .,U .,11" ~6..,u..,,t ,I,.
"-
00-'"
"
" " "s.
: "
"
-'"
-"
"-
"'"200"'--'1
--
•
.
-
• . . . . . . .
.300"
". . . .
m~z#
" . . . .
.
"
"
-'-4oo"
- o. . . . . . .
"-
;'"
"
.
. ' ~. 6 o ".
.
.
.
.
.
0'
Fig. 1. FAB mass spectrum o f corynomycolic acids f r o m C. diphtheriae. The matrix was triethanolamine.
Results and discussion
Corynomycolic acids from C. diphtheriae Corynomycolic acids are the simplest form of mycolic acids. Their carbon number is relatively low (from 26 to 36). The most abundant corynomycolic acid from Corynebacterium diphtheriae was identified many years ago [13] as the C-32 compound, 1, formed biosynthetically by condensation between 2 tool of palmitic acid [14] but several other specie were also detected [2]. In particular, both the main chain R 1 and the branched chain R 2 may contain one double bond. Negative FAB-MS of the corynomycolic acids from C. diphtheriae shows different ions due to the presence of several sets of homologues containing zero to two double bonds (Fig. 1). In the upper mass range, the main deprotonated molecular ions are seen at m/z 495, 493, 491 corresponding to C-32 specie with zero, one and two double bonds, respectively. Other molecular
specie are observed at m/z 467 and 465 (C-30 specie with zero and one double bond). Less abundant ions are detected for the minor homologues, namely C-34 specie with zero to two double bonds and C-28 specie with zero or one double bond. In the medium mass range, fragments ions are observed mainly at m/z 255 (deprotonated hexadecanoic acid) and m/z 253 (deprotonated hexadecenoic acid). Less abundant ions correspond to deprotonated octadecanoic, octadecenoic and tetradecanoic acids (m/z 283, 281 and 227, respectively). The CAD-MIKE spectrum of each deprotonated molecular ion shows characteristic and intense fragmentations due to the cleavage of C2-C3 bond. A likely mechanism is depicted in Scheme 1. It produces the enolic form of a deprotonated fatty acid (ion a) and a fatty aldehyde as a neutral compound via a retroClaisen-like process. The mass of ion a indicates the number of both carbon atoms and-unsaturations of the side chain R2. Complementary information concerning the main chain R1 are
34
2 CH
/
/'-J\ 11, R 1-CH
C=O
-II,
is an indication that the following isomers are present:
0(-)
R1-CHO + R2-OH=C\
o®
CIIH23-CH(OH)-CH-(CI4H29)-COOH OH
C13H2~--CH(OH)--CH--(CI2H25)--COOH
H
C15H31--CH(OH)--CH--(C,oHu)--COOH
ion a
In addition to this abundant fragmentation, the CAD/MIKE spectra of the (M-H)- ions from corynomycolic acids exhibit a large number of weaker peaks which can be attributed to charge remote fragmentation processes. Figure 3a shows the cleavage pattern of the saturated C-32 specie 1. This representation is magnified by ten times to visualize weak signals. The very abundant ion of at m/z 255 is due to ion a. Subsequent loss of water gives the ion of rnTz 237. The charge remote fragmentation process gives regularly spaced signals 14 ainu apart corresponding to the formal expulsion of alkanes of increasing length (alkenes plus one hydrogen molecule). The C3-C4 bond cleavage (near the hydroxyl group) gives the more abundant ion of at m/z 283. These charge-remote fragmentations are useful for characterizing additional features on the aliphatic chains. In the spectrum of the ion of m/z 491, which corresponds to di-unsaturated C-32 corynomycolic acids (Fig. 3b), the mass of ion a (m/z 253) indicates that both chains R~
R1-CH(OH)-CH(R2)-COOH i
R 1 = n-C15 H31
R2 = n-C14 H29
2
R 1 = CH3-(CH2)5-CH=CH-(CH2) 7
R2=CH3"(CH2)5CH=CH'(CH2) 6
3
R1 = CH3-(CH2)5-CH=CH-(CH2) 7
R2=n'C13 H17
Scheme 1.
obtained from the mass difference between ion a and the molecular specie. Such fragment ion] are accompanied by weaker ones due to the consecutive loss of water. Thus, whereas all the C32 homologues fragment into two C-16 moieties, others give a more complicated pattern. For example, the ion of m/z 439, corresponding to the minor C-28 saturated corynomycolic acids, cleaves into deprotonated hexadecanoic, tetradecanoic and dodecanoic acids (to give ions of m/z 255, 227, 199, respectively) (Fig. 2). This
.
.
.
.
.
.
~
.
.
.
.
Fig. 2. CAD-MIKE spectrum of the deprotonated C-28 corynomycolic acid at m / z 439.
.
.
.
.
.
460
.
.
.
.
.
35
a
b
419 9.35
C
I
mlz Fig. 3. CAD-MIKE spectra of deprotonated C-32 corynomycolic acids: (a) saturated specie at m/z 495; Co) di-unsaturated specie at m/z 491; (c) mono-unsaturated specie at m/z 493. The intensity of all signals was magnified ten times.
and R 2 contain an unsaturation. The chargeremote fragmentation signals exhibit a characteristic pattern for a double bond: three weak signals surrounded by two stronger ones at m / z 419 and 363 due to the slower cleavage of both
vinylic and double bonds compared to allylic fragmentations. This indicates that the double bonds are situated between the 7th and 8th carbon atoms from the methyl end (compound 2). The characteristic pattern indicating the posi-
36
mycolic acids because most of the interesting structural features are only located in the main chains, which are expelled as neutrals under FAB ionization. Thus a direct study on intact mycolic acids cannot provide clear information about the position of double bonds, branches. A different strategy must be used.
tion of the double bond is also seen in the spectrum from m/z 493 (Fig. 3c) due to monounsaturated C-32 corynomycolic acids 3. The mass of ion a (m/z 255) indicated that the double bond is located on the main chain R r The lower abundance of the ions located between those formed by allylic cleavages at m / z 421 and 365 are enhanced by the superimposition of regularly spaced signals arising from the cleavage of the saturated chain. This pattern indicates that both chains cleave in parallel. A more complex and quite uninterpretable pattern is observed from the mono-unsaturated C-30 specie (not shown); this is due to the presence of two chain isomers, as detected by the formation of two different ions a and, probably, to several double bonds isomers_ The location of double bonds in the side chain would be easily located by recording the CAD/MIKE spectra of ion source fragments a. Unfortunately, this is not useful for the study ~f
a
4
Mfmycolic acids from M. fallax: analysis via a chemical retro-Claisen cleavage Mycobacterium fallax synthesizes a variety of unsaturated very long chain mycolic acids [12]. Whereas the di-unsaturated specie (M 1 and M2) have been clearly identified by chemical methods, some doubt remains about the position of the middle double bond in the tri-unsaturated fraction M 3. From the complex mixture of homologues and isomers, it has been established both by chemical cleavages and electron ioniza-
767
48.7.
23i.,, 293 100
200
300
400
A^AA.AAAAA^A o .
loo
~o
.
.
"
.
.
.
.
'
,~o ............
~o
"-
7oo
A
Fig. 4. CAD-MIKE spectra of two carboxylate anions from the dissociative electron capture ionization of pentafluorobenzyl ester derivatives of a tri-unsaturated fatty acids mixture from M. fallax. These acids arose from the oxidation/retro-Claisen chemical cleavage of M3-mycolic acids: (a) spectra from m / z 767 (C53 H~ O'~); (b) spectra from m/z 753 (Cs~ Hv O-2). ~- A ~ indicates the position of the characteristic pattern for the double bonds.
37 tion mass spectra of bis-silyloxy derivatives, that an even number of methylene units separate the remote double bond from the middle one. An isomer bearing four methylene groups between the two unsaturations was clearly identified. It was not possible, however, to infer the presence or the absence of isomers exhibiting an ethylene group inserted between the two double bonds. In a previous paper, we described the chargeremote cleavage of polyunsaturated fatty acids possessing an ethylene interrupted double bond sequence [8]. This type of structure induces a characteristic pattern due to a single intense signal (due to allylic cleavage between two consecutive double bonds) flanked by two sequences of three weak signals (due to vinylic and n-bond cleavages). Our aim was to try to detect such a sequence in the M 3 mycolic acids. As pointed out previously, it was not possible to detect clearly the fragmentation pattern of the main chain by analysing intact mycolic acids. Our strategy was to cleave chemically mycolic acids between the C2 and C3 bonds, prior to mass spectrometric analysis. An oxidation step, before or after the cleavage would produce fatty acids possessing all the carbon atoms of the main chain R r Although such a cleavage can be done by pyrolysis at 300°C giving the main chain as an aldehyde [15], we prefered a softer method. We described earlier that long chain/3keto fatty acid esters are easily cleaved by a retro-Claisen reaction occurring in the presence of potassium carbonate and a-trehalose [16]. This sugar provides assistance to this reaction: the 6'-hydroxyl group induces an intramolecular nucleophilic attack on the /3-ketoester group from an intermediary trehalose-/3-keto fatty acid ester formed by a transesterification reaction. This leads to a fast and easy cleavage of the molecule into two acid esters. This cleavage method was applied to mycolic acids from M. fallax after the preliminary oxidation of their /3-hydroxyl groups into /3-ketogroups. The long-chain fatty acid sugar esters obtained by the retro-Claisen reaction were converted into methyl esters and then fractionated by silver ion chromatography. The tri-unsaturated specie (which arose from M3-mycolic acids)
were derivatized as pentafluorobenzylesters. They were directly analyzed by mass spectrometry using electron capture ionization to generate gas-phase carboxylate anions. The CAD/MIKE spectrum of each isobaric carboxylate anion from the C50-C60 atom range was recorded. Two representative spectra are presented in Fig. 4. Spectrum a corresponds to C52 H99 COOspecie of m/z 767. A characteristic pattern of three intense peaks flanked by two smaller sets of three peaks is clearly seen. This corresponds to the structure already described for the M 3mycolic acids in which two double bonds are separated by four methylene groups. The remaining double bond is detected at lower mass range, but its pattern is partly obscured by the presence of several isomers. The main component from these C-53 specie is thus
CH3--(CH2)I4--CH=CH--(CH2)4--CH --CH--(CH2)t2--CH=CH--(CH2)15~COOSpectrum b corresponds to C5~ H97 COOspecie at m/z 753. The expected pattern from an ethylene-interrupted double bond sequence is seen. The 19th peak from that of the parent ion is particularly strong and two sets of three smaller peaks surrounded by two higher ones can be seen on both sides of this major peak. This characteristic pattern, however, is partly complicated by the superimposition of spectra from other isomers. The remaining double bond is clearly seen at lower masses (abundant 33th and 37th peaks flanking three smaller ones). The main structure of the ion is
CH3--(CH2)Is--CH--CH--(CH2)2--CH --CH~(CH2)I2--CH--CH--(CH2)15--COOConclusion
The structural determination of mycolic acids by charge remote fragmentation needs a chemical chain cleavage before mass spectrometric analysis, and several characteristic patterns for special arrangement of double bonds are clearly detected. Thus, a new isomer of M3-mycolic acids from M. fallax was identified. Some spec-
38 tra, h o w e v e r , are o b s c u r e d by t h e presence o f t o o m a n y isomers. A n e f f e c t i v e c h r o m a t o g r a p h i c s e p a r a t i o n o f i s o m e r s will c e r t a i n l y i m p r o v e these analysis, a n d such w o r k is in progress.
References 1 J. Asselineau (1966) The Bacterial Lipids, Holden-Day, San Francisco. 2 M.D. Collins, M. Goodfellow and D.E. Minnikin (1982) J. Gcn. Microb. 128, 129--149. 3 K.B. Tomer, F.W. Crow and M.L. Gross (1983) J. Am. Chem. Soc. 105, 5487--5488. 4 N.J. Jensen and M.L. Gross (1986) Lipids, 21, 362-365. 5 K.B. Tomer, N.J. Jensen and M.L. Gross (1986) Anal. Chem. 58, 2429--2433. 6 N.J. Jensen, K.B. Tomer and M.L. Gross (1985) J. Am. Chem. Soc. 107, 1863--1867. 7 V.H. Wysocki, M.E. Bier and R.G. Cooks (1988) Org. Mass Spectrom. 23, 627--633.
8
9 10
11 12 13 14 15 16
H. Aurelle, M. Treilhou, D. Prom~, A. Savagnac and J.C. Prom6 (1987) Rapid Commun. Mass Spectrom. 1 : 3, 65--67. J.C. Prom~, H. Aurelle, F. Couderc and A. Savagnac (1987) Rapid Commun. Mass Spectrom. 1 : 4, 50--51. F. Couderc, H. Aurelle, D. Prom~, A. Savagnac and J.C. Prom~ (1988) Biomed. Environ. Mass Spcctrom. 16, 317--321. J. Asselineau (1961) Biochim. Biophys. Acta 54, 359-360. E. Rafidinarivo, J.C. Prom~ and V. Levy-Frebault (1985) Chem. Phys. Lipids 36, 215--228. E. Lederer and J. Pudles (1951) Bull. Soc. Chim. Biol. 33, 1003--1010. Gastambide-Odier and E. Lederer (1960) Biochemistry 333,285--295. J. Asselineau and E. Lederer (1950) Nature 116, 728-729. H. Aurelle and J.C. Prom~ (1980) Tetrahedron Lett. 21, 3277--3279.
31
Structure determination of mycolic acids by using charge remote fragmentation Arlette Savagnac, H616ne Aurelle, Christiane Casas, Francois Couderc, Pierre Gavard, Danielle Prom6 and Jean-Claude Prom6 Centre de Recherche de Biochimie et G~n&tiqueCeilulaires C.N.R.S., 118 Route de Narbonne 31062 Toulouse Cedex (France)
(Received December 5th, 1988; revised and accepted February 2rid, 1989) The collision-induced remote site fragmentation process of closed-shell ions, such as carboxylate anions, is a very potent analytical tool for the structural determination of fatty acids. This leads to an easy location of branch points, double bonds, cyclopropane rings and other functional groups. Although corynomycolicacid mixtures from Corynebacterium diphtheriae can be directly analyzed by negative-ion fast atom bombardment combined with collisionally activated decomposition spectra, mycolic acid mixtures from mycobacteria need a preliminary chemical cleavage. They are oxidized to /~-keto esters and then submitted to a retro-Clalsen reaction. The resulting fatty acids were then converted into pentafluorobenzyl derivatives and introduced directly into a high pressure ion source working in the negative ion mode. The resulting gas phase carboxylate anions are activated to decompose by collision with helium atoms. When applied to M3-mycolicacids from Mycobacterium failax, this method allows for the characterization of a new tri-unsaturated mycolicacid, which has the middle and the remote double bonds separated by two methylene groups. Keywords: mycolicacids; mycobaeteria; corynebaeteria; mass spectrometry; negative ions; remote site fragmentation.
Introduction Mycolic acids (very long a-alkyl-/I-hydroxy fatty acids) are the most characteristic components in the cell wall lipids o f "acid fast" bacteria such as Mycobacteria and related taxa (Nocardia, Corynebacteria, Rhodococcus) [1-2]. Despite numerous efforts, their structures remain only partially determined. This is due to the difficulty in isolating a pure c o m p o u n d from the complex mixture o f isomers and homologues. Recently, Gross and coworkers have proposed a new mass spectrometric method for the structural determination o f fatty acids, [3--5]. This mass spectrometry process is called "charge remote fragmentation", and it occurs when the internal energy o f "closed-shell" ions (such as carboxylate anions) is raised up by collision with Correspondence to: J.-C. Prom6.
neutral atoms [6--7]. The electric charges of such gas-phase ions remain localized during the fragmentation process, thus avoiding isomerisation of an aliphatic chain before any carboncarbon cleavage takes place. In the original procedure, gas-phase ions are produced by fast atom bombardment. We recently described an improvement to this technique that provides a powerful analytical tool for the study o f complex mixtures [8--10]. In this method gas-phase carboxylate anions are produced by dissociative electron capture o f pentafluorobenzyl esters of fatty acids (PFB derivatives). This modification allows GC separation before ionization and increases the ionization yield to a large extent to provide sensitive and fast determinations. The aim o f this paper is to describe a strategy for structural determination o f mycolic acids, using the charge remote fragmentation process. Two kinds o f mycolic acids will be studied: corynomycolic acids, from C o r y n e b a c t e r i u m
0009-3084/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
32
diphtheriae [11], which are short-chain mycotic acids, and M 3 mycotic acids from Mycobacterium failax, which possess a very long aliphatic chain with three double bonds [12]. In the latter study, the authors were unable to locate precisely the intermediary double bond in all isomers by chemical degradations, because of the complexity of the mycotic acid mixture. Experimental Corynomycolic acids and mycolic acids were extracted and purified from C. diphtheriae and M. fallax as described [11,12]. The mass spectrometer was a reverse geometry ZAB-HS instrument (VG-analytical, Manchester). A high pressure ion source filled with methane was used for electron capture negative ionization. The electron energy was 50 eV. Alternatively, negative FAB-ionization in a triethanolamine matrix was used to produce carboxylate anions from free fatty acids. Helium was introduced into the collision cell located in the second field free area in such a way that a signal arising from a carboxyiate anion was reduced to approximately 30% of its original value. CAD/MIKE spectra were collected by the data system (VG-2035). Scan speed was 800 eV/s.
Oxidation o f mycolic acid methyl esters Crude a-mycotic acids from M. fallax (400 mg) were dissolved in 5 ml anhydrous methylene chloride and added under stirring to a mixture of 400 mg pyridinium chlorochromate, 150 mg calcium carbonate and 10 ml methylene chloride. After 2 h at 40°C, the mixture was extracted with diethyiether, washed and the corresponding /1-ketoesters were purified by siticic acid column chromatography. The yield was 90%. Infrared carbonyl stretching frequencies at 1700 and 1740 cm -1 were observed.
Retro-Claisen reaction assisted by a-trehalose The O.keto~sters (350 mg) were added to a mixture of 180 mg anhydrous potassium carbonate, 600 mg anhydrous a-trehalose in 30 ml
dimethylformamide. The mixture was stirred at 80°C for 6 h under vacuum (125 Torr). After cooling, the mixture was acidified and extracted with chloroform. The mixture of a-trehalose esters was hydrolyzed with 5%0 KOH in methanol/water 1:1 (60°C, 2 h). After acidification and extraction with diethyl ether, the fatty acids were methylated with ethereal diazomethane. Long chain ketones resulting from the degradation of unreacted/~-ketoesters (40%0) were separated from the fatty acid methyl esters (60%) by silicic acid chromatography.
Fractionation o f fatty acid methyl esters by silver nitrate chromatography Chromatography on silver nitrate impregnated silicic acid column allows the separation of fatty acid methyl ester mixtures according both to the number of double bonds and their steric environment. The fatty acid methyl esters resulting from the retro-Claisen reaction (130 rag) were fractionated on 30 g of freshly prepared silver nitrate impregnated siticic acid. Successive elutions with increasing amounts of diethylether in hexane gave four main fractions. The first fraction consisted of a mixture of straight chain C-22 and C-24 fatty acid esters, as identified by GC/MS. The two subsequent fractions contained di-unsaturated long-chain fatty acids as identified by mass spectrometry (direct inlet, electron impact). The fourth fraction contained tri-unsaturated fatty acid methyl esters (direct inlet) corresponding to what was expected from the main chain of the M3-mycotic acids, namely m/z 824 (C56), 810 (C55), 796 (C54), 782 (C53) and 768 (C52) in the approximate ratio 2 : 1 : 7 : 1 : 6 (the carbon number of each fatty acid is indicated between brackets).
Synthesis o f PFB derivatives Less than 1 mg of free fatty acids was mixed with pentafluorobenzyibromide (2 ~l), diisopropylethylamine (2 /A), acetonitrile (50 ~l) and methanol (10 ~). The mixture was stirred at room temperature for 1 h. An aliquot (1 /A) was then directly introduced into the crucible of the direct inlet probe of the mass spectrometer.
33 . ,,.,
255
/
253 \
!227
283 281 [[ ,t[J..t I1' .,U .,11" ~6..,u..,,t ,I,.
"-
00-'"
"
" " "s.
: "
"
-'"
-"
"-
"'"200"'--'1
--
•
.
-
• . . . . . . .
.300"
". . . .
m~z#
" . . . .
.
"
"
-'-4oo"
- o. . . . . . .
"-
;'"
"
.
. ' ~. 6 o ".
.
.
.
.
.
0'
Fig. 1. FAB mass spectrum o f corynomycolic acids f r o m C. diphtheriae. The matrix was triethanolamine.
Results and discussion
Corynomycolic acids from C. diphtheriae Corynomycolic acids are the simplest form of mycolic acids. Their carbon number is relatively low (from 26 to 36). The most abundant corynomycolic acid from Corynebacterium diphtheriae was identified many years ago [13] as the C-32 compound, 1, formed biosynthetically by condensation between 2 tool of palmitic acid [14] but several other specie were also detected [2]. In particular, both the main chain R 1 and the branched chain R 2 may contain one double bond. Negative FAB-MS of the corynomycolic acids from C. diphtheriae shows different ions due to the presence of several sets of homologues containing zero to two double bonds (Fig. 1). In the upper mass range, the main deprotonated molecular ions are seen at m/z 495, 493, 491 corresponding to C-32 specie with zero, one and two double bonds, respectively. Other molecular
specie are observed at m/z 467 and 465 (C-30 specie with zero and one double bond). Less abundant ions are detected for the minor homologues, namely C-34 specie with zero to two double bonds and C-28 specie with zero or one double bond. In the medium mass range, fragments ions are observed mainly at m/z 255 (deprotonated hexadecanoic acid) and m/z 253 (deprotonated hexadecenoic acid). Less abundant ions correspond to deprotonated octadecanoic, octadecenoic and tetradecanoic acids (m/z 283, 281 and 227, respectively). The CAD-MIKE spectrum of each deprotonated molecular ion shows characteristic and intense fragmentations due to the cleavage of C2-C3 bond. A likely mechanism is depicted in Scheme 1. It produces the enolic form of a deprotonated fatty acid (ion a) and a fatty aldehyde as a neutral compound via a retroClaisen-like process. The mass of ion a indicates the number of both carbon atoms and-unsaturations of the side chain R2. Complementary information concerning the main chain R1 are
34
2 CH
/
/'-J\ 11, R 1-CH
C=O
-II,
is an indication that the following isomers are present:
0(-)
R1-CHO + R2-OH=C\
o®
CIIH23-CH(OH)-CH-(CI4H29)-COOH OH
C13H2~--CH(OH)--CH--(CI2H25)--COOH
H
C15H31--CH(OH)--CH--(C,oHu)--COOH
ion a
In addition to this abundant fragmentation, the CAD/MIKE spectra of the (M-H)- ions from corynomycolic acids exhibit a large number of weaker peaks which can be attributed to charge remote fragmentation processes. Figure 3a shows the cleavage pattern of the saturated C-32 specie 1. This representation is magnified by ten times to visualize weak signals. The very abundant ion of at m/z 255 is due to ion a. Subsequent loss of water gives the ion of rnTz 237. The charge remote fragmentation process gives regularly spaced signals 14 ainu apart corresponding to the formal expulsion of alkanes of increasing length (alkenes plus one hydrogen molecule). The C3-C4 bond cleavage (near the hydroxyl group) gives the more abundant ion of at m/z 283. These charge-remote fragmentations are useful for characterizing additional features on the aliphatic chains. In the spectrum of the ion of m/z 491, which corresponds to di-unsaturated C-32 corynomycolic acids (Fig. 3b), the mass of ion a (m/z 253) indicates that both chains R~
R1-CH(OH)-CH(R2)-COOH i
R 1 = n-C15 H31
R2 = n-C14 H29
2
R 1 = CH3-(CH2)5-CH=CH-(CH2) 7
R2=CH3"(CH2)5CH=CH'(CH2) 6
3
R1 = CH3-(CH2)5-CH=CH-(CH2) 7
R2=n'C13 H17
Scheme 1.
obtained from the mass difference between ion a and the molecular specie. Such fragment ion] are accompanied by weaker ones due to the consecutive loss of water. Thus, whereas all the C32 homologues fragment into two C-16 moieties, others give a more complicated pattern. For example, the ion of m/z 439, corresponding to the minor C-28 saturated corynomycolic acids, cleaves into deprotonated hexadecanoic, tetradecanoic and dodecanoic acids (to give ions of m/z 255, 227, 199, respectively) (Fig. 2). This
.
.
.
.
.
.
~
.
.
.
.
Fig. 2. CAD-MIKE spectrum of the deprotonated C-28 corynomycolic acid at m / z 439.
.
.
.
.
.
460
.
.
.
.
.
35
a
b
419 9.35
C
I
mlz Fig. 3. CAD-MIKE spectra of deprotonated C-32 corynomycolic acids: (a) saturated specie at m/z 495; Co) di-unsaturated specie at m/z 491; (c) mono-unsaturated specie at m/z 493. The intensity of all signals was magnified ten times.
and R 2 contain an unsaturation. The chargeremote fragmentation signals exhibit a characteristic pattern for a double bond: three weak signals surrounded by two stronger ones at m / z 419 and 363 due to the slower cleavage of both
vinylic and double bonds compared to allylic fragmentations. This indicates that the double bonds are situated between the 7th and 8th carbon atoms from the methyl end (compound 2). The characteristic pattern indicating the posi-
36
mycolic acids because most of the interesting structural features are only located in the main chains, which are expelled as neutrals under FAB ionization. Thus a direct study on intact mycolic acids cannot provide clear information about the position of double bonds, branches. A different strategy must be used.
tion of the double bond is also seen in the spectrum from m/z 493 (Fig. 3c) due to monounsaturated C-32 corynomycolic acids 3. The mass of ion a (m/z 255) indicated that the double bond is located on the main chain R r The lower abundance of the ions located between those formed by allylic cleavages at m / z 421 and 365 are enhanced by the superimposition of regularly spaced signals arising from the cleavage of the saturated chain. This pattern indicates that both chains cleave in parallel. A more complex and quite uninterpretable pattern is observed from the mono-unsaturated C-30 specie (not shown); this is due to the presence of two chain isomers, as detected by the formation of two different ions a and, probably, to several double bonds isomers_ The location of double bonds in the side chain would be easily located by recording the CAD/MIKE spectra of ion source fragments a. Unfortunately, this is not useful for the study ~f
a
4
Mfmycolic acids from M. fallax: analysis via a chemical retro-Claisen cleavage Mycobacterium fallax synthesizes a variety of unsaturated very long chain mycolic acids [12]. Whereas the di-unsaturated specie (M 1 and M2) have been clearly identified by chemical methods, some doubt remains about the position of the middle double bond in the tri-unsaturated fraction M 3. From the complex mixture of homologues and isomers, it has been established both by chemical cleavages and electron ioniza-
767
48.7.
23i.,, 293 100
200
300
400
A^AA.AAAAA^A o .
loo
~o
.
.
"
.
.
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'
,~o ............
~o
"-
7oo
A
Fig. 4. CAD-MIKE spectra of two carboxylate anions from the dissociative electron capture ionization of pentafluorobenzyl ester derivatives of a tri-unsaturated fatty acids mixture from M. fallax. These acids arose from the oxidation/retro-Claisen chemical cleavage of M3-mycolic acids: (a) spectra from m / z 767 (C53 H~ O'~); (b) spectra from m/z 753 (Cs~ Hv O-2). ~- A ~ indicates the position of the characteristic pattern for the double bonds.
37 tion mass spectra of bis-silyloxy derivatives, that an even number of methylene units separate the remote double bond from the middle one. An isomer bearing four methylene groups between the two unsaturations was clearly identified. It was not possible, however, to infer the presence or the absence of isomers exhibiting an ethylene group inserted between the two double bonds. In a previous paper, we described the chargeremote cleavage of polyunsaturated fatty acids possessing an ethylene interrupted double bond sequence [8]. This type of structure induces a characteristic pattern due to a single intense signal (due to allylic cleavage between two consecutive double bonds) flanked by two sequences of three weak signals (due to vinylic and n-bond cleavages). Our aim was to try to detect such a sequence in the M 3 mycolic acids. As pointed out previously, it was not possible to detect clearly the fragmentation pattern of the main chain by analysing intact mycolic acids. Our strategy was to cleave chemically mycolic acids between the C2 and C3 bonds, prior to mass spectrometric analysis. An oxidation step, before or after the cleavage would produce fatty acids possessing all the carbon atoms of the main chain R r Although such a cleavage can be done by pyrolysis at 300°C giving the main chain as an aldehyde [15], we prefered a softer method. We described earlier that long chain/3keto fatty acid esters are easily cleaved by a retro-Claisen reaction occurring in the presence of potassium carbonate and a-trehalose [16]. This sugar provides assistance to this reaction: the 6'-hydroxyl group induces an intramolecular nucleophilic attack on the /3-ketoester group from an intermediary trehalose-/3-keto fatty acid ester formed by a transesterification reaction. This leads to a fast and easy cleavage of the molecule into two acid esters. This cleavage method was applied to mycolic acids from M. fallax after the preliminary oxidation of their /3-hydroxyl groups into /3-ketogroups. The long-chain fatty acid sugar esters obtained by the retro-Claisen reaction were converted into methyl esters and then fractionated by silver ion chromatography. The tri-unsaturated specie (which arose from M3-mycolic acids)
were derivatized as pentafluorobenzylesters. They were directly analyzed by mass spectrometry using electron capture ionization to generate gas-phase carboxylate anions. The CAD/MIKE spectrum of each isobaric carboxylate anion from the C50-C60 atom range was recorded. Two representative spectra are presented in Fig. 4. Spectrum a corresponds to C52 H99 COOspecie of m/z 767. A characteristic pattern of three intense peaks flanked by two smaller sets of three peaks is clearly seen. This corresponds to the structure already described for the M 3mycolic acids in which two double bonds are separated by four methylene groups. The remaining double bond is detected at lower mass range, but its pattern is partly obscured by the presence of several isomers. The main component from these C-53 specie is thus
CH3--(CH2)I4--CH=CH--(CH2)4--CH --CH--(CH2)t2--CH=CH--(CH2)15~COOSpectrum b corresponds to C5~ H97 COOspecie at m/z 753. The expected pattern from an ethylene-interrupted double bond sequence is seen. The 19th peak from that of the parent ion is particularly strong and two sets of three smaller peaks surrounded by two higher ones can be seen on both sides of this major peak. This characteristic pattern, however, is partly complicated by the superimposition of spectra from other isomers. The remaining double bond is clearly seen at lower masses (abundant 33th and 37th peaks flanking three smaller ones). The main structure of the ion is
CH3--(CH2)Is--CH--CH--(CH2)2--CH --CH~(CH2)I2--CH--CH--(CH2)15--COOConclusion
The structural determination of mycolic acids by charge remote fragmentation needs a chemical chain cleavage before mass spectrometric analysis, and several characteristic patterns for special arrangement of double bonds are clearly detected. Thus, a new isomer of M3-mycolic acids from M. fallax was identified. Some spec-
38 tra, h o w e v e r , are o b s c u r e d by t h e presence o f t o o m a n y isomers. A n e f f e c t i v e c h r o m a t o g r a p h i c s e p a r a t i o n o f i s o m e r s will c e r t a i n l y i m p r o v e these analysis, a n d such w o r k is in progress.
References 1 J. Asselineau (1966) The Bacterial Lipids, Holden-Day, San Francisco. 2 M.D. Collins, M. Goodfellow and D.E. Minnikin (1982) J. Gcn. Microb. 128, 129--149. 3 K.B. Tomer, F.W. Crow and M.L. Gross (1983) J. Am. Chem. Soc. 105, 5487--5488. 4 N.J. Jensen and M.L. Gross (1986) Lipids, 21, 362-365. 5 K.B. Tomer, N.J. Jensen and M.L. Gross (1986) Anal. Chem. 58, 2429--2433. 6 N.J. Jensen, K.B. Tomer and M.L. Gross (1985) J. Am. Chem. Soc. 107, 1863--1867. 7 V.H. Wysocki, M.E. Bier and R.G. Cooks (1988) Org. Mass Spectrom. 23, 627--633.
8
9 10
11 12 13 14 15 16
H. Aurelle, M. Treilhou, D. Prom~, A. Savagnac and J.C. Prom6 (1987) Rapid Commun. Mass Spectrom. 1 : 3, 65--67. J.C. Prom~, H. Aurelle, F. Couderc and A. Savagnac (1987) Rapid Commun. Mass Spectrom. 1 : 4, 50--51. F. Couderc, H. Aurelle, D. Prom~, A. Savagnac and J.C. Prom~ (1988) Biomed. Environ. Mass Spcctrom. 16, 317--321. J. Asselineau (1961) Biochim. Biophys. Acta 54, 359-360. E. Rafidinarivo, J.C. Prom~ and V. Levy-Frebault (1985) Chem. Phys. Lipids 36, 215--228. E. Lederer and J. Pudles (1951) Bull. Soc. Chim. Biol. 33, 1003--1010. Gastambide-Odier and E. Lederer (1960) Biochemistry 333,285--295. J. Asselineau and E. Lederer (1950) Nature 116, 728-729. H. Aurelle and J.C. Prom~ (1980) Tetrahedron Lett. 21, 3277--3279.