Mass Spectra of Acetylenic Fatty Acid Methyl Esters and Derivatives 1 R. KLEIMAN, M.B. BOHANNON, F.D, GUNSTONE,2 and J.A. BARVE, 2 Northern Regional Research Center, ARS, USDA, Peoria, Illinois 61604 ABSTRACT

MATERIALS AND METHODS

A series of isomeric methyl octadecynoates was analyzed by mass spectrometry; each isomer gave a unique spectrum. The characteristic ions were those resulting from a McLafferty rearrangement of the allenic sites or of the a l r e a d y - r e a r r a n g e d allenic sites. The acetylenic esters were also subjected to oxymercuration whereupon a carbonyl group was formed at either of the original actylenic carbon atoms providing two oxostearates. Further reaction with NaBH 4 formed h y d r o x y esters which, a f t e r silylation, gave diagnostic mass spectra indicative of the triple bond location. Applied to esters with both double and triple bonds, this procedure permitted differentiation between the two' types of unsaturation. Methoxyl groups marked the original double bond locations and hydroxyls did so for triple bonds.

Preparation of the series of octadecynoic acids has been previously reported (6). Each acid was reacted with diazomethane to form the methyl ester. Methyl trans-lO,16-heptadecadien-8-ynoate and methyl 17-octadecen-9ynoate were gifts from R.G. Powell, and a quantity of methyl stearolate was from R.O. Butterfield for exploratory studies. Methyl c r e p e n y n a t e (cis-9-octadecen-12-ynoate) was isolated from methyl esters of Crepis alpina seed oil (7). Oxymercuration was carried out as previously described (5), except that a large excess of sodium borohydride was used. IR analyses were performed with a PerkinElmer Infracord 137 in 1 mm NaCI ceils with CHC13 as the solvent. G a s c h r o m a t o g r a p h y - m a s s spectrometry (GC-MS) was conducted as before (8) except that the Packard 7401 gas chromatograph was replaced by a Bendix 2600 instrument for sample introduction into the mass spectrometer during the later stages of this work. Columns were either glass 6 ft x 1/4 in. packed with 3% Silar-5cp on Gas Chrom Q (Applied Science Laboratories, Inc., State College, PA) or stainless steel 4 f t x 1/8 in. packed with 3% Dexsil 300 on Gas Chrom Q.

I NTRODUCTION

Mass spectrometric analyses of esters containing triple bonds have been reported (1-4). Although these reports present the pathways for ion formation, none present results from a complete series of esters of one chain length. We analyzed by mass spectrometry an almost complete series of methyl octadecynoates (all but the 3,4 and 16,17 isomers). We found that each mass spectrum was unique and that all but two include the same types of ion fragments. The spectra are complex, and the complexity is greater when olefinic bonds accompany the acetylenic bonds. By applying oxymercuration procedures (5) to monoacetylenic and ene-yne esters, both double and triple bonds form derivatives which can be used to give definitive mass spectra that locate and differentiate the two types of unsaturation.

RESULTS AND DISCUSSION

The mass spectra of the series of methyl octadecynoates analyzed are in general agreement with those published (1-3). The basic mass spectral pattern is one of cleavage with McLafferty rearrangement either of the acetylenic bond or of the isomeric allenes found by rearrangement. Therefore, electron bombardment of each octadecynoate should produce four characteristic ions (Table I). F o r example, the mass spectrum of methyl 9-octadecynoate (methyl stearolate) showed ions at m/e 196 [CH3OCO(CH2)7-CH=C=CH2. ~] (A), m/e 210 [CHaOCO(CH2)7-CH=CH-CH=CH2 ~] (B), m/e 152 [H2C=C=CH-(CH2)7-CH3 *] (C), and m/e 166 [H2C=CH-CH=CH-(CH2)7-CH3 | (D) (Table I). Ions with 32 mass units (CHaOH) less 1Presented at the 48th Fall Meeting of the Ameri- than ions A and B are also present. These can Oil Chemists' Society, Philadelphia, PA, Septemfragments are the same as those reported by ber 29-October 2, 1974. 2Chemistry Department, the University of St. Bohlmann et al. (1) and may well be formed in the way he postulated, but we find the relative Andrews, Scotland. 599

600

R. KLEIMAN, M.B. BOHANNON, F.D. GUNSTONE, AND J.A. BARVE TABLE 1 Characteristic Ions (and Relative Intensities) from Methyl Octadecynoates

Triple bond position

n

m

A

B

C

D

A-32

B-32

4 5 6 7 8 9 10 11 12 13 14 15

2 3 4 5 6 7 8 9 10 ll 12 13

12 11 10 9 8 7 6 5 4 3 2 1

126(69) 140(100) 154(59) 168(24) 182(30) 196(13) 210(6) 224(5) 238(3) 252(2) 266(-) 280(-)

140(13) 154(1) 168(2) 182(4) 196(18) 210(26) 224(14) 238(13) 252(5) 266(-) 280(-) 294(1)

222(-) 208(1) 194(-) 180(2) 166(17) 152(38) 138(59) 124(89) 110(100) 96(100) 82(100) 68(100)

236(-) 222(-) 208(-) 194(-) 180(8) 166(15) 152(14) 138(19) 124(35) 110(25) 96(57) 82(82)

94(23) 108(13) 122(26) 136(33) 150(81) 164(24) 178(10) 192(4) 206(3) 220(1) 234(-) 248(-)

108(25) 122(5) 136(5) ' 150(11) 164(28) 178(25) 192(11) 206(6) 220(2) 234(-) 248(-) 262(-)

CH30~C-(CH2)n-~C-(CH2)m-CH3 A = CH3ooC-(CH2)n.CH=C=CH 2

B = CH30~C-(CH2)n-CH=CH-CH=CH 2

C = H2C=C=CH-(CH2)m.CH 3

D = H2 C=CH-CH=CH-(CH2)m-CH 3

intensity of these ions compared to the base of double bonds is more pronounced in this peak to be lower. However, our spectrum of molecule than in esters with acetylenic bonds methyl 9-octadecynoates agrees well with the farther up the chain. The spectrum of the 2,3 spectrum reported by Odham and Stenhagen isomer does exhibit a small m/e 235 (M-59) ion (4). which, presumably, represents the loss of Ions containing the terminal part of the molecule (Table I, C and D) are the most CH30..L:-. Methyl 17-octadecynoate also does abundant when the triple bond is close to this not show ions indicative of the triple bond part of the molecule. Types A and A-32 are the position. Its spectrum is understandably much most intense of the characteristic ions when the like a saturated methyl ester in containing acetylenic bond is near the ester function. In intense ions o f m/e 74 and m/e 87. Three esters containing b o t h triple and fact, when the triple bond is in the 4,5 to 6,7 positions, C and D ions are not detected, and double bonds were analyzed by mass specthe spectra of the 14,15 and 15,16 isomers do trometry without derivatization. The spectrum not show ions of type A and B. A small amount o f m e t h y l crepenynate (cis-9-octadecen-12o f ions [ ( M ) - (CH2-C-=C-(CH2)x-CH3) ] is ynoate) shows a prominent peak at m/e 236 O~ found in esters with 5,6 thru 15,16 triple (29%) which, most likely, represents CH30..t2bonds. For example, methyl 15-octadecynoates (CH2)7-HC=CH-CH2-HC=C=CH 2 . . The analoproduces an ion of m/e 227 gous fragment (m/e 238) from methyl 12[M-(CH2-C-=C-CH2-CH3)] with relative in- octadecynoate is found at only 3% relative tensity of 6% and methyl 11-octadecynoates intensity level (Table I). The most intense ion one of m/e 171 [M-(CH2-C-~----C-(CH2)s-CH3)] in the 12-octadecynoate is the hydrocarbon 5%. Other ions, which are common to all esters fragment o f m/e 110. The comparable C8 in this series, are M(294), M-31, M-74, 87, and fragment in methyl crepenynate, which would 74. require cleavage between the double and triple Esters with the two extreme triple bond bonds, is not found in significant amounts. The positions (2,3 and 17,18) do not conform to position of the double bond is not readily disthe general pattern. The mass spectrum of cernible from the mass spectrum of methyl methyl 2-octadecynoates would be expected to crepenynate. Ions produced from methyl 17contain abundant m/e 98 and 112 ions. Instead, octadecen-9-ynoate are those expected from m/e 100, 140, 154, and 168 are more promi- analogy with methyl 9-octadecynoate. Fragnent. The last three suggest that the movement ments A and B, m/e 196 and 210, are the same LIPIDS, VOL. 11, NO. 8

601

MASS SPECTRA OF ACETYLENIC ESTERS TABLE I1 Major Ions of Silylated Hydroxystearates from Methyl Octadecynoates Triple bond position

n

m

E

F

G

H

R

R'

2 4 5 6 7 8 9 10 11 12 13 14 15 17

0 2 3 4 5 6 7 8 9 10 11 12 13 15

14 12 11 10 9 8 7 6 5 4 3 2 1

161(1) 189(-) 203(40) 217(73) 231(78) 245(64) 259(59) 273(87) 287(25) 301(51) 315(43) 329(11) 343(8) 371(3)

175(100) 203(100) 217(100) 231(100) 245(100) 259(68) 273(52) 287(83) 301(33) 315(62) 329(36) 343(35) 357(32)

313(17) 285(41) 271(65) 257(60) 243(66) 229(52) 215(88) 201(100) 187(49) 173(100) 159(100) 145(100) 131(100) 103(1)

327(-) 299(-) 285(18) 271(45) 257(61) 243(44) 229(80) 215(85) 201(56) 187(75) 173(73) 159(42) 145(35) 117(100)

132(10) 160(1) 174(3) 188(11) 202(9) 216(12) 230(21) 244(13) 258(5) 272(10) 286(5) 300(4) 314(2) 342(9)

146(2) 174(4) 188(14) 202(13) 216(13) 230(30) 244(8) 258(13) 272(10) 286(9) 300(0 314(8) 328(9) 356(-)

O: C

CH30" --(CH2)n--C=C--(CH2)m-CH3

E

eOTMS O CH30~C-(CH2)n-LH

•OTMS

~OTMS o..

l!

F = CH30"C -(CH2 )n+ 1-CH

G = HC -(CH2)m--CH3

eOTMS II H = HC -(CH2)m+I-CH3

eOTMS R = CH30~.C-(CH2) ~ n

R'= CH30~C--(CH2 )n+ 1

%(MS

as in 9-octadecynoate and fragments C and D, m/e 164 and 150, are two mass units less. The spectrum of the conjugated ester, methyl trans10,16-heptadecadien-8-ynoate, is not readily interpreted. Almost all its ions are of low mass with no distinct ions indicative of unsaturated sites.

spectra. Addition of excess NaBH 4 resulted in a mixture of methyl 9- and 10-hydroxystearates w i t h small amounts of unreacted methyl stearolate and 9- and 10-oxostearates. This reaction sequence was affected in all methyl octadecynoates. The major ions were determ i n e d f o r t h e silylated hydroxystearates formed from each member of the series (Table Derivatives of Monoacetylenic Esters II). The fragmentation pattern is one of aThe reaction of mercuric acetate with triple cleavage on each side of the carbon atom bonds to produce oxo derivatives has long been bonded to the silylated hydroxyl group (11) known (9). The mass spectra of methyl oxo- and a smaller rearrangement ion (12). Each stearates formed from methyl octadecynoates, derivatized octadecynoate produces up to four though definitive, are complicated (10). How- major characteristic ions and up to two minor ever, reduction of the keto-esters with NaBH 4 characteristic ions (Table II). Ions 32 mass units and silylation produce trimethylsiloxy deriva- (CH3OH) less than fragments E and F (Table tives which, upon analysis by mass spectrom- II) are also found. Analogous to oxymercuraetry, produce spectra that define the position tion of olefinic bonds (13), when the triple of the oxygen function (11). We used this bond is close to the ester group, the hydroxyl scheme to locate acetylenic bonds in fatty group forms only on the carbon atom farther esters. from this functional group. Therefore, only Oxymercuration of methyl stearolate pro- 3 - h y d r o x y s t e a r a t e is formed from 2-octad u c e d b o t h 9- and 10-oxostearates. The decynoate and 5-hydroxystearate from 4-octaproducts were identified by mass spectrometry, decynoate (Table II). The fragments are either n u c l e a r magnetic resonance, and infrared hydrocarbons (G and H) or oxygen-containing LIPIDS, VOL. 11, NO. 8

602

R. KLEIMAN,M.B. BOHANNON, F.D. GUNSTONE, AND J.A. BARVE

log

c, ~

DO

I

: /]l I ,,1

2Sl

1~8~:::C.~CH_ICfl2Is_CH 3

51

.

I

273

-a 245

tl[ll[ rl.L,l .... ~

"-"

201 215

273 211 4| I~.

se!

80 120 160 200 MaSS OTIS Or

... )-tCH ,)~H-~CH,I,-~II--CH, ~.lv

,,! il

ILl tl .....

iiO'

' 'i6i'

'mE2QO , q ~i*

Mars

onls OCHI ~ -~CH,l, -i-~-i-{CHzl, .i-~H.-.ClII Cll:eO-- 2 1 3 ~ ' ~ 245 " " ' ~ 55

M-{32+151

ii 2BI

2SllA>~'~lSll " ~ 5|

240 280 | [,} ~--(CU,), C930"

321

360

~'O'llilS )--(CH2) I CfllO

It')

320 ll|

....

FIG. 3. Mass spectrum of silylated oxymercuration FIG. 1. Top: Mass spectrum of underivatized products of methyl 17-octadecen-9-ynoate. methyl 10-octadecynoate. Bottom: Spectrum of silylated methyl 10- and ll-hydroxystearates derived 0%c-ICH2Is-C~C--CN=CN--ICH214-CH=ClI2 from methyl 10-octadecynoate. See Table II for R and R'. CH3O/ 159

'"t881 100i

i -

T

250

411'

i| 4( o

ol0

95

,,, JLLLJ .....160 I0 120

40

317 -90 I

40 IO 120 110 200 @ OCII~ OflllS MIU ~C-t CH/ l, "~H'O'[cII,I,.~'H-~CH,I4--CH, CHIO 201-~258 345~ ~IS -32 O~

240

286

Or,Ha I

320

360

0Ills I

N~'-~CII,II~CIIII,~IJI~GtZ~I--CII -32

FIG. 2. Mass spectrum of silylated oxymercurafion products of methyl crepenynate. (E, R, F a n d R'). The former predominates when the OTMS group is close to the 6o-methyl group, and the A-containing ions are dominant when the functional group is nearer to the ester function. Mass spectra of underivatized and d e r i v a t i z e d m e t h y l 10-octadecynoate are reproduced in Figure 1. Ester with Ene-yne Structures

Oxymercuration in methanol and reduction with NaBH4 result in two isomeric methoxy esters for each double bond and two isomeric hydroxy esters for each triple bond. In other words, from an ester with one olefinic and one LIPIDS, VOL. 11, NO. 8

200 MISS

240

280

320

310

400

O OTM$ OCll OCHs 9~ I I I CHONf~C-[C''I'~H'I'CH' 'IJ'~H--CH' -32 243

-32 127

-32 -32 211 15

FIG. 4. Mass spectrum of oxymercuration products of methyl trans-10,16-heptadecadien-8-ynoate. acetylenic bond, four different isomers are formed from this reaction. However, after silylation, these reaction products are so similar that GC does not completely separate them and they elute as one broad peak. While spectra taken at different points on the peak show changes in the ratio of the ions, the characteristic ions of all isomers are still present and they can be used to distinguish between double and triple bonds and to establish their location. For example, the mass spectrum of derivatized methyl crepenynate (Fig. 2) clearly locates the original triple bond (m/e 173 and 187) and the double bond (m/e 215 and 201). In the same manner, Figure 3 shows the fragmentation pattern of the oxymercuration

MASS SPECTRA OF ACETYLENIC ESTERS products of methyl 17-octadecen-9-ynoate. The ions resulting from s-cleavage at the carbon attached to the trimethylsiloxy group are m/e 259 and 273 (fragments containing the ester function) and 245 and 259 (fragments with the terminal end of the molecule). The fragment with m/e 245 could be construed as the ion C H 3 0 ~ C - ( C H 2 ) 6 - H C O T M S *,

which

would

locate t h e triple bond at the 8,9 position; however, this position is ruled out since no rearrangement ion occurs at m/e 216, whereas rearrangement ions are found for a sflyloxy group at both the 9 and 10 positions (m/e 230 and 244). In addition, significant peaks found at m/e 227 (259-32) and m/e 213 (245-32) are formed by loss of methanol from the original ion. The derivatized olefinic group in this molecule is located by the large m/e 59 found in the spectrum. The fragmentation pattern from derivatized m e t h y l t r a n s - 10,16-heptadecadien-8-ynoate (Fig. 4) clearly shows the presence of a sflylated hydroxyl group at the ninth carbon atom (m/e 259) and a methoxyl at positions 11 and 16 (m/e 159 and 59). However, these ions define the location of only one of the carbons at each of the unsaturated sites. Obviously, the conjugated nature of the original ester directs the derivatization to only one of the two possible carbon positions. If the relative position of the formed hydroxyl and methoxyl groups prove to

603

be the same for all conjugated ene-yne structures, then the location of the unsaturation could be estabhshed. At this time, we have not investigated other conjugated esters. REFERENCES 1. Bohlmann, F., D. Schumann, H. Bethke, and C. Zdero, Chem. Ber. 100:3706 (196"/). 2. Groff, T.M., H. Rakoff, and R.T. Holman, Ark. Kern/ 29:179 (1968). 3. Sun, K.IC and R.T. Holman, JAOCS 45:810 (1968).

4. Odham, G, and E. Stenhagen, "Biochemical Applications of Mass Spectrometry," Edited by George R. Waller, Wiley-Interscience, New York, NY, 1972, p. 223. 5. Abley, P., F.J. McQuillin, D.E. Minnikin, IC Kusamran, K. Maskena, and N. Polgar, Chem. Commun. 1970:348. 6. Barve, J.A., and F.D. Gunstone, Chem. Phys. Lipids 7:311 (1971). 7. Spencer, G.F., R. Kleiman, F.R. Earle, and I.A. Wolff, Anal. Chem. 41:1874 (1969). 8. Kleiman, R., and G.F. Spencer, ]AOCS 50:31 (1973)o 9. Myddleton, W.W., R.G. Berchem, and A.W. Barrett, J. Am. Chem. Soc. 49:2264 (1927). 10. Ryhage, R., and E. Stenhagen, Ark. Kemi 15:545 (1960). 11. Eglinton, G., D.H. Hunneman, and A. McCormick, Org. Mass Spectrom.1:593 (1968). 12. Richter, W.L, and A.L. Burlingame, Chem. Commun. 1968:1158. 13. Gunstone, F.D., and R.P. In#is, Chem. Phys. Lipids 10:73 (1973). [ Received March 18, 1976]

LIPIDS, VOL. 11, NO. 8

Mass spectra of acetylenic fatty acid methyl esters and derivatives.

Mass Spectra of Acetylenic Fatty Acid Methyl Esters and Derivatives 1 R. KLEIMAN, M.B. BOHANNON, F.D, GUNSTONE,2 and J.A. BARVE, 2 Northern Regional R...
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