Determination of double-bond position in some unsaturated terpenes

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Anal. Chem. 1993, 65, 2528-2533

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Determination of Double-Bond Position in Some Unsaturated Terpenes and Other Branched Compounds by Alkylthiolation A. B. Attygalle,' G. N. Jham,+and J. Meinwald Baker Laboratory, Department of Chemistry, Cornell University, Ithaca, New York 14853

The electron-impact mass spectra of a,&bis(methylthio) derivatives of certain terpenes and other compounds with branched alkenyl groups contain diagnostic peaks that can be used for locating the position of the double bond in the parent compound. In contrast to the spectra of dimethyl disulfide (DMDS) derivatives obtained from compounds containing CH=CH type double bonds, which show two predominant fragment ions, most of the compounds examined in the current investigation showed only one predominant fragment ion, arising from that part of the molecule which possesses the more substituted carbon of the original double bond. For example, all the derivatives from compounds with an isopropylidene moiety showed a base peak at m/z89 which can be attributed to the formation of a [(CH&C=SCH3]+ fragment. The application of the DMDS procedure to naturally occurring terpenes is discussed. INTRODUCTION Determination of the positions of carbon-carbon double bonds in naturally occurringcompounds is a classical problem, the solutions of which have been reviewed recently.'" One frequently used method entails the addition of dimethyl disulfide (DMDS) to the double bond to give a dimethyl disulfide adduct.5 The electron-impact mass spectrum (EIMS) of these adducts shows a prominent pair of fragment ions due to the preferential cleavage of the carbon-carbon bond linking the carbon atoms bearing the two methylthio substituents. The mlz values of these two ions define the position of the original double bond. This technique has been used extensively for the location of double bonds in simple alkenes,5galkadienes and trienes,'~~ acetates,QJOfatty acid methyl esters,11-15 alcohols,14 aldet

Permanent address: Universidade Federal de ViGosa, ViGosa, MG

36570,Brazil.

(1) Hogge, L. R.; Millar, J. G. In Advances in Chromatography; Giddinm, J. C., Grushka, E., Brown, P. R., Eds.; Marcel Dekker: New York, f987;Vd. 27,p 299. (2)Schmitz, B.; Klein, R. A. Chem. Phy. Lipids 1986,39, 285-311. (3)Anderegg, R. J. Mass Spectrorn. Reu. 1988,7,395-424. (4)Attygalle, A. B.;Morgan, E. D. Angew. Chem., Int. Ed. Engl. 1988,

27,460-478. (5)Francis, G. W.;Veland, K. J. Chromatogr. 1981,219,379-384. (6) Scribe, P.; Pepe, C.; Barouxis, A,; Fuche, Ch.; Dagaut, J.; Saliot, A. Analusis 1990,18,284-288. (7)Takano, 1.; Kuwahara, Y.; Howard, R. W.; Suzuki, T. Agric. Biol. Chem. 1989,53,1413-1415. (8)Carlson, D. A.; Roan, C.; Yost, R. A.; Hector, J. Anal. Chem. 1989, 61,1564-1571. (9)Buser, H. R.; Am, H.; Guerin, P.; Rauscher, S.Anal. Chem. 1983, 55,818-822. (10)Vincenti, M.; Gugliemetti, G.; Cassani, G.; Tonini, C. Anal. Chem. 1987,59,694-699. (11)Christie, W. W.;Brechany, E. Y.; Shukla, V. K. S.Lipids 1989, 24,116-120. (12)Scribe, P.; Guezennec, J.; Dagaut, J.; Pepe, C.; Saliot, A. Anal. Chem. 1988,60,928-931. 0003-2700/93/0385-2528$04.00/0

hydes,14and k e t ~ n e s . ~The ~ J ~method is particularly useful because only nanogram amounts of unknown material is required and because it can be applied directly to mixtures. Despite the great analytical potential of DMDS derivatives, however, this procedure has not been applied to terpenes or other compounds containing tri- or tetrasubstituted double bonds. In this paper we report the use of DMDS derivatives for the determination of the position of a double bond in a number of terpenes and branched-chain compounds containing, in addition to an isolated double bond, other functionalities such as hydroxyl, ester, ketone, or a,& unsaturated aldehyde groups.

EXPERIMENTAL SECTION Reagents. (+)-Sabinene, (lS)-(-)-@pinene, (-)-isopulegol, (S)-(-)-&citronellol,(*) chrysanthemyl alcohol (mixture of cis and trans isomers), (S)-(-)-perillaldehyde, dihydrocarveol, 3-methyl-2-buten-l-ol,6-methyl-5-hepten-2-one, (a-7-tetradecenylacetate, citral (from which nerd and geranial were isolated by micropreparative gas chromatography),and dimethyl disulfide were from Aldrich Chemical Co. 4,7-Dimethyl-6-octen-3-one was available in our laboratory. (S)-(-)-@-Citronellol was converted to its acetate by using acetyl chloride and triethylamine; the product was about 80% pure. (-)-Isopulegol was oxidized to (+)-isopulegonewith K2Cr20,/H2SOd. All compounds were about 97% pure (GC). Derivatization. Hexane (100pL),DMDS(100pL),and5pL of I2 in ether (5%) were added to each compound (100 pg) or mixture (200 pg), and the reaction mixture was allowed to stand in the dark for 18 h at room temperature. The solution was decolorized with 1-2 drops of sodium thiosulfate solution (5%), and the products were extracted into 200 pL of hexane. The extractionwas repeated twice (2 X 300pL),and the organic layers were combined and evaporated to dryness under Nz. The residue was redissolved in 500 pL of hexane and examined by GC/MS and GC/FTIR. For nerd and geranial, a reaction time of only 3.5 h was used. Short reaction times (4.5h) were also used when the derivatizations were conducted with 10-100 ng samples. Five ants (Acanthomyops clauiger) were frozen and decapitated. The heads were crushed and extracted twice with 20 pL of hexane. The combined organic layers were derivatized as described above. The essential oil of Dicerandra immaculata was obtained as described previously for a related species.'* The oil was dissolved in dichloromethane ( 5 % ) ,and a 10-pL aliquot was derivatized as described above. Instrumentation. GC/MS analyses were performed on a Hewlett-Packard (HP) 5890gas chromatographcoupled to a 5870 mass selective detector (ionization energy 70 eV; source tem(13)Yamamoto, K.; Shibahara, A.; Nakayama, T.;Kajimoto, G. Chem. Phys. Lipids 1991,39-50. (14)Leonhardt, B. A.; DeVilbis, E. D. J.Chrornatogr. 1985,322,484490. (15)Dunkelblum, E.; Tan, S. H.; Silk, P. J. J. Chem. Ecol. 1985,11, 265-277. (16)Murata, Y.; Yeh, H. J. C.; Pannel, L. K.; Jones, T. H.; Fales, H. M.; Mason, R. T. J. Nut. Prod. 1991,54,233-240. (17)Schulz, S.;Francke, W.; Boppre, M. Biol. Chem. Hoppe-Seyler 1988,369,633-638. (18)Eisner, T.; McCormick, K. D.; Sakaino, M.; Eisner, M.; Smedley, S. R.; Aneshansley, D. J.; Deyrup, M.; Myers, R. L.; Meinwald, J. Chemoecology 1990,1,30-37. 0 1893 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993

2129

Scheme I

I

10

A+

\

,ScH,

'

R/

B+

10

.::~

m/z 61 '.

n

I

Group11

I

mlz 248

mlz 187

u IO

H/c=scH3

D+ 50

perature 250 OC) or a Finnigan ion trap detector (ITD 800; ionization energy 70 eV; manifold temperature 200 "C), using a 25 m X 0.22 mm fused-silicacolumn coated with DB-5stationary phase (J&W Scientific). For GC/MS analyses the oven temperature was held at 60 OC for 3 min and increased to 250 OC at 10 OC/min. GC/IR analyses were performed on an HP 5890 gas chromatograph coupled to a HP 5965A infrared detector (cell temperature 265 "C), using a 25 m X 0.33 mm fused-silicacolumn coated with DB-5(J&W Scientific). Samples were introduced by splitless injection; the injection temperature was 250 "C, and the split vent was kept closed for 0.5 min. Helium was the carrier gas for all systems, at a column head pressure of 50 kPa for the 0.33-mm4.d. capillaries and 80 kPA for those with 0.22-mm i.d. columns.

40

30 20

IO 5

RESULTS AND DISCUSSION The derivatives formed by addition of DMDS to terpenes and other branched-chain unsaturated compounds used in this study can be divided into two groups as shown in Scheme

15

25

loo 0

1

~

89

I

75

I

I

I. The iodine-catalyzed addition of DMDS to a double bond follows the mechanism outlined in Scheme I. The reaction proceeds smoothly at ambient temperatures under mild conditions in nonpolar solvents. The expected DMDS derivatives were observed for all the compounds tested in this study; for some compounds essentially quantitative conversion was achieved. For example, the gas chromatogram of the product derived from (-)-isopulegol contained only one major peak (Figure 1). In some chromatograms, peaks due to other sulfur-containing byproducts were also observed (these peaks were readily recognized by their mass spectra; an ion containing a sulfur atom shows a characteristic isotope cluster). However, unambiguous recognition of the desired DMDS adduct peak was readily achieved by GC/MS analysis. The mass spectra of all the adducts showed recognizable molecular ions (Table I); even the spectra of alcohols and acetates showed molecular ions of moderate intensity (316%). Interestingly, the rates of derivatization of the compounds used in this study was generally slower than those of linear unsaturated compounds. For example, when a 1:l mixture of isopulegone and (2)-7-tetradecenylacetate was allowed to react with DMDS, a considerable amount (2040%) of unreacted ketone was still present after 24 h, while the unsaturated acetate was complete consumed. As a rule, the mass spectra of DMDS adducts of linear alkenes, alcohols, esters, and aldehydes usually show two intense ions arising from the cleavage of the carbon-carbon bond bearing the two methylthio substituents. The mass spectra of the adducts obtained from di- and trisubstituted double bonds of the branched-chain compounds in the present study were significantly different from those of linear compounds. The fragment ions that may be expected are illustrated in Scheme I. However, of all the compounds tested, only one example, the derivative of 4,7-dimethyl-6-octen-3-

min

35

Reconstructed total Ion chromatogram (ROC, lowermost profile), and three selected-ion retrieval chromatograms for m/z 187, 61, and 248, obtained by OC/MS analysis of the product of DMDS addition to (-)-isopuiegol. About 10-20 ng of isopulegol was used for the derivatization: DB-bcoated 30 m X 0.22 mm column, held at 60 O C for 3 min and Increased to 250 O C at 10 OC/mIn. Figure 1.

159 i

0

' '

30

1l~I~~.11

I I "

80

I ,,

1 1 ,

" " " I " "

130

t

180

'

' I " '

I "

230

mlz Figure 2. Electron-impact mass 4,7dimethyl-8-octen-3-one.

spectrum of the DMDS adduct of

Scheme I1

'1 89 ( 1 0 0 )

w

159(34)

one, showed prominent peaks for both ions expected from the cleavage of the bond between the carbon atoms attached to the methylthio groups (Figure 2; Scheme 11). The peaks at mlz 89 and 159 represent A+ and B+, respectively. The signal at mlz 201 can be attributed to a loss of CH3S from the molecular ion [in fact, a loss 47 mass units from the molecular ion is commonly observed in most DMDS adduct spectra (Tables I and 1111. The fragment at mlz 111represents a loss of CH3SH from the m/z 159 ion. The spectra of other derivatives usually showed a prominent peak for only one of the expected ions, which infact was the base peak in most spectra. From the data presented in Tables I and 11, we can infer that, in the mass spectra of DMDS derivatives of group 1 and group I1 type compounds, the fragment ion bearing positive charge at the more substituted carbon atom is more intense (A+ and C+) than the less substituted ions (B+ and D+). This is not unexpected since

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993

~~~

Table I. Mass Spectrometric and Gas-Phase Infrared Data of DMDS Derivatives of Group I Type Compounds GC/MS data of deriv mass spectrum mlz (% ,re1 intena) name (MW) and atruct of parent compd (S)-(p)-citr0nellol(156)

P

O

RT (min)

19.9

M+

A+

B+

M+ M+ - 47 M+ - 48 (47+ 48)

250 (IO)89 (100) 161 (4) 203 (1) 202 (0) 155 (1)

other significant ions

41

IR data of deriv (cm-1)

55 (E),67 (lo), 3665 (W), 2970 (a), 2930 (a), 2884 (m), 69 (15),81 (lo),88 (15), 1050 (w) 90 (7),91 (e), 95 (IS), 252 (1)

H

(S)-(p)-citroneltd acetate (198) 20.9

QOAC

292 (9) 89 (100) 203 (2) 245 (2) 244 (0) 197 (0) 41 (20),43 (30),55 (12), 2971 (m), 2931 (m), 1760 (a), 1232 (a) 61 (8),67(9),81 ( W , 90 (8),91 (7),95 ( W , 137 (7),143 (5),204 (8), 294 (1)

(*) -chrysanthemyl alcohol (154) 19.1

248 (3) 89 (100) 159 (4) 201 (14)200 (1) 153 (3) 41 (2% 43 (19),45 (7), 3660 (w), 3614 (w), 55 (15),59 (12),61 (101, 2974 (a), 2886 (m), 1032 (w) 67 (11),69(15),77 (8), 79 (15),81 (26),90 (61, 91 (15),93 (12),95 (12), 115 (9),123 (15),203 (l), 250 (1) gmethyC5-hepten-2-0ne(126) 16.8 220 (8) 89 (100)131 (9) 173 (1) 172 (0) 125 (6) 41 (17),43 (52),45 (6), 2981 (a), 2970 (m), 2831 (a), 1729 (8) 49 (8),55 (8),83 (11), 90 (6),91 (5),132 (14), 222 (1)

%""

t

4,7dimethyl-Socten-3-one (126)17.8

t

3-methyl-2-buten-1-d (86) (CY)&=CHCHpH

neral(152) $HO

geranial(152) &CHO

A a The

248 (10)89 (100)159 (31) 201 (11) 200 (2) 153 (16) 40 (lo),41 (17),42 (13), 2977 (a), 2932 (m), 1719 (m) 43 (9),45 (14),47(101, 49 (lo),53 (7),55 (14), 57 (27),59 (7),90(20), 91 (7),111 (46),121 (ll), 161 (2),203 (1)

180 (11) 89 (100) 91 (7) 133 (0) 132 (0) 85 (9) 41 (28),43 (9),45 (131, 3662 (w), 3562 (w, b), 49 (l8),55 (22),61 (13), 2977 (a), 2932 (a), 1056 (m) 69 (7),75 (g), 88 (W, 90 (7),103 @A115 (U, 182 (1) 37.916 246 (1) 89 (100) 157 (1) 199 (2) 198 (4) 151 (9) 45 (14),53 (12),55 (W, 2977 (m), 2931 (m), 67 (12),69 (20),78 (12), 2836 (w), 1697 (a), 81 (17),91 (12),94 (ll), 1620 (w), 1109 (w) 109 (7) 16.2

37.886 246 (1) 89 (100)157 (1) 199 (1) 198 (3) 151 (7) 45 (1U,53 (lo),55 (9), 2977 (m), 2931 (m), 2836 (w), 1697 (a), 67 (9),69 (16),78 (9), 1619 (w), 1109 (w) 81 (14),91 (lo),94 (8), 109 (6)

following abbreviations are used: a, strong; m, medium; w, weak, b, broad. The oven temperature increase was 4 OC/min.

A+ and C+ are expected to be more stable than B+ and D+, respectively. In most of our mass spectra, the peaks due B+ and D+are either weak or entirely absent. These peaks, even when present are of little diagnostic value since their intensities are often very small. The spectra of derivatives of group I type compounds, which contain an isopropylidene moiety, were particularly simple and informative. All of these spectra show a base peak a t ml2 89, which can be attributed to the ion [(CH3)&=SCH31+. The interpretation of spectra of derivatives of group I1 type compounds is not quite as straightforward as those of group I, since diagnostically less useful peaks show significant intensity. However, the peak representing C+, which some-

times was the base peak, was always a prominent signal. A peak depicting a loss of 66 mass units from the C+ ion, which may be due to loss of the elements of water and thiomethanol, was observed in the spectra of group I1 type alcohols, (-)isopulegol and dihydrocarveol. For the derivative of dihydrocarveol, this peak is the base peak [mlz 121 (loo)]. This is not surprising, since the elimination of H20 and CHsSH from C+ may yield a conjugated species. From the mass spectral data in Table I, it is evident that the DMDS procedure is a convenient method for establishing the structure of alkylidene moieties in natural products. In particular, this procedure offers considerable advantages over ozonolysis, a method often used for such determinations.19

ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993

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~~

Table 11. Maes Spectrometric and Infrared Data of DMDS Derivatives of Group I1 Type Compounds GC/MS data of deriv maas spectrum m / z ( % , re1 intens) name (MW) and struct RTQ M+ other of parent compd (min) M+ C+ D+ M+ - 47 M+ - 48 (47+ 48) significant ions

IR data of derivb (cm-1)

(+) - isopulegone (152)

19.4, 246 (3) 185 (61) 61 (25) 199 (10)198 (77) 151 (30) 53 (17),55 (22),65 (12), 2965 (a), 2931 (a), 19.7 67 (40),69 (12),75 (44), 1720 (a), 1197 (w) 77 (ll), 79 (E),81 (27), 87 (9),93 (26),95 (18), 101 (13).109 (100).110 (10). 111 (3),i34 (io),195 (7), 137 (9),141 (8),155 (l), 186 (7),187 (l), 200 (2) (S)-isopulegd(154) 20.9 248 (10)187 (100)61 (19) 201 (1) 200 (3) 153 (4) 41 (36),43(1% 45 3630 (w),3473 (w, b), 53 (12),55 (24),67 (20), 2961 (a), 2926 (a), 69 (26),75 (21),81(39), 2881 (m), 1050 (w) 87 (12),93 (29),95 (20), 101 (ll),109 (12),121 (29), 124 (4),135 (ll),169 (34), 171 (2),189 (51,250(1) dihydrocarved (154) 21.1, 248 (16) 187 (99) 61 (43) 201 (1) 200 (1) 153 (1) 41 (36),43 (28),45(141, 3659 (w), 2931 (a), 21.2, 53 (13),55 (28),57 (9), 2881 (m), 1042 (w) 21.25 67 (17),69 (29),75 (16), 79 (20),81 (18),87 (19), 93 (41),95 (19),107 (lo), 121 (loo),122 (lo), 123 (2), P O H 135 (24),137 (2),139 (32), 169 (36),171 (2),188 (ll), 189 (5), 250 (2) (S)- (-) -perillaldehyde (150) 22.3 244 (4) 183 (72) 61 (81) 197 (12) 196 (54) 149 (16) 41 (40),45(20)s 47 (lo), 2931 (w), 2803 (w), 51 (9),53 (26),55 (ll), 1706 (a), 1649 (w), 59 (lo),65 (16),67 (20), 1166 (w) 73 (9),75 (loo),77 (35), 79 (55), 81 (13),87 (18), 88 (25),89 (16),91 (36), 93 (17),101 (19),105 (20), 107 (29),109 (12),120 (ll), 134 (12),135 (711,137(4), 148 (28),198 (5)

P-

0 Some diastereomeric derivatives gave two or more GC peaks. For such compounds, retention times corresponding to each GC peak are presented. The mass spectra of diastereoisomers are virtually identical. The data corresponding to the first-eluting isomer are presented. * The following abbreviations are used s, strong; m, medium; w, weak, b, broad.

Ozonolysis, with subsequent determination of the products by gas chromatography, is not an ideal technique, since small fragment molecules such as formaldehyde, acetaldehyde, acetone, and butanone are not easily separated from solvent peaks.20 Furthermore, for the successful application of the ozonolysis procedure, each unknown compound in a mixture must be isolated in pure form; it is difficult to establish which carbonyl fragment originates from which alkene if ozonolysis is performed on an unseparated mixture. One of the main advantages of the DMDS procedure therefore, is its direct applicability to mixtures. This advantage has been well demonstrated by numerous applications of the method to mixtures of linear unsaturated compounds in crude pheromone mixtures. It is also effective with very small samples. Thus, the technique is ideal when only nanogram quantities of material are available. Although for convenience, and to obtain enough product for IR spectra, we customarily used 100 r g of material for each derivatization, we can apply the method successfully to nanograms of material. Thus, readily interpretable mass spectra of the DMDS derivative were obtained by derivatizing 5-10-ng samples of 6-methyl-5hepten-2-one. When mass chromatographic searches for expected fragments were conducted on reconstructed total ion chromatograms, the presence of even a few nanograms of derivatized substrate could be revealed. The chromatograms shown in Figure 1were obtained by derivatizing about 10-20 (19)Beroza, M.; Bierl, B. A. Anal. Chem. 1966,38,19761977. (20)Attygalle, A. B.; Morgan, E. D. Anal. Chem. 1983,55,1379-1384.

ng of isopulegol. The sensitivity, could be further enhanced by using the selective-ion-monitoring mode of the mass spectrometer, searching for the ions A+ and C+. Since the DMDS addition reaction usually creates a new asymmetric center (by a stereospecific trans mechanism21), we can expect to observe a mixture of diastereoisomers when optically active compounds are derivatized. Diastereomeric mixtures are usually separable by capillary GC. The chromatogram obtained from the DMDS derivative of (+)isopulegone showed two closely eluting peaks of similar intensity. The mass and infrared spectra corresponding to both GC peaks were nearly identical, indicating that they do indeed represent diastereoisomers (Table 11). The derivative of dihydrocarveol (supplied as mixture of stereoisomers) separated into three GC peaks with very similar spectra under GC/MS and GC/IR conditions. Nevertheless, all diastereomeric DMDS derivatives were not resolvable on the DB-5 capillarycolumn. On this column,for example,the derivatives obtained from (-)-isopulegol, (SI-(-)-perillaldehyde, and (S)citronellol and its acetate eluted as single peaks. This is not surprising, since the stationary phase OV-1, which is only slightly less polar than DB-5, is known to afford only poor resolution for diastereomeric DMDS derivatives.10 Double bonds conjugatedto a carbonylgroup were observed to be resistant to DMDS addition. Leonhardt and Devilbid' also reported that methyl esters with a conjugated double (21)Caaeiro, M. C.;Fisher, C. L.; Kim, J. K. J. Org. Chem. 1988,50, 43904393.

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993

Scheme I11

9= CHO

100

CHO

-

1

240 SCH,

I;, , ,

perillaldehyde

GCIMS data

RT peak 1

2

(min)

IR dataa

mass spectrum mlr (% ,re1 intens)

14.02 41 (66),43 (55),49 (lo), 53 (9),55 (30),57 (15), 67 (16),69 (60),71 (16), 88 (18),89 (76),95 (loo), 112 (21),113 (go), 202 (M+, 16) 14.13 41 (70),43 (60), 49 (ll), 53 (lo),55 (30),57 (16), 67 (17),69(63),71 (16), 88 (19),89 (74),95 (loo), 112 (23),113 (94), 202 (M+, 17)

(cm-1)

2958 (s), 2932 (s), 2880 (m), 1117 (e)

Scheme IV

h

w

,

, 201 , , , , , , ,

80

113

bond fail to form DMDS derivatives. Our experience with the DMDS reaction confirms this observation. In the present study, some of the compounds we used were doubly unsaturated with one conjugated and one isolated double bond. For example, after the derivatization, (29-per perillaldehyde produced a product in which the conjugated double bond was still present (Scheme 111). Its mass spectrum showed a loss of 61 mass units from the molecular ion [m/z 183 (72;C+)], indicating the terminal methylene group of the parent compound. The resistance of conjugated double bonds to DMDS addition was further seen from the reactions of neral and geranial (Table I). The GCIIR data of DMDS derivatives were useful in recognizing the product peaks when functional groups such as carbonyl and hydroxyl groups were present in the original compound. For example,the presence of an unreacted double bond, conjugated to the aldehyde group, in the derivatives of perillaldehyde, neral, and geranial was established by the characteristic IR absorptions. The absorptions due to the aldehyde and conjugated C=C group of perillaldehyde, neral, and geranial at 1707 ( 8 ) and 1645 (w), 1698 (s) and 1620 (w), and 1698 ( 8 ) and 1622 (w) cm-I, respectively, were virtually unchanged in those of the derivatives (Tables I and 111, confirmingthe proposed structures. However, no significant infrared absorptions diagnostic of the methylthio moieties are evident. The IR data were particularly helpful, however, in proposing structures for the byproduct peaks observed in the derivatization. For example, the gas chromatogram obtained from the reaction products derived from (S)-pcitronellol showed a pair of closely eluting peaks with retention

, , ,I,

, ,

130

Scheme V

CHO= % SCH,

2959 (s), 2932 (s), 2875 (m), 1120 ( 8 )

Resolution, 8 cm-l.

89

'eo ,

, ,;

180 230 mlz Figure 3. Electron-Impact mass spectrum of the DMDS derivative of citronellal obtained from a crude extract of an lndivldual head of A. claviger.

30

Table 111. Analytical Data of Two Byproducts Found in and DMDS the Reaction Mixture of (5')-@-Citronellol

I , ,

citronellal (mol. wt. 154)

89

derivative (mol. wt. 248)

times shorter than that of the main product. Virtually identical spectral data were obtained by GCIMS and GC/IR for the compounds represented by these two byproduct peaks (Table 111). We propose that the two compounds are diastereoisomers of the oxepane structure shown in Scheme IV. The IR spectra confirmedthe absenceof a hydroxylgroup and indicated a C-0 by the strong absorption at 1114 cm-l. Two prominent peaks a t m/z 113 and 89 in the mass spectra further corroborated the proposed structure. The formation of these byproducts can be readily rationalized on the basis of either direct participation of the hydroxyl group in the addition process or displacement of one methylthio group subsequent to DMDS addition. Citronellal, from the mandibular glands of the ant Acanthomyops clauiger,has been identified as one of the compounds which evokes alarmldefense behavior in this insect.22 We applied the DMDS method to confirm the presence of citronellal amongmany other glandular constituents in a crude extract obtained from head of this ant. The GC peak corresponding to the derivative was readily identified by obtaining ion retrieval chrotomatograms for mlz 89 and 248. As seen from the mass spectrum in Figure 3, the DMDS derivative of citronellal fragments in a manner characteristic of derivatives of group I type compounds, to give a base peak at m/z 89 (SchemeV). The infrared spectrum of the derivative showed a strong absorption for the aldehyde group at 1760 cm-l, confirming the identification. Moreover,the mass and infrared spectra as well as the gas chromatographic retention time of the DMDS derivative were indistinguishable from those obtained from an authentic sample of citronellal derivatized with DMDS. Although the DMDS method worked fine for group I compounds, and reasonably well for group I1compounds, the mass spectra of DMDS derivatives of another group, i.e., compounds with the double bond of interest as a methylene group exocylic to a ring, did not show spectral peaks useful for locating the double bond. For example, the position of the double bond in the parent compound was not directly evident from the mass spectra of DMDS derivatives of (+)sabinene and (-)-@-pinene(Table IV). The identification of these two monoterpene hydrocarbons in a mixture is not straightforward since the underivatized compounds have not (22) Chada,M. S.;Eisner,T.;Monro,A.;Meinwald, J.J.InsectPhysiol. 1962,8, 175-179.

ANALYTICAL CHEMISTRY, VOL. 65, NO. 18, SEPTEMBER 15, 1993

Table IV. GC/MS Data of the DMDS Derivatives of Authentic (+)-Sabineneand (-)-&Pinene GC/MS data derivatized RT mass spectrum of DMDS deriv compd (min) m/z (% ,re1 intens) (+) - sabinene

18.9

41 (21),43 (lo), 45 (a), 61 (25), 65 (9),69 (lo),77 (19),79 (28), 91 (loo),92 (27),93 (361,107(13), 116 (ll),134 (14),135 (30), 182 (lo),183 (28),230 (M+,21)

19.4

41 (43),43 45 (8),49 (lo), 53 (lo),55 (lo),61 (18),69(161, 77 (21),79 (51),89 (59),91 (37), 92 (14),93 (661,105(a), 107 (30),119 (8,135(881, 136 (ll),183 (100)

II

(-) -p-pinene

only similar mass spectra but also coelute on DB-5 stationary phase. The DMDS derivatives of sabinene and @-pinene, however, show significantlydifferent retention times and mass spectra. Thus, DMDS derivatization offers a way of unambiguously differentiating the two compounds. By applying this method, we able to establish the presence of both sabinene and &pinene in the essential oil of a mint plant (Dicerandra immaculata). The mass spectra and retention times of the DMDS derivatives of the two candidate compounds from the natural product sample were virtually identical to those of the DMDS derivatives obtained from authentic sabinene and @-pinene.

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From the results of this study, it is possible to draw some generalizations about the mass spectra of the DMDS derivatives of three types of compounds containing substituted alkenyl groups. Although the formation of DMDS adducts followed by gas chromatographic/massspectrometric analysis is not applicable to all compounds with substituted alkenyl groups, the technique is nevertheless useful in locating the position of double bonds in many terpenes and other natural and synthetic compounds. The procedure is particularly useful when onlynanogram amounts of material are available, and when mixtures of unsaturated compounds must be characterized. The production of only one major fragment ion from substrates containing a trisubstituted double bond is clearly a deviation from the usual observation of two prominent ions from derivatives of previously studied less substituted compounds. The awareness of this new fragmentation pattern will be useful when the mass spectra of DMDS derivatives of unknowns are analyzed.

ACKNOWLEDGMENT We thank K. D. McCormick for a sample of D. immaculata essential oil. This work was supported in part by collaborative grants from the National ScienceFoundation (INT 92-92380) and the Brazilian Government (CNPQ), as well as by the National Institutes of Health (AI 12020). Financial support for G.N.J. was provided by the Brazilian Government (CNPQ 201555/91-3).

RECEIVED for review December 7, 1992. Accepted June 14, 1993.