Isolation and Identification of Toxic Agents from Plants - ACS

10 Isolation and Identification of Toxic Agents from Plants MARTIN JACOBSON

Downloaded by MONASH UNIV on May 4, 2015 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch010

Biologically Active Natural Products Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Methods, techniques, and instrumentation for the improved, rapid isolation and identification of physiologically active plant constituents are available today that were lacking 25, 10, or even 5 years ago. The time period between the isolation of a pure compound and its complete identification has often been frustratingly protracted, but that period has recently been narrowed. Perhaps one of the classical examples of the lengthy time span between isolation and complete structural determination is the case of the well known insect toxicant pyrethrum, whose active components—the pyrethrins, cinerins, and jasmolins—were isolated in 1910-1916 by Staudinger and Ruzicka (1), in 1944 by LaForge and Barthel (2), and in 1965 by Godin et al. (3), respectively; however, their complete structures were not obtained until years after they were isolated. No attempt has been made here to present a comprehensive review of a l l of the methods useful for isolating and identifying pesticidal toxicants from plants. Rather those methods that are most generally useful and those pesticidal compounds that are perhaps of major interest are treated. These include pyrethrum, rotenoids, nicotine and nicotinoids, other well-known alkaloids such as those from Veratrum, unsaturated isobutylamides, bitter substances (especially insect feeding deterrents), fungal peptides, gossypol, mycotoxins, and insect growth regulators. The first section treats the isolation and general methods of structure elucidation for the toxicants in each of these categories; the second section cites additional information for each of the most significant methods. For further general reference your attention is directed to the two recent volumes by Nakanishi et al. (4_,5) entitled "Natural Products Chemistry" and to "Naturally Occurring Insecticides" (6) edited by Jacobson and Crosby. Pyrethrum The term "pyrethrum" usually refers to the dried flower heads

153 In Host Plant Resistance to Pests; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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or to an e x t r a c t o f the d r i e d heads of Chrysanthemum c i n e r a r i i f o l i u m V i s , (family Compositae). The a c t i v e components are t o x i c to house f l i e s , mosquitoes, cockroaches, l i c e , and a number of stored product s p e c i e s . I s o l a t i o n . P a r t i t i o n o f a petroleum ether s o l u t i o n of p e t r o leum ether e x t r a c t from d r i e d flower heads between the hydrocarbon and nitromethane, followed by passage o f the nitromethane s o l u t i o n through a short column of a c t i v a t e d c h a r c o a l gives an a c t i v e conc e n t r a t e of 90% p u r i t y or above (7). Molecular d i s t i l l a t i o n can then be used to p u r i f y and d e c o l o r i z e the concentrate (8). The i n s e c t i c i d a l a c t i v i t y o f pyrethrum i s a t t r i b u t e d to the a c t i o n of 6 c o n s t i t u e n t s , namely p y r e t h r i n s I and I I , c i n e r i n s I and I I , and jasmolins I and I I . P y r e t h r i n I and c i n e r i n I are f a i r l y e a s i l y separated from p y r e t h r i n I I and c i n e r i n I I by column chromatography on alumina or s i l i c a g e l , and good s e p a r a t i o n may a l s o be obtained by paper and t h i n - l a y e r chromatography (TLC) (9). I d e n t i f i c a t i o n . The p y r e t h r i n s and c i n e r i n s form c r y s t a l l i n e 2,4-dinitrophenylhydrazones and are f u r t h e r i d e n t i f i e d by degradat i o n , b y d r o g e n o l y s i s , o z o n o l y s i s , u l t r a v i o l e t (UV), i n f r a r e d (IR), and n u c l e a r magnetic resonance (NMR) spectroscopy, and o p t i c a l r o t a t i o n (9). The j a s m o l i n s are i d e n t i f i e d by UV, IR, and NMR spectroscopy (10). Rotenone and Rotenoids T h i s group of compounds comprises mainly rotenone, though i t i n c l u d e s minor amounts of deguelin, tephrosin, t o x i c a r o l , sumat r o l , malaccol, and e l l i p t o n e . The m a t e r i a l s are obtained from v a r i o u s p a r t s ( u s u a l l y the r o o t s ) o f species of D e r r i s , Lonchocarpus, Tephrosia, and Mundulea (family Leguminosae). They a c t as contact o r stomach poisons f o r aphids, house f l i e s , and v a r i o u s species of chewing i n s e c t s . I s o l a t i o n . The a c t i v e compounds are obtained by e x t r a c t i n g the crushed, d r i e d root w i t h ether or methylene c h l o r i d e , followed by overnight c o o l i n g of a carbon t e t r a c h l o r i d e s o l u t i o n of the concentrate at 0°. The carbon t e t r a c h l o r i d e s o l v a t e of rotenone c r y s t a l l i z e s out and i s f i l t e r e d o f f and taken up i n t r i c h l o r o ethylene. When t h i s s o l u t i o n i s d i l u t e d w i t h methanol, n e a r l y pure rotenone separates out and can be f u r t h e r p u r i f i e d by c r y s t a l l i z a t i o n from t r i c h l o r o e t h y l e n e . I s o l a t i o n of rotenone from seeds i s s i m p l i f i e d i f the seeds are d e f a t t e d with petroleum ether p r i o r to e x t r a c t i o n w i t h ether (11). The r e s o l u t i o n of r o t e n o i d mixtures can be e f f e c t e d by column chromatography, but the use of a l k a l i n e alumina must be avoided s i n c e i t causes r a c e m i z a t i o n ; n e u t r a l or a c i d grades of adsorbents must be used. Countercurrent d i s t r i b u t i o n (CCD) i s u s e f u l f o r d e a l i n g w i t h i n t r a c t a b l e gums, and paper, TLC, and

In Host Plant Resistance to Pests; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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high-performance l i q u i d chromatography (11,12).

(HPLC) have also been used

I d e n t i f i c a t i o n . The s t r u c t u r e s are i d e n t i f i e d by chemical degradation and by the use of IR and NMR spectroscopy, and o p t i c a l r o t a t o r y d i s p e r s i o n (ORD). The o p t i c a l l y a c t i v e forms of rotenone are much more t o x i c to i n s e c t s than the racemic form (13).


N i c o t i n e and N i c o t i n o i d s N i c o t i n e and n o r n i c o t i n e occur i n the leaves o f the tobacco p l a n t , N i c o t i a n a tabacum L. Although these compounds were used to a c o n s i d e r a b l e extent as i n s e c t stomach poisons before the ad vent of DDT, they are seldom used as i n s e c t i c i d e s today i n the United States. Anabasine, obtained from the roots of Anabasis a p h y l l a L. and N. glauca Graham (tree tobacco), i s e x t e n s i v e l y used i n the Soviet Union to combat t h r i p s , mites, aphids, l e a f hoppers, s a w f l i e s , l i c e , and mosquitoes (14). N. tabacum and N. glauca are members of the f a m i l y Solanaceae, and A. a p h y l l a i s i n the f a m i l y Chenopodiaceae. I s o l a t i o n . The t e r t i a r y amine, n i c o t i n e , was f i r s t obtained i n pure form i n 1828 by steam d i s t i l l i n g the b a s i f i e d ether ex t r a c t of tobacco leaves (15). Although n i c o t i n e sometimes occurs i n the p l a n t i n the f r e e s t a t e , i t i s u s u a l l y present as the monoa c i d i c base of c i t r i c or m a l i c a c i d (16). Anabasine i s obtained by mixing ground tree tobacco w i t h ether and d i s t i l l i n g the a l k a l i n e e x t r a c t . Since t h i s compound and n o r n i c o t i n e have a secondary amino Ν group, they may be separated from n i c o t i n e by c o n v e r t i n g them to N - d e r i v a t i v e s . I d e n t i f i c a t i o n . The s t r u c t u r e s of n i c o t i n e , n o r n i c o t i n e , and anabasine were determined by degrading the molecules w i t h a c i d s , as w e l l as by TLC and ORD. Natural nicotine i s levorotatory; d e x t r o r o t a t o r y n i c o t i n e i s much l e s s t o x i c to i n s e c t s (16). Other A l k a l o i d s The major i n s e c t i c i d a l a l k a l o i d s other than those from t o bacco are j e r v i n e , veratramine, cevine, genuine, zygadenine, and p r o t o v e r i n e ; they are present i n many s p e c i e s of Veratrum and Zygadenus, and i n s a b a d i l l a (Schoenocaulon o f f i c i n a l e A. Gray), members of the f a m i l y L i l i a c e a e . I s o l a t i o n . The a l k a l o i d s are e x t r a c t e d from the powdered p l a n t with a c i d i c a l c o h o l o r an o r g a n i c s o l v e n t w i t h o r without ammonia. The i n d i v i d u a l a l k a l o i d s are separated by f r a c t i o n a l c r y s t a l l i z a t i o n , chromatography on alumina, s i l i c a g e l , k i e s e l guhr, or an ion-exchange r e s i n * or by CCD (17).


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I d e n t i f i c a t i o n . The veratrum a l k a l o i d s are i d e n t i f i e d by i n f r a r e d and NMR spectroscopy, and by mass spectrometry (MS).


Unsaturated Isobutylamides A number of i n s e c t i c i d a l isobutylamides of unsaturated, a l i p h a t i c , s t r a i g h t - c h a i n C-10 to C-18 acids have been i s o l a t e d from p l a n t s of the f a m i l i e s Compositae and Rutaceae. Although most of these compounds, which are h i g h l y t o x i c to f l i e s and mosquitoes, have been i d e n t i f i e d , and i n some cases s y n t h e s i z e d , others have been only p a r t i a l l y c h a r a c t e r i z e d . They possess two p r o p e r t i e s i n common w i t h one another and with the p y r e t h r i n s pungency, and r a p i d knockdown and k i l l of f l y i n g i n s e c t s . Examples of these compounds are a n a c y c l i n from Anacyclus pyrethrum DC, s p i l a n t h o l from S p i l a n t h e s spp., a f f i n i n from H e l i o p s i s longipes (A. Gray) Blake, s c a b r i n and h e l i o p s i n from H. scabra Dunal., e c h i n a c e i n from Echinacea a n g u s t i f o l i a DC, and h e r c u l i n from Zanthoxylum c l a v a - h e r c u l i s L. (18). I s o l a t i o n . These compounds are i s o l a t e d by the same procedures a p p l i c a b l e to the p y r e t h r i n s ; namely, by p a r t i t i o n of a petroleum ether e x t r a c t of the p l a n t between t h i s s o l v e n t and nitromethane followed by passage of the nitromethane s o l u t i o n through a c t i v a t e d charcoal and e i t h e r high-vacuum d i s t i l l a t i o n of the f i l t r a t e or column chromatography on alumina or s i l i c a g e l

(18). I d e n t i f i c a t i o n . The s t r u c t u r e s are determined by h y d r o l y s i s to the component amine and a c i d m o i e t i e s and by UV, IR, and NMR spectroscopy, and MS (18). Bitter

Substances

The limonoid b i t t e r p r i n c i p l e s are a c l a s s of C 2 6 t r i t e r p e n e s b e l i e v e d to a r i s e i n p l a n t s of the Meliaceae and Rutaceae f a m i l i e s as o x i d a t i o n products o f t e t r a c y c l i c t r i t e r p e n e s . They i n c l u d e such i n s e c t i c i d e s and feeding d e t e r r e n t s as l i m o n i n , n o m i l i n , melianone, nimbin, a z i d i r o n e , nimbalide, and s a l a n n i n ; melianone occurs i n M e l i a azedarach L. and the others occur i n neem, A z a d i r a c h t a i n d i c a (L.) Juss. Other limonoid b i t t e r p r i n c i p l e s are r a t h e r p l e n t i f u l among the Simaroubaceae. Limonoids o c c u r r i n g among the Meliaceae show, i n general, much g r e a t e r s t r u c t u r a l v a r i a t i o n and complexity than those i n the Rutaceae. The chemist r y of the b i t t e r substances was w e l l reviewed by Korte (19) i n 1959 and by Dreyer (20) i n 1968. I s o l a t i o n . Column chromatography of the f l a v o n o i d s and pept i d e s w i t h v a r i o u s types of packings has been compared (21). The most r e c e n t l y described method f o r s e p a r a t i n g f l a v o n o i d s and p i g ments from c i t r u s o i l s i n v o l v e s g e l permeation chromatography


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followed by t h i n - l a y e r chromatography (22). One o f the most potent i n s e c t f e e d i n g d e t e r r e n t s , a z a d i r a c h t i n , occurs i n v a r i o u s p a r t s (mainly the seed k e r n e l s ) of A. i n d i c a . Although f i r s t i s o l a t e d from neem i n 1968 (23,24) by tedious column chromatography o f the ethanol e x t r a c t , i t was ob tained i n much higher y i e l d by Nakanishi's group a t Columbia U n i v e r s i t y (25) i n 1974 by means of a s i n g l e column chromatogra phy of the e x t r a c t on s i l i c a g e l followed by p r e p a r a t i v e t h i n l a y e r chromatography. I d e n t i f i c a t i o n . Limonin, the major limonoid i n c i t r u s seeds, has been known f o r over 100 years and, as a reasonably a c c e s s i b l e m a t e r i a l , has been the most e x t e n s i v e l y s t u d i e d member o f the limonoids. I t s s t r u c t u r e determination was reported i n 1960 and confirmed soon afterward by X-ray c r y s t a l l o g r a p h i c s t u d i e s on the iodoacetate of e p i l i m o n o l . The s t r u c t u r e determination o f many limonoids subsequently i s o l a t e d has followed the same p a t t e r n , that i s , degradation, IR, and NMR spectroscopy, and MS, as w e l l as ORD (20). The determination o f the exceedingly complex s t r u c t u r e o f a z a d i r a c h t i n would not have been p o s s i b l e without the simultane ous use of p a r t i a l l y r e l a x e d F o u r i e r transform and continuous wave decoupling carbon-13 NMR techniques (25). Toxic Fungal Peptides Isolation. I n s e c t i c i d a l peptides such as p h a l l o i d i n , p h a l l o i n , amanitin, and amanin were i s o l a t e d from Amanita pantherina (Fr.) Quelet (deadly a g a r i c ) and A. muscaria ( F r . ) S. F. Gray ( f l y a g a r i c ) as described by Wieland (26) and Eugster (27). The mushrooms were steeped i n methanol, the steepate was evaporated to a small volume, and the p r e c i p i t a t e d i n o r g a n i c sub stances were f i l t e r e d o f f . The f i l t r a t e was f r e e d o f s o l v e n t , d i s s o l v e d i n water, t r e a t e d with l e a d acetate and f i l t e r e d ; the f i l t r a t e , f r e e d o f l e a d with s u l f u r i c a c i d , was s a t u r a t e d with ammonium s u l f a t e a t pH 4 to p r e c i p i t a t e the p e p t i d e s . These toxic peptides are separable by p a r t i t i o n chromatography f o l l o w i n g t h e i r s e p a r a t i o n i n t o l i p o p h i l i c and h y d r o p h i l i c f r a c t i o n s by treatment with methyl e t h y l ketone-acetone-water (20:6:5). Paper and TLC are a l s o s a t i s f a c t o r y . Amanitin i s separable i n t o i t s α-, 3 - , and γ- forms (26). The peptides o f A. muscaria, which are h i g h l y t o x i c to house f l i e s and mosquitoes, may a l s o be i s o l a t e d by chromatography of an a l c o h o l i c e x t r a c t on a column o f Dowex 50 o r Amberlite IR-120 ( H ) , washing f i r s t with water and then with 2N formic a c i d . P u r i f i c a t i o n on Amberlite IRC-50(H ) y i e l d s i b o t e n i c a c i d and muscazone; d e c a r b o x y l a t i o n of i b o t e n i c a c i d gives muscinol. E l e c t r o p h o r e s i s may a l s o be used. An improved method g i v i n g l a r g e r y i e l d s o f the t o x i c compounds i n v o l v e s chromatography of a butanol e x t r a c t of the fungus on alumina, i n which water-saturated +


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butanol i s used as s o l v e n t (27). I d e n t i f i c a t i o n . The peptides a r e i d e n t i f i e d by degradation, by UV, IR, and NMR spectroscopy, and by MS (27). Gossypol


Gossypol i s a yellow c o l o r i n g matter o c c u r r i n g i n species o f the genus Gossypium that i s r e s p o n s i b l e f o r the r e s i s t a n c e o f c e r t a i n v a r i e t i e s o f cotton, G. hirsutum L. (family Malvaceae) to cotton i n s e c t s such as the b o l l w e e v i l . I t i s a l s o t o x i c to r a t s , guinea p i g s , and r a b b i t s , but not to c a t t l e f e e d i n g on cottonseed meal. I s o l a t i o n . Cottonseed k e r n e l s are e x t r a c t e d with petroleum ether to remove the o i l , and the gossypol i s then e x t r a c t e d with ether. A d d i t i o n o f a,cetic a c i d to the ether s o l u t i o n gives a c r y s t a l l i n e g o s s y p o l - a c e t i c a c i d complex (28). I d e n t i f i c a t i o n . Gossypol was i d e n t i f i e d by degradation and combination o f v a r i o u s forms o f spectroscopy, i n c l u d i n g MS (28). Mycotoxins The most important o f the mycotoxins today i s a f l a t o x i n , which i s found i n peanuts, cottonseed, and s t o r e d products contaminated w i t h the common mold A s p e r g i l l u s f l a v u s L i n k . Soon a f t e r attempts were made to i s o l a t e a f l a t o x i n i t became c l e a r that a t l e a s t 4 major a f l a t o x i n s , as determined by t h i n - l a y e r chromatography o f the b l u e - and g r e e n - f l u o r e s c i n g compounds, were involved; these were designated a f l a t o x i n s B-^, G^, and G 2 . At l e a s t 8 minor a f l a t o x i n s a r e now known. The a f l a t o x i n s a r e h i g h l y t o x i c to warm-blooded animals feeding on contaminated f o o d s t u f f s ; they are a l s o c a r c i n o g e n i c . I s o l a t i o n . A r a p i d procedure f o r s e p a r a t i n g the a f l a t o x i n s i n r o a s t e d peanuts u t i l i z e s t h i n - l a y e r p l a t e s coated w i t h Adsorbo s i l No. 1 and the s o l v e n t system benzene-ethanol-water (40:6:3) (29). E s p e c i a l l y recommended f o r the s o l v e n t e x t r a c t i o n o f a f l a toxins from mold-damaged commodities i s the use o f the azeotrope of 2-propanol-water (87.7:12.3) (30). I d e n t i f i c a t i o n . The a f l a t o x i n s are i d e n t i f i e d by chemical degradation, UV, IR, and NMR spectroscopy, and fluorometry. An e x c e l l e n t , e n t e r t a i n i n g d i s c u s s i o n o f the e n t i r e a f l a t o x i n problem, i n c l u d i n g methods f o r i s o l a t i o n and i d e n t i f i c a t i o n , appeared t h i s year i n a r e p o r t by G o l d b l a t t (31). Insect Growth Regulators ( J u v e n i l e Hormones)


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Many species of p l a n t s have been extracted and t e s t e d f o r the presence of j u v e n i l e hormone (JH)-mimicking a c t i v i t y (see references 32^ and 33 f o r recent reviews). These compounds have profound e f f e c t s on many species of i n s e c t s when a p p l i e d to the l a r v a l or pupal stage; i f a d u l t s do develop from such species they are almost always deformed i n one or s e v e r a l ways and may be incapable of feeding and(or) reproducing. The c l a s s i c a l example of a p l a n t - d e r i v e d i n s e c t juvenoid i s the s o - c a l l e d "paper f a c t o r " (juvabione) reported by Slama and Williams (34) i n 1965 and subsequently i s o l a t e d by Bowers e t a l . (35) from the wood of balsam f i r , Abies balsamea (L.) M i l l , (family Pinaceae). Juvabione was i d e n t i f i e d as the methyl e s t e r of todomatuic a c i d f o l l o w i n g chromatographic i s o l a t i o n . I s o l a t i o n . A more recent example of a juvenoid obtained from a p l a n t i s echinolone [(+)-(E)-10-hydroxy-4,10-dimethyl-4,lldodecadien-2-one], i s o l a t e d from the roots of the common American coneflower, Echinacea a n g u s t i f o l i a DC ( f a m i l y Compositae) (36). Echinolone was obtained i n pure form by p a r t i t i o n i n g a pentane ext r a c t of the roots between pentane and nitromethane, s e p a r a t i n g the pentane-soluble p o r t i o n i n t o n e u t r a l , a c i d i c , and b a s i c f r a c t i o n s , and chromatographing the n e u t r a l f r a c t i o n on successive columns of F l o r i s i l , s i l i c a g e l , and s i l v e r n i t r a t e - c o a t e d s i l i c a g e l p r i o r to p r e p a r a t i v e gas chromatography. Identification. Echinolone was i d e n t i f i e d by IR and NMR spectroscopy, MS, microozonolysis, and ORD measurements (36). Insect Growth Regulators

(Molting Hormones)

The best-known examples of these compounds are the p o l y hydroxy s t e r o l ecdysone and i t s analogs i s o l a t e d from i n s e c t s and p l a n t s (37). Treatment of i n s e c t pupae with these compounds causes g r e a t l y a c c e l e r a t e d growth and development that r e s u l t i n premature breaking of diapause and, i n some cases, g i a n t a d u l t s , c o n d i t i o n s that are detrimental to the normal l i f e of the i n s e c t . Isolation. Since the polyhydroxy s t e r o l s are h i g h l y s o l u b l e i n p o l a r s o l v e n t s and s p a r i n g l y s o l u b l e i n nonpolar s o l v e n t s , the p l a n t m a t e r i a l s are extracted with a l c o h o l s . P u r i f i c a t i o n of the e x t r a c t s i s obtained by a combination of p a r t i t i o n , l i q u i d chromatography, p r e p a r a t i v e TLC, and c r y s t a l l i z a t i o n . CCD i s a l s o used. A mixture of e c d y s t e r o l s that i s d i f f i c u l t to separate may be a c e t y l a t e d to g i v e a mixture of acetates whose s e p a r a t i o n may be much e a s i e r , a f f o r d i n g pure acetates from which the f r e e ecdys t e r o l s can be regenerated by h y d r o l y s i s (38). The i s o l a t i o n of e c d y s t e r o l s i s now most commonly c a r r i e d out by TLC on s i l i c a gel-coated p l a t e s o r by column chromatography on Amberlite XAD-2 with gradient e l u t i o n by using 30 to 70% ethanol while monitoring the eluates by absorption at 254 nm (38).


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A s i m p l i f i e d procedure f o r f r a c t i o n a t i n g p l a n t m a t e r i a l s by s e q u e n t i a l e x t r a c t i o n w i t h methanol-chloroform-water and phenola c e t i c acid-water mixtures gives water-soluble, low molecular weight compounds w i t h l i t t l e chemical damage (39). Identification. S t r u c t u r a l e l u c i d a t i o n of ecdysone w i t h UV, IR, and NMR spectroscopy r e s u l t e d i n a p a r t i a l s t r u c t u r e , which was completed only with the help of i t s X-ray d i f f r a c t i o n p a t t e r n and MS. A comprehensive review of ecdysone chemistry i s that by Horn (37).


A d d i t i o n a l Information on Methods of I s o l a t i o n and

Identification

Thin-Layer Chromatography. New procedures f o r s e p a r a t i n g compounds by programmed m u l t i p l e development TLC e q u i v a l e n t to 5,000-8,000 t h e o r e t i c a l p l a t e s have r e c e n t l y been d e s c r i b e d (40). The usual s o l v e n t system i s e t h y l acetate-ethylene d i c h l o r i d e (1:10). The most recent book on TLC i s that e n t i t l e d "HPTLC. High Performance Thin Layer Chromatography, e d i t e d by Z l a t k i s and K a i s e r , which j u s t appeared (41). 11

High-Performance L i q u i d Chromatography (HPLC). Hostettmann et a l . (42) have very r e c e n t l y reported a method f o r o b t a i n i n g pure compounds d i r e c t l y from crude p l a n t e x t r a c t s by f i l t r a t i o n of a hexane e x t r a c t through a column of s i l i c a g e l with e t h e r hexane (1:1) followed by p r e p a r a t i v e HPLC on s i l i c a g e l w i t h ether-hexane (1:9) and two runs with e t h y l acetate-hexane (1:4). This procedure r e q u i r e d only 2-3 hours compared w i t h conventional column chromatography, which r e q u i r e d 2 weeks. No degradation of unstable compounds was observed. HPLC on s i l v e r n i t r a t e - c o a t e d s i l i c a g e l (43) and a reverse phase method on μ Bondapak C-18 (44) i s of value i n s e p a r a t i n g c i s and trans isomers of unsaturated compounds. I n f r a r e d Spectroscopy. The a p p l i c a t i o n of IR spectroscopy to the i d e n t i f i c a t i o n of n a t u r a l products was reported by Cole (45) i n 1956. The use o f F o u r i e r transform IR i n research has brought new and extended c a p a b i l i t i e s i n s p e c t r a l s e n s i t i v i t y and resolution. V i b r a t i o n a l frequency assignments were p u b l i s h e d i n book form by S i l v e r s t e i n e t a l . (46) i n 1974, by Bellamy (47) i n 1975, and by Pearse and Gaydon (48) i n 1976. NMR Spectroscopy. In 1939, Linus P a u l i n g (49) published a review on the c o n f i g u r a t i o n and e l e c t r o n i c s t r u c t u r e o f molecules w i t h a p p l i c a t i o n to n a t u r a l products. T h i s may be considered the forerunner of the use o f NMR f o r the s t r u c t u r a l determination of n a t u r a l products. In 1965, Jackman (50) reviewed the use of NMR spectroscopy f o r determining e m p i r i c a l formulae, the c l a s s e s of


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protons i n a molecule, the sequence of groups i n a molecule, r e l a t i v e stereochemistry, and conformation as a p p l i e d to n a t u r a l products. 23 The use of C NMR spectroscopy has helped immensely i n determining n a t u r a l products s t r u c t u r e , s i n c e the -^C n u c l e i are u s u a l l y completely decoupled from a l l o f the n u c l e i by the use of double resonance. The spectrum i s thus simply a s e r i e s o f s i n g l e t s corresponding to each v a r i e t y of carbon atom present. Textbooks on NMR spectroscopy o r i e n t e d toward organic chemists are those by Stothers (51) and by Levy and Nelson (52), both publ i s h e d i n 1972, by Levy (53) i n 1974 and 1975, and by Muellen and Pregosin (54), published i n 1977. Mass Spectrometry. In 1966, Biemann (55) published a review of MS as a p p l i e d to n a t u r a l products, e s p e c i a l l y v a r i o u s types of alkaloids. S t e r o i d s have been very thoroughly i n v e s t i g a t e d by t h i s technique by D j e r a s s i ' s group, and the i n f o r m a t i o n gained has helped considerably with the complicated e l e c t r o n impact i n duced fragmentation r e a c t i o n s o f organic molecules. The p o l y c y c l i c nature o f many p h y s i o l o g i c a l l y a c t i v e n a t u r a l products p r a c t i c a l l y excludes simple fragmentation processes and r e q u i r e s cleavage of a number o f bonds f o r the production o f a fragment. However, c o n s u l t a t i o n of a v a i l a b l e t a b l e s o f f r e q u e n t l y encountered fragment ions and c o l l e c t i o n s o f mass s p e c t r a f o r the c l a s s of compound under c o n s i d e r a t i o n makes s t r u c t u r a l determination much e a s i e r . F i e l d d e s o r p t i o n and e l e c t r o n impact MS are recent innovations of considerable importance. An e x c e l l e n t book f o r a i d i n i n t e r p r e t i n g mass s p e c t r a i s that published by M c L a f f e r t y (56) i n 1973. A novel r e p r e s e n t a t i o n of data o b t a i n a b l e from doublef o c u s s i n g mass spectrometers has been developed. I t can d i s p l a y on a s i n g l e three-dimensional s u r f a c e the normal mass spectrum together with peaks due to a l l metastable t r a n s i t i o n s o c c u r r i n g i n the instrument. Developed by Drs. Macdonald and Lacey, o f the C.S.I.R.O. D i v i s i o n of Entomology i n Canberra, A u s t r a l i a , t h i s three-dimensional r e p r e s e n t a t i o n i s more s e n s i t i v e to molecular s t r u c t u r e than any o f the two-dimensional r e p r e s e n t a t i o n s commonly used by mass s p e c t r o m e t r i s t s (57). An e x c e l l e n t review o f m i c r o a n a l y t i c a l methodology u s e f u l i n i d e n t i f y i n g unsaturated compounds i s that published i n 1975 by Beroza (58). I t includes b r i e f d i s c u s s i o n s of s p e c t r a l a n a l y s i s , chemical reagents u s e f u l i n determining f u n c t i o n a l groups, ozonol y s i s to determine double bond p o s i t i o n , and carbon-skeleton chromatography. Disclaimer The use of trade or p r o p r i e t a r y names does not n e c e s s a r i l y imply the endorsement of these products by the U.S. Department of Agriculture.


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