105Supplementary references
4.4 POLYACETYLENES
174 Organic acids, lipids and related compounds
very sensitive to impurities. The advantage of mass spectral studies is that they may well reveal trace amounts of isomers in what otherwise appear to be pure GLC fractions (Misra and Ghosh, 1991).
(d) Ethylene
The identification of this simple hydrocarbon (CH2=CH2) is of especial interest to plant physiologists since it has been recognized to be an impor- tant natural growth regulator. Ethylene produced by plants is measured using a GLC apparatus set up for gas analysis. Since only very small amounts of ethylene are produced by plant tissues, it is essential that the GLC recorder used is operating at the maximum level of sensitivity.
Before analysis, ethylene can be condensed in a liquid oxygen trap, or by passing it on to a column of silica gel impregnated with mercuric perchlorate (Phan, 1965).
Originally, GLC was carried out on a column of 30% silicone oil 550 coated on Firebrick (60-80 mesh) (Meighet al., 1960). Galliard et al. (968) have employed a stainless steel column 050cmX 3mm) packed with 10%
Triton x-305 on NAW Chromosorb G (80-100 mesh), operating isother- mally at room temperature with a flame ionization detector. The same authors removed polar volatile compounds from the gas samples by employing a pre-column 07cm X 4mm) packed with 20% diglycerol on celite and fitted with a back-flushing device. Using this procedure, 0.03 p.p.m. ethylene can be detected.
Muir and Richter (1972) recommended GLC on a column of Porapak, at a temperature of 80°C. In a typical run on this support, the authors found oxygen emerging after 1.5 min, methane at 2 min and ethylene at 4 min.
Although ethylene clearly separates from related hydrocarbons (e.g.
methane, ethane, propylene) on most column stationary phases, it is as well to confirm its identification in natural plant vapours by GLC on at least two different types of column, e.g. on Porapak together with silicic acid (80-100 mesh) or alumina Fj (80-100 mesh) as the contrasting phase.
Conclusive identification of ethylene in a new plant source really requires GLC-MS analysis as well (Wardet al., 1978).
Polyacetylenes
Straight chain hydrocarbon CH3-CH==CH-(C==C)3- CH =CH-CH=CH2
Alcohols CH2=CH-CHOH-(C==C)2CH2CH=CH(CH~7H
Falcarinol
175
Ketone
Ester
Aromatic
Furans
Fatty acid
HOCH~HOH-CH=CH-(C==Ch-CH==CH-CH3 Safynol
CH2==CH-CO-(C=C)2C~CH=CH(CH2)~
Falcarinone
CH3- (C==C)3 -CH=CH-COzMe Dehydromatricaria ester
PhC~-C==C-C== CH
o-c=
C-CH,PhCarlina oxide
CH,CH,cH
=CH-C=C-CO~CH=CHCo,H
Wyerone acid
CH3- (CH2)7-C
==
C-(CHz)~02H Stearolic acidFig. 4.7 Structures of some representative polyacetylenes.
terized by standard phytochemical techniques. Indeed, over 1000 polyacetylenes are now known as plant products. Only a few are simple hydrocarbon derivatives; most have additional functional groups and are either alcohols, ketones, acids, esters, aromatics or furans. Some typical structures found among the polyacetylenes are indicated in Fig. 4.7.
Polyacetylenes have a taxonomically interesting distribution pattern in higher plant families; they occur regularly in only five families, namely the Campanulaceae, Compositae, Araliaceae, Pittosporaceae and Umbelliferae. The former two and latter three families are especially closely linked in other ways (Sorensen, 1968). Acetylenic acids (e.g.
stearolic acid) have a rather different distribution from the other polyacetylenes and are found inSantalum, Santalaceae and other families in the Santalales and also in certain Malvales, where they occur in associa- tion with cyclopropene fatty acids (Bu'Lock, 1966). Acetylenics are also found in the higher fungi, in two families of the Basidiomycetes, the
176 Organic acids, lipids and related compounds
Agaricaceae and Polyporaceae. The fungal compounds are slightly differ- ent in having a chain length mainly between CIl and C14, whereas the higher plant acetylenes are mostly C14 to C18compounds.
In their biosynthesis, the polyacetylenes are probably formed from the corresponding fatty acid, via an olefinic intermediate by successive dehydrogenations, followed by other modifications (Bu'Lock, 1966). If acetylenes have an overall function, it is most likely as toxins in either plant-animal or plant-plant interactions. Thus, some are highly poison- ous, e.g. those found in the roots of the water dropwort, Oenanthe crocata and of fool's parsley,Aethusa cynapium,while others in fungi have antibi- otic activity. Also, two acetylenes, wyerone acid in broad bean, Vida faba, and safynol in safflower, Carthamus tinctorius, have been implicated as natural phytoalexins and are toxic to micro-organisms which attack these plants.
A comprehensive review of the chemistry and chemotaxonomy of the polyacetylenes is available (Bohlmannet al., 1973).
4.4.2 Recommended techniques (a) Thin layer chromatography
Fresh tissue is extracted by maceration with ether in the presence of activated alumina and left to extract for at least 24h at 4°C in the dark. The decanted solution is dried over Na2S04and taken to dryness at 15°C. The samples, in ether, are then chromatographed on either Al203or silica gel plates in benzene-ehloroform (10:1),pentane-ether (9:1)or chloroform- methanol (9: 1). On spraying the plates with 0.4% isatin in conc.H2S04,
and heating, acetylenes appear as brown or green spots. They will appear as yellow spots on plates sprayed with 1% KMn04in 2% aqueous Na2C03•
Ifsilica gel plates with fluorescent indicator are used (HF254 ), acetylenes can be simply detected by their quenching action.
For the TLC separation of acetylenes with closely related structures, chromatography on silica gel plates treated with 5-10% caffeine is recom- mended (Lam and Hansen, 1990).
(b) Spectroscopy
The most characteristic property of polyacetylenes is their UV spectrum.
Almost all compounds show a series of three or more very intense peaks in the region 200-320 nm (see Fig. 4.8) and UV spectroscopy is widely used as a means of preliminary detection. For example, in screening plants of the Campanulaceae (leaves and roots) for polyacetylenes, Bentleyet al.
(1969) depended on UV monitoring of crude ether extracts for their detec- tion. Thus, two compounds in the root of the bellflower, Campanula
Polyacetylenes 177 Table 4.4 Ultraviolet spectral maxima of some polyacetylenes of the Umbelliferae Polyacetylene
Falcarinol Falcarinolone Falcarinone Falcarindione Aethusin
UV maxima (nm) 229,240,254
259,274,290
208,246,260,274,291 245,253,268,285,303
210,248,264,280,294,313,334
Source
root of domestic carrot caraway,Carum carvi Falcaria vulgaris caraway,Carum carvi root of Fool's parsley,
Aethusa cynapium
Data are from Bohlmannet al. (961). Details of the distribution of polyacetylenes in the Umbelliferae canbefound in Bohlmann (971).
250 300
Wavelength (nm)
350
Fig. 4.8 Ultraviolet spectrum of a polyacetylene. Absorption spectrum of CH3(C=C)3CH=CH-e0zMe (from Sorensen,1968).
178 Organic acids, lipids and related compounds
glomerata after preliminary purification on silica gel in ether-petroleum, had Amax 252, 266 and 288nm and 277, 294 and 313nrn respectively. UV spectra of some pure polyacetylenes found in the Umbelliferae are given in Table 4.4, to further illustrate the range and number of spectral maxima.
Unfortunately, UV spectroscopy is not a conclusive test for presence or absence of acetylenes, since just a few compounds fail to give a series of intense peaks, showing instead only a single broad band. In such cases, IR spectra may be measured, since there is a characteristic band at about 2200cm-1for the acetylenic triple bond. Even so, substances with a triple bond adjacent to two conjugated double bonds cannot be clearly identi- fied by either UV or IR measurements (Sorensen, 1968). Two other spec- tral procedures, NMR and MS, are now widely employed for structural identification of polyacetylenes. Even these techniques are not completely unequivocal in indicating acetylenic substituents.
Most polyacetylenes can be isolated in reasonably stable form and the carrot root compound, falcarinol, can even be purified by gas chroma- tography using a column temperature of 192°C and a liquid phase of 5% Dow II silicone oil on 60-80 mesh Chromosorb W (Crosby and Aharonson, 1967). Some, however, are much more unstable. A ketoaldehyde present in parsnip seed was obtained as a colourless oil, which resinified within 2 days, even when kept in the dark at -40°C (Joneset al., 1966).
HPLC is a gentler procedure for acetylenic separation and purification.
Ithas been used, for example, to separate wyerone and other acetylenic phytoalexins in infected bean leaves (Mansfieldet al.,1980). Reverse phase separations were carried out on an ODS Hypersil column, eluted with a gradient of MeOH-5% HC02H (7: 13) and MeOH-MeCN-5% HC02H (4: 1: 5) and monitored by UV detection at 330nm.
4.4.3 Practical experiments (a) The polyacetylenes
of
the carrotThe domestic carrot contains four acetylenes, the major one being falcarinol (Bentleyet al.,1969). This compound is weakly toxic, producing neurotoxic symptoms on injection into mice (Crosby and Aharonson, 1967). However, the quantities in carrot are so low (2mgkg-1carrot) that it does not present a dietary hazard. The structure advanced by Crosby and Aharonson for their 'carotatoxin' was later revised by Bentleyet al.
(1969) to that of the already known polyacetylene, falcarinol. This com- pound can be easily isolated from carrot (and from roots of a number of related umbellifers) and detected by its UV spectrum.
(b) Procedure
Sulphur compounds 179
(1) Three medium-sized fresh carrots are cut into small pieces, covered with ether and stored in the refrigerator for at least 24h.
(2) The ether is then decanted, the extract dried over MgS04and evapo- rated to dryness at below 35°C. The residue is taken up in a small volume of n-hexane and applied as a streak to a narrow (5 X 20 em) and full size (20 x 20cm) silica gel plate.
(3) The plates are developed in benzene-ehloroform (10:1) for 1 h. The smaller plate is then sprayed with 0.4% isatin in conc.H2S04 and heated at 110°C for 5 min. The most intense brown spot is due to fa1carinol and the position of the major band on the unsprayed plate can be determined by comparison with the marker plate.
(4) The band is then scraped off, eluted with ether (3m1) and the UV spectrum measured. Several intense peaks should be obtained, corre- sponding in position to those of fa1carinol (Table 4.4). Extra peaks may also be present due to contamination with related acetylenes. Ifthis is so, fa1carinol can be purified by re-running it on silica gel in pentane-ether (9:1).
(c) Other sources of polyacetylenes
Another plant which can be used as a convenient source of acetylenics is the water dropwort, Oenanthe crocata, which grows abundantly near ditches and streams in temperate regions of the Northern hemisphere.
The roots are poisonous and the plant has been described as the 'five finger death' because of their shape. Fresh roots can be collected, and after careful washing, extracted in the same way as in the carrot experiment above. A TLC plate of a concentrated extract developed in pure chloro- form will show the presence of several acetylenes after spraying with isatin in cone.H2S04• One of the major toxins, a ketone, has spectral maxima at 210, 249, 266, 291, 314 and 336nm.
4.5 SULPHUR COMPOUNDS