105Supplementary references
3.3 DITERPENOIDS AND GIBBERELLINS .1 Chemistry and distribution
The diterpenoids (see Fig. 3.7) comprise a chemically heterogenous group of compounds, all with a C20carbon skeleton based on four isoprene units;
most are of very limited distribution. Probably the only universally dis- tributed diterpene is the acyclic parent compound of the series, phytol, which is present as the ester attachment in the molecule of chlorophyll (see Chapter 5). Three classes of diterpenoids will be considered here:
resin diterpenes, toxic diterpenes and the gibberellins.
The resin diterpenes include compounds such as abietic and agathic acids, found in both modem and fossil plant resins (Thomas, 1970). These resin compounds have a protective function in nature in that they are exuded from wood of trees or the latex of herbaceous plants. Abietic acid is widespread in gymnosperm resins, especially in Pinus. The various
Diterpenoids and gibberellins 125
HO
Phytol
Agathic acid
i ~ OAc"OH Grayanotoxin-l
Hardwickic acid
4JyQ
CO0HO OH
Me C02H CH2
Gibberellic acid Fig. 3.7 Structure of some diterpenoids.
'copal' resins of legume trees contain a range of different diterpenes (Ponsinetet al., 1968), the dicyclic terpene hardwickic acid being but one example. A group of toxic diterpenes are the grayanotoxins, typified by grayanotoxin-1 (see Fig. 3.7), which occur in the leaves of manyRhododen- dron andKalmiaspecies. They are responsible for the poisonous nature of the foliage of these plants.
The third class of diterpenoids to be mentioned are the gibberellins, a group of hormones which generally stimulate growth and which are known to be widespread in plants. Gibberellic acid (abbreviated as GA3) is the most familiar gibberellin, but in fact over one hundred compounds in the series have been found to date. Chemically, they are closely related and thus difficult to separate and distinguish. The only really satisfactory method of determination is by GLC-MS.
The chemistry and natural distribution of diterpenes has been reviewed
126 The terpenoids
by Hanson 0968, 1972). There are many other reviews of the plant gibberellins (e.g. MacMillan, 1983) and a monograph on their physiology and biochemistry (Crozier, 1983).
3.3.2 Recommended techniques (a) Diterpenes
In general, these compounds are separated by GLC and TLC using the same methods as for the lower terpenes. However, diterpenes are less volatile than the sesquiterpenes and slightly different GLC techniques may be required in some cases. Further identification is largely based on IR and mass spectra.
GLC, for example, was used by Aplin et al. (963) for surveying the distribution of eleven diterpene hydrocarbons in the Podocarpaceae.
These authors extracted dried leaf tissue (20-40g) in a Soxhlet with ether for 12-18h. After concentration, the residue was dissolved in benzene and chromatographed on an alumina column (de-activated with 5% acetic acid) packed with light petroleum (b.p. 6O-80°C). Elution with the latter solvent gave the diterpenes in the initial fractions and these were then submitted to GLC on two different columns. The first was a 350cm col- umn of embacel with 3% E301 silicone oil, which was operated at 160°C.
The second was a 120cm column of presiliconized Gas Chrom P 000-120 mesh) coated with 1% E 301 silicone oil at 140°C. Typical retention times on the first column (in min) were: rimuene 16.0, cupressene 17.8, isophyllocladene 20.9, isokaurene 23.0, phyllocladene 25.0 and kaurene 26.3. Thus, closely related pairs of isomers were satisfactorily separated and characterized by this procedure.
Similar procedures are used for the TLC of diterpenes as for other terpenoids. Demole and Lederer (958) for example, separated phytol(RF
35), isophytol (50), geranyl-linalol (44) and phytyl acetate (66) on silica gel in n-hexane-ethyl acetate 07:3). More recently, TLC on silica gel-AgN03 00: 1) with light petroleum as solvent has been exploited (Norin and Westfelt, 1963). Detection is by the standard procedures (conc.H2S04, antimony chloride reagent, 0.2% KMn04etc.).
When screening a series of plants for diterpenoids, a simple TLC proce- dure would normally be used, but HPLC might be advantageous, particu- larly in the case of oxygenated derivatives. Thus, Ganjianet al. (1980) were able to detect three biologically active diterpenes in a single dried leaf weighing 45 mg ofRabdosia umbrosusby HPLC. The methanol extract was injected into a Zorbax ODS CIS column (25 X 0.46cm), which was eluted with methanol-water (9: 11) and monitored at 230nm. For preparative work, diterpenoids are routinely purified by column chromatography on either silica or alumina (Croteau and Ronald, 1983).
(b) Gibberellins
Diterpenoids and gibberellins
127In view of the importance of these substances as ·plant hormones, much effort has been devoted to their isolation and estimation in plant tissues.
There are, however, one hundred known gibberellins and no single TLC system is yet available for their resolution. Identification by TLC alone is therefore not to be relied on and other methods (see below) must be used in conjunction with TLC.
At least thirty solvents have been applied to the separation of gibberellins on layers of inactive or activated silica gel (Kaldewey, 1969).
Two generally useful ones, on activated plates, are benzene-butanol- acetic acid (70:25:5) and benzene-acetic acid-water (50:19:31, upper layer); the time of development is approximately 1 h. TLC-electrophoresis on silica gel G at 3.8-5V cm-1has also been found useful (Schneideret ai., 1965). The standard method of detection is by spraying plates with H2S04- water (7:3) and then heating at 120°C; gibberellins appear as UV fluorescent yellow-green spots.
For GLC, gibberellins must first be converted to their methyl esters or trimethylsilyl ethers. They may be separated on columns of 5% SE-30, 5%
SE-52 and 5% OV-22, all on DMC5-treated Chromosorb W (Perez and Lachman, 1971). Alternatively, columns of 2% QF-1 or 2% SE-33 may be used (Durleyet ai.,1971).
For reliable gibberellin identifications, combined GLC-MS is the technique of choice. In this procedure, the hormones are separated gas- chromatographically as their trimethylsilyl ethers, and then the mass spectra are automatically determined directly on the separated compo- nents as they emerge from the GLC column. GLC-MS can be applied to crude extracts or to 5~gsamples of impure gibberellins and known com- pounds can be identified without recourse to authentic samples (which are often inaccessible), as long as reference mass spectra are available (Binkset ai., 1968) (see Table 3.6). For gibberellin conjugates (e.g. gibberel- lic acid glucoside or glucose ester), it is best to identify them by GLC-MS of the permethyl ethers (Rivieret ai.,1981).
The HPLC of plant gibberellins has been developed, but there are difficulties in detecting them because, generally, they do not absorb in UV.
One solution is to separate them as their benzyl esters (Reeve and Crozier, 1978); another is to depend on their end absorption between 200 and 210nm, when it is necessary to avoid solvents that are not transparent at these wavelengths.Inthe latter case, separations have been achieved on reverse-phase C18 silica columns using methanol-o.8% aqueous H3P04
and detection at 205nm (MacMillan, 1983).
The quantitative determination of gibberellins in plants is difficult and no ideal procedure that has been tested sufficiently is yet available. One approach is to employ an elaborate 6-stage clean-up procedure, involving
128 The terpenoids
Table 3.6 Mass spectral fragmentation patterns of gibberellins AJ-A~; as their methylester trimethylsilyl ethers
M- M- M- M- M- m/z
Name M+ 15 28 31/32 59/60 89/90 207/208
AI 506 491 447 416 +
A2 508 493 476 449 418
A3 504 489 473 445 414 +
A4 418 403 386 358 328
As 416 401 357 +
Ab 432 417 373 +
A7 416 401 384 356 326
AB 594 579 535 504 +
AIO 420 405 389 361 331
AD 492 477 460 432 400
AI4 448 433 416 388 358 358
Alb 506 491 475 416
AI7 492 477 460 432 401 +
AlB 536 521 504 477 446 446 +
AI9 462 447 434 431 402 373 373 +
A20 418 403 387 359 +
A21 462 447 430 403 371 +
A22 504 489 472 444 414 +
A23 550 535 522 519 491 461 461 +
Note: A few gibberellins show other fragments atmlz130,mlz 147, M-103,M-134/5and M-193/4. Data from Binksetal. (968).
several HPLC separations and finally GLC-MS with selective ion moni- toring (e.g. Yamaguchi et al., 1982). Another is to use a very sensitive analytical procedure, such as immuno-assay, which will work on rela- tively crude extracts. The latter procedure is expensive, since animals are needed for producing the antisera, but it has the advantage that it can be applied to quite small amounts of plant tissue (Weiler, 1983).
When identifying such a well known substance as gibberellic acid itself, many of the more complicated procedures mentioned above can be avoided. Simple methods can be applied, but careful comparison with authentic material at all stages is essential. The procedures used by Sircar et al. (1970) for identifying GA3 in petals of Cassia fistulosa at a level of Smg kg-Jfresh weight are illustrative. The material was isolated by extraction with acetone and purified by bicarbonate fractionation and column chromatography on silica gel with chloroform as eluent.
GA3 was identified by Rrcomparison on paper and TLC, IR and mass
Triterpenoids and steroids 129 spectra and by spectrofluorimetry. Finally, identity was confirmed by bioassay using the lettuce hypocotyl, a-amylase and dwarf rice leaf sheath tests.
3.4 TRITERPENOIDS AND STEROIDS