O w e n H. Wheeler
Puerto Rico Nuclear Center University of Puerto Rico Mayaguez, Puerto Rico 00708
I
I I
The Girard Reagents
Acids and bases can be extracted from mixtures of natural products by using aqueous base or acid (1). Aldehydes and ketones are important constituents of essential oils in which they occur mixed with hydrocarbons, carbinols, and esters (8). most of the important steroids have structures containing keto groups (3). Recovery of the carbonyl components of such mixtures can be achieved if the aldehydes and ketones are converted to water soluble derivatives. The only general reagents available for preparing such derivatives are the quaternary ammonium hydrazides (I and 11), known as the Girard reagents (4,6). Their acid hydrazide group reacts with aldehydes and ketones affording hydrazones which are soluble in water by virtue of the quaternary ammonium function (111).
The Girard-T reagent (trimethylammonium a c e thydradde chloride, I) and the Girard-P reagent (pyridinium acethydrazide chloride, 11) were first prepared by Andr6 Girard and George Sandulesco, (6),by reacting ethyl ohloracetate with either trimethylamine or pyridine to form the corresponding quaternary ammonium acetate esters (IV). These afforded the Girard reagents on treatment with hydrazine. Although the reagents were little used by their discoverers, they made the details of the preparation of the Girard-T reagent available to Reichstein prior to publication, and his use of this reagent was instrumental in the isolation of many adrenocortical hormones from beef adrenal glands (7). Aldehydes and ketones react on heating with the Girard reagents in methanol in the presence of acetic acid to form the hydrazones (111). Unreacted noncarbonyl containing compounds are usually removed by partially neutralizing the acetic acid, diluting with water, and extracting with ether. The carbonyls are then recovered from the aqueous layer by ether extraction after heating the solution with dilute mineral acid to hydrolyze the hydrazones. Reichstein achieved a primary separation of the adrenocortical steroids by first removing the more reactive a, @-unsaturated ketones with Girard-T reagent and then reacting the less reactive saturated ketones with more reagent. A further separation was obtained by reacting each of the ketone fractions separately with Girard-T reagent and then hydrolyzing the hydrazones at decreasing pH, when the saturated ketones were first liberated (7). A later kinetic study (8),carried out by titrating the un-
reacted Girard-T reagent iodometrically (9), has wnfirmed that a,@-unsaturatedketones react more readily but that their hydrazones are hydrolyzed less rapidly. The Girard-T and P reagents have been widely used to separate ketones from such essential oils as vetiver and lavender essences. The a- and @-vetivonescan be separated using the fact that the n-isomer (V) was regenerated more readily from its hydrazone than the conjugated @-isomer. a-Ionone (TI) has also been sep-
arated from dihydro-a-ionone (VII) by hydrolyzing the Girard-P hydrazone of the latter at pH 5. More recently Girard-T reagent has been used on a microscale to concentrate and separate the carbonyls in orange essence oil, prior to analysis by gas chromatography (10). The ketones were conveniently regenerated by treating the Girard solution with formaldehyde (11). The concrete essence obtained by the petroleum ether extraction of jasmine flowers was repeatedly investigated by two groups of Swiss workers for the isolation of a ketolactone which occurs together with the principal constituents, which are jasmone (VIII) and methyl jasmonate (IX). The ketolactone was r e covered by first reacting with Girard-T or P reagent and then extracting the ketone fraction with sodium bicarbonate. Its content in oils from diierent sources varied from 4 to 32%, and it was shown to be 2-(5-hydroxy-cis-pent-2-eny1)-3-ketocyclopentylaceticacid lactone (X) (18).
Many chemical reactions involving ketones result in the formation of a mixture of ketone or non-ketonic compound as reactant or product. These mixtures can usually be separated by using a Girard reagent. Typically, the nnreacted starting material left in the reaction of a ketone with a Grignard reagent or with sodium Volume 45, Number 6, June 1968
/
435
acetylide can be removed by treatment with Girard r e agent. a-Ionone (VI) has been separated from its reduction product, or-ionol, by employing Girard-T r e agent. Methyl geraniol (XI, R = CH20H), on Oppenauer oxidation, formed the corresponding aldehyde (XI, R = CHO), which condensed with the acetone present to afford pseudoirone (XI, R = CH= CHOCHa). This compound was purified by using Girard-P reagent, and the purified ketone cyclized with acid to a mixture which was principally or-irone (XII), recovered through a Girard-T separation (6).
Aldehydes react very readily with the Girard reagents, in some cases without the addition of acetic acid. Girard and Sandulesco reported that Girard hydrazones of aldehydes were difficult to hydrolyze (6),but this is not substantiated by later work which indicated that hydrolysis occurred at room temperature (13). Some a l d e hydes are polymerized under the acid conditions of hydrolysis, but can be regenerated safely with formalde hyde (11). Low molecular weight aldehydes are not easy to extract from the aqueous solution, but can be recovered by precipitation as their 2-4-dinitrophenylhydrazones. An interesting preparative use of Girard-T reagent involved the hydrogenation of benzonitrile (PhCN) over a Raney nickel catalyst in the presence of this reagent when phenylacetaldehyde (PhCH,CHO) was obtained as its Girard-T derivative (14). Secondary reactions may occur during a Girard extraction. 8-(2, 6, 6-Trimethylcyclohex-2-enyl)octa-3, 4, 7-trien-2-one (XIII)
the fox-glove plant, which forms insoluble digitonides with 3 p-hydroxysteroids. Pregnone-3p,2Ocr-diol (XVI) has been isolated in this manner.
XVI
One synthetic route to androsterone (XVII) involved oxidizing cholesteryl acetate dibromide (XVIII) with chromium trioxide in acetic acid. The neutral fraction of the products contained a variety of products from which dehydroepiandrosterone (androst-5-en-36-01-17one) acetate (XIX, R = Ac) and pregnenolone (pregn5-en-3 6-01-20-one) acetate (XX) were separated using Girard-T reagent. Numerous other applications have been reported of the use of Girard reagents in separating steroid ketones from nonketonic compounds (5).
@
Br Br
HO-'
XVlI
rearranged to the corresponding fully conjugate ketone under the normal conditions of Girard separation, but formed the unrearranged hydrazone on reaction with Girard-P reagent in the absence of acetic acid. In addition, sterically hindered ketones such as benzophenone and camphor do not react with the Girard reagents, and appear in the "non-ketonic" fraction in such a separati%. The principal use of Girard reagents has been that for which they were originally prepared, namely in the steroid field (5). Mare and stallion urine contain estrogenic steroids. These are present in part as glucuronides which are hydrolyzed with acid or enzymatically. The phenolic estrogens are commonly removed by washing an organic solution with sodium hydroxide, and are then recovered on acidifying the alkaline extract. The phenolic fraction is separated by reaction with Girard reagent into estrone (XIV) and estradiol, the corresponding carbinol. The neutral steroids can also be separated into a ketonic fraction, largely pregnanedione (XV) and related compounds. The nonketonic fraction may be further separated by treatment with a digitonin, a steroid sapogenin found in 436
/
Journal of Chemical Education
XWI
RO XIX
A d
\
XX
In studies of the completeness of reaction of various steroid ketones with Girard-T reagent using Amberlite IRC-50 (H) resin as acid catalyst, (16), it was found that 3- and A4-3-ketones reacted completely, whereas 20- and 17-ketones reacted less completely. 11-Ketones are sterically hindered and do not react with Girard reagents. As a result a steroid which only possesses an 11-keto group appears in the "nonketonic" fraction in a Girard separation. Secondary reactions can also occur in some Girard treatments of steroids. Thus, the compound, 3-keto5-hydroxy-14iso-17-iso-21-norpregnan-1920-dioicacid 20 ethyl ester (XXI), underwent elimination of the 5hydroxyl group and decarboxylation of the 19-carboxyl function to afford 3-keto-14iso-19-nor-A4-etiocholanic
acid ethyl ester (XXII) on acidifying its Girard-T reagent at room temperature.
&& C02Et
/
H
xxn
XXI
A Girard separation is often used to assist in separating the complex mixture of steroids found in human urine. Such a separation is usually combined with the prior removal of phenolic steroids by extracting with base, and a fractionation of the 3a and 38 hydroxysteroids using digitonin. The amount of 17-ketosteroids excreted is a useful indication of the metabolic functioning of the body. These stemids are usually extracted with Girard-T reagent and determined colorometrically using m-dinitrohenzene in alkaline solution (the Zimmerman reaction) (6). The urine of a girl suffering from a corticoadrenal tumor contained ten times the normal amount of etiocholan-3a-01-17-one (XXIII) and one hundred times the normal amount of
xxv dehydroepiandrosterone (XIX, R = H). In many metabolic studies, it is convenient to administer a carbon-14 or tritium labeled steroid and follow the radioactivity in the subsequent isolation of the products. Thus, during pregnancy estradiol-16-14C is converted to labeled estriol (XXIV) and estrone (XIV). When labeled estradiol was administered to a patient with breast cancer, labeled 2-methoxyestmne (XXV) was isolated from the urine, and this accounted for 8% of the total radioactivity. Biochemical synthesis can also he carried out in vitro, and an ovarian tissue culture converted testosterone-3-14C (XXVI) to labeled estradial (XXVII) and androst-4-en-3, 17-dione (XXVIII). In all these examples the crude steroid fraction was separated into phenolic and uonphenolic fractions by extraction with sodium hydroxide. These portions were then separately treated with Girard-T reagent to affordketonic and nonketonic fractions.
o
w XXVIII
Excellent paper chromatographic separations of steroid ketones can he achieved by running the compounds in the form of their Girard-T derivatives. As little as 20 pg of steroid suffice and the chmmatograms are developed with n-butanol saturated with water. The positions of the derivatives are revealed by spraying the paper with iodoplatinate solution (16). Girard-T hydrazones, being quaternary salts, can also he separated by electrophoresis. The supporting electrolyte is usually 0.05 M sodium borate and the papers are suhsequently sprayed with Kraut-Dragendorff reagent (bismuth subiodide). Both methods afford qualitative means of identifying steroid ketones. Girard-T hydrazones have been found to undergo polamgraphic reduction (17). The derivatives of A'-3-ketosteroids have half-wave reduction potentials of - 1.25 v, and those of 17- and 20-ketones at -1.45 v. 3-Hydmxy-As-stemids have been analyzed polarographically after first converting them by an Oppenauer oxidation to the corresponding A4-3-keto-steroids. By using special mirocells, amounts as small as 1 pg (0.003 pmoles) of ketostemids can be detected in samples of urine or blood. With the advent of gas chromatography, the Girard reagents are finding less use in the separation of carbony1 components from essential oils. However, they are still widely employed in synthetic organic chemistry, and are extensively employed in the separation of both large and small amounts of steroid ketones, as well as for their qualitative identification and quantitative analysis. Literature Cited (1) O'CONNOR, R., J. CKEMEDUC.,42, 492 (1965). (2) GUENTHER, E., "The Essential Oils," Van Nostrand Co., 1949. Vol. -11.~0.~305. -~ - ,(3) FIESEE; LIF., AND FIESER, M., "Steroids," Reinhold Puhlishing Co., 1959. (4) WHEELER, 0 . H., Chem. Revs., 62,205 (1962). (5) WHEELER, 0. H., "Girard and Related Reagents," HoldenDay, Inc., San Francisco, 1967. (6) GIRARD,A., AND SANDULESCO, G., Helv. Chim. Acta, 19, 1095 (1936). . . (7) R E I C H ~ ~T., I NHelv , Chim. Acta, 19, 1107 (1936). (8) WHEELER, 0. H., AND ROSADO, O., Tetrahedron, 18, 477 (1962). . . (9) WHEELER, 0. H., GAIND,V., AND ROSADO, O., J. erg. C h a . , 26, 3537 (1961). D. F., MENDELSOHN, J. M., AND RONSIVALLI, L. (10) GODBOIS, J.. Anal. Chem.. 37. 1776 (196.51. , , (11) TEITELBAUM, C. L , J. Or$ C h . , 23, 646 (1958). A. V., AND DEMOLE, E., Helv. (12) NAVES,Y. R., GRAMPOLOFF, Chim. Aeta. 49. 1006 (1963). (13) LEDERER, E., AND NACHMAIS, G., Bull. Soe. Chim. Fmnee, 400 (1949). R.. Compt. Rendu, 253, 134 (14) GAIFFE,A., AND POLLAUD, (1961). ~ -- -,~ (15) LINDNER, H. R., Biochem. & Bwuhvs. . . Ada.. 38.. 362 (1960). . . (16) ZAFFBRONI, A., BURTON,R. B., AND KEUTMANN, E. H., J . Biol. Chem., 177, 109 (1949). (17) HERSHBERY, E. B., WOLFE,J. K., AND F T E S EL.~ F.,J . A m . Chem. Soc., 62,3516 (1940). ~~
A~
~
~
~
. .
. .
. ~.
Volume 45, Number 6, June 1968
/
437
