condensation iiicorporatiiig carboii-1-1 iiito carboiis side chain, which were then lost iri but recovered as isolated lignin. PIIENVLALAXINE, CINXAMIC ACID AND ACETATETO WHEAT However, after feeding acetate t o both species virtually inactive DHCX and DHSA were isolated AND MAPLE (Table I), and as these contain all nine skeletal carSp. activity --hlaterial recoveredof lignin bons, t h e above possibility was eliminated. The alSp. activity monomer (mpc./mmole) (mpc./ ternative explanation is t h a t acetate has been incod mmole) Compound corporated into some artifact associated with t h e administereda Name Wheat hlaple Wheat Maple isolated lignins, which does not yield vanillin atid i . - ~ ' h e n y ~ a ~ a n i n e - ~Klason ilignin '12 1 21.1 si+~b 222b e 1 4 Dioxane lignin :iY.n 1lj.3 400h 1i1+ syringaldehyde. These results underline the difVanillin-NUIId 29 C, 23 0 57OC .1HC ficulties involved if isolated lignins are employed Syringaldehyde- 2 8 , 8 10.7 SOO' 220c in lignification experiments. KBH~ Caution is needed when comparing t h e degrec uf ~ i n n a i i i i cacii1~3-Cld Klason lignin 30.0 .. 3.'ib , , Dioxane lifinin 2z.3 .. Zii8h . utilization of such differerit compounds as acetate Vanillin-NBHd 2O.!J , :il L and substituted phenylpropanes. There niay he Syringaldrhydc- 2 8 . 8 , . liil considerahlc difference in pool sizes, of course, aiid N1311d feeding of equivalent amounts of t h e two types of iwetic acid 2-c" filasnn ligniri :IX 1 7. I 7sh 1)imane lipnin a.7 12 o 12iih compouiid has been shown in this Laboratoryi1 Vanillin-XBHd 0.17 0 62 2 li 12 to yield data which may riot be closely coinparable. Syrinxaldehyde0 :Xi 1 2% 5.3 19.5 But even allowing for this, the present results inSBHd Wheat plants (equivalent to ca. 4..5 g. dry weight) were dicate that incorporation of acetate into t h e isofed 4.0 pc. carbon-14 in 0.079 mmole of the compound. lable aromatic ring or side chain of lignin is slight Maple twigs ( d r y wt. ca. 7.5 9 . ) received 6.5 pc. carbon-I4 Acknowledgments.-Microanalyses reported Calculated on the basis of the average in 0.10 mmole. number of carbon atoms per lignin molecule equalling 10.5 here were clone by LIr. J. A. Baign6e and analyses of (see text). Results are multiplied by 9/7 t o correct for carbon-14 by Mr. John Dyck. The technical asloss of two skeletal carbons during oxidation. S B H 3 M - sistance of Mr. J. P. Shyluk throughout t h e investinitrobenzoylhydrazone. gation is gratefully acknowledged. (21) J E. Watkin a n d A C Neish, unpublished d a t a Two possible explanations suggest themselves for this. I t could have been due to a c6,Cl C f SASKATOON, SASK., CANADA TABLE I\'
1 and 2 of the R AIIIOACTIVITY OF ISOLATEDLIGXIXS AND L I G N ~OXIDAN 1'10sPRODUCTS AFTER ADMINISTRATIONOF LABELED t h e oxidation
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[ ~ O S I R I I ~ ICU1N PROM TIIE I)EPARTMEST OF CIIEMISTKY A S D
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B O T A N Y , USIVERSITY OF CALIFORNIA, L O S
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Gibberellins from Flowering Plants. I. Isolation and Properties of a Gibberellin from Phaseolus vulgaris L.' IjY C l l l l R L E S
\VEST '1ND
BERSARD0 . P I I I K N E Y
RECEIVED OCTOBER 13, 1958
A procedure is dcscribctl f m the isolation of a gibberellin, beaii factor 11, from acetone-watcr extracts of immature bean seed (Phaseolus vulgaris L. ). Bean factor I1 call be distinguished from the fungal gibberellins, gibberellic acid, gibberellin AI and gibbcrellin A*, on the basis of its differential biological activity for dwarf maize mutants dwarf-1 and dwarf-5, its chromatographic behavior and its infrared spectrum. Preliminary chemical studies suggest t h a t bean factor I1 has a carboxylic acid and a lactone functional group, as well as sites of unsaturation not in conjugation with either of these.
The gibberellins, which are potent plant growth regulators,? were isolated as a crude crystalline mixture from culture filtrates of t h e vegetative stage of the fungus Gibberella fujikuroz' (Saw.) UTr. by Yabuta and Sumiki in 1938.3 Since that time four different gibberellins have been isolated in the pure state from t h a t source, either as a free acid or methyl ester: gibberellic acid (C19H2206) ; 4 gibberellin A (gibberellin A,) (C19H2406)'; gibberellin (1) This project \ras supported in p a r t b y grants from hZcrck and Co., Inc., S a t i o n a l Science Foundation ( G r a n t 352ti), and Research Corporation. 12) B. B. Stowe a n d T. Yamaki, , 1 1 1 u . Reg. Plant Physiol., 8 , 181 (1957). (8) T. Y a b u t a and li. Suiniki, J . A g r . C h e w . SOC.J a p a n , 14, l52G (1038). (4) P. J. Curtis a u d B. E. Cross. C h e m . I n d . (London), 1086 (1954). ( 5 ) F.H. Stodola, K . B. Raper, I). I. Fennell, H. F. Conway, V. E. Sohns, C. T. Langford and R. W. Jackson, Arch. Biochent. Biophrs., 54, 240 (19541; F. 13. Stodola, G E. N. Nelson and D. J. Spence, ibid., 66, 438 (1957).
Az (C1&?606) ;6 arid gibberellin Ah ( C I ~ H Z Oor~ C19) H2405) .' A structural formula has been assigned to gibberellic acid by Cross, et aZ.* Gibberellin
(6) N.Takahashi, H. Kitamllra, A . K a n a r a n d a . Y. Seta, XI. Takai, S. T a m u r a a n d Y . Sumiki, Bull. A g r . Citem. Soc. J a p a n , 19, 287 (1955).
( 7 ) K. Takahashi, I ' . Seta, H. Kitarnura a n d Y . Sumiki, ; b i d , 21, 306 (1957) (8) T 3 E Cross, J F Grove, J . hIachIillan a n d T. I' C . hlulholland, Prur. Cheni. Soc., 221 (1958).
May 20, 1959
GIBBERELLINS FROM FLOWERING PLANTS
2425
A1 has been shown to be identical with one of the isomers resulting when ring A is saturated.p A considerable body of evidence has been accumulated to support the idea t h a t gibberellins or gibberellin-like substances act as natural growth regulators or hormones in flowering plants. Extracts of many seeds,1°-12plant vegetative and plant tissue cultures1* are capable of inducing a growth response in plants indistinguishable from the response to gibberellin treatment. A t least some of the active substances in these extracts seem to differ chemically from gibberellin AI,gibberellin A2 and gibberellic acid as evidenced by chromatographic studies. l o Recently Macmillan and Suter have reported the isolation of gibberellin A1 from extracts of runner bean seed (Phaseolus multiflorus L.).19 Otherwise, none of the substances responsible for the biological activity of the plant extracts has been identified. Extracts of immature bean seen (Phaseolus vulgaris L.) were found to be relatively active in the bioassay for gibberellin-like substances, and the active principle of these extracts was found to be similar in chromatographic behavior to the fungal gibberellins.l0 For these reasons this source was selected for investigation of the gibberellin-like substances of flowering plants. Two crystalline compounds, called for convenience bean factor I and bean factor II,20have been isolated in small quantities from this source. Bean factor I is identical with gibberellin A1 in many of its properties and very similar to it in others. However, a positive identification of this factor must await the isolation of additional material and will be reported later. Bean factor I1 has biological, physical and chemical properties which distinguish it from the other reported gibberellins; its isolation and properties are the subject of this paper. T h e procedure employed for the isolation of bean factor I1 involved a number of purification steps. The gibberellin-like substances were detected throughout by means of a bioassay based on the growth response of dwarf mutants of maize. Immature bean seed was extracted with a mixture of acetone and water (1:l). The active material was adsorbed on charcoal from aqueous solution and subsequently eluted with acetone in a step patterned after the isolation of gibberellic acid from fungal filtrates.21 The eluate was chromatographed on a silicic acid: celite colunin developed
with mixtures of ethyl acetate and chloroform. Assay of the fractions from this column on different maize mutants, dwarf-1 (d-1) and dwarf-5 (d-5) revealed that a group of the early fractions was relatively active in stimulating d-5 growth but only slightly active for d-1, whereas a later group of fractions was relatively active for both mutants. The differential activity of the early fractions was unique since the fungal gibberellins and the gibberellin-like substances from flowering plants tested previously had been found to have approximately equal activity for both mutants. Thus, there were a t least two different gibberellin-like substances present in the bean extract, and one of these differed from the fungal gibberellins in its biological properties. Subsequent purification steps, which were carried out separately for the two groups of fractions, included chromatography on a charcoal : celite column developed with mixtures of acetone and water and a countercurrent distribution between the two phases of a n-butyl alcohol and aqueous ammonia buffer (pH 8) mixture. Approximately two milligrams of bean factor I , which was equally active on d-1 and d-5 and approximately two mg. of bean factor 11, which was a t least ten times more active for d-5 than d-1, were crystallized from the material recovered from the countercurrent distributions. A comparison of the properties of bean factor I1 and those of the fungal gibberellins suggests t h a t they possess many structural features in common. Figure 1 illustrates the many similarities in the
(9) J. F. Grove, P. W. Jeffs and T. P C. Lfulholland, J . Chem. Soc.. 1236 (1958). (10) B. 0. Phinney, C . A. West, M. Kitzel and P. h l . Neely, Pmc. N Q ~A.c n d . Sci.. L'. S. 43, 398 (1957). (11) M. B. Ritzel, Plant Physioi. (Suppl.), 3 2 , xxxi (1957). (12) A . Lang, J. A. Sandoval and A . Bedri, Proc. N u t i . Acnd. Sci. U . S . , 43, 960 (1937). ( 1 3 ) hf. Radley, N o L w e , 178, 1070 (1956). (14) A. J . RIcComb a n d D. J . C a r r , ibid., 181, 1348 (1F)S81. (15) R.Bunsow, J. Penner and R . Harder, h ' n l z ~ v w i s s . ,46, 46 (1958). (16) F. Lona, Atenec P a r m e n s e , 28, 111 (19.57). (17) VI. Radley, A U K Botany ( L c n d o n ) , 22, 297 (19,581. (18) I,. G. Nickell, Science, 1 2 8 , 88 (1958). (19) J MaciLlillan and R. J. S u t e r , Nnfurwiss., 46, 46 (1958). (20) T h e problem of a suitable nomenclature for newly discovered members of this class of plant growth regulators is under discussion with other workers in t h e field. (21) A. Borrow, P. W. Brian, V. E . Chester, P. J. Curtis, H. G. Hemming. C. Henehan, E. G. Jeffreys, P. B. Lloyd, I S Nixon, G . L. F. Norris a n d M. Radley, J , Sci. Food Agr., 6 , 340 (1955).
Frequency (cm.-I).
I
I
L 1800
I
1600
1400
I
1200
I
1000
I
800
Fig. 1.-Infrared spectra of gibberellic acid (upper curve) and bean factor I1 (lower curve). Samples were prepared by pressing approximately 300 g r a m s OF the compound and 50 mg. of potassiuni bromide into a small pellet. Spectra were obtained oil a Model 21, Perkin-Elmer recording infrared spectrophotometer.
infrared absorption spectra for bean factor I1 and gibberellic acid b u t also shows that distinct differences do exist. Table I summarizes the absorption maxima in the frequency range 1700 to 1800 cm.-l for gibberellic acid, gibberellin AI, and bean factor I1 and the methyl esters of these compounds. The positions of the maxima are consistent with the presence of a carboxylic acid
TARLE,
I
INFRARED ABSORPTIOSMAXIMA, 1700-1800 c h i . ' Supporting medium
Compound
Absorption maxima
1745 (broad) 1720 (shoulder) 1740 1717 1750 (broad) 1717 (shoulder) 176d 1730
Gibberellic acid
KBr
(;ibberellin AI
KRr
Bean factor I1
KBr
Gibberellic acid methyl ester
KBr
Gibberellin A I methyl ester
CHCla
1785 1726
Bean factor I1 rneth?-l ester
CHC13
1760 172j
and a y-lactone in bean factor I1 of types which are assigned to the fungal gibberellins. Other properties deterinimed for bean factor IT and the fungal gibberellins are collected in Table 11. Hydrogenation studies carried out on 100 to 200 microgram saniples in a 1I:arburg-Rancroft respirometer with palladium on powdered charcoal as catalyst suggest t h a t bean factor I1 and gibberellic acid possess the same degree of unsaturation; however, these results should be considered tentative because of the sample size and the lack of material for replication. The infrared spectra of both bean factor I1 and of gibberellic acid show two weak absorption maxima between 1600 and 1700 crn.-', which is again suggestive of two sites of unsaturation. TABLE I1 COMPARISON OF PROPERTIES OF FUXGAL GIBBERELLINS AND BEANFACTOR I1 Gibb. acld
Gibb. AI
Gibb.
Az
Hean fact. I1
Paper chromatographya (Rp,b) )t-Butyl a l ~ o h o l - a ~ ~ ~ monkc (1.0) 1 . O l . S 2.0 Pyridine-amyl alcoliol-materd (1.0) 1 . n t . 1 1.2 Rclativc activity on maize mutaIits d- 1 (100) 20 -15 d-5 (100) "0 1 90 Iluoresccnce in H2SOa BlueS o n e Xl'une None green Ratio of the According t o the procedure described.'O distance from the origin the compound had migrated t o the distance a gibberellic acid control sample had migrated on the same chromatogram. Gibberellic acid has a n average Rr of 0.25 in the butyl alcohol-ammonia system and 0.58 i n the pyridine-amyl alcohol-water system. Upper phase of a mixture of n-butyl alcohol and 1.5 ,If ammonium Iiydroxide (3: 1 ) . Upper phase of a mixture of pyriditiee n-amyl ;~lcoliol-water ( 3 5 : 3 5 : 30).
The failure of sulfuric acid solutions of bean factor I1 to show the characteristic blue-green fluorescence seen with gibberellic acid indicates the absence of the unsaturated ring substituted with hydroxyl and lactone functional groups of the type attributed to ring A of gibberellic acid. Gibberellin which differs from gibberellic acid in t h a t ring -1 is saturated, does not fluoresce appreciably when dissolved in concentrated sulfuric
acid. X inethanolic solution of bean factor I1 showed only a slight end absorption and no absorption niaxiniurn above 220 mp. Thus, unsaturated sites in conjugation do not appear to he present. The differential biological activity of bean factor I1 for the single-gene dwarf mutants of maize which respond to treatment with the fungal gibberellins may have implications concerning the biosynthetic pathway leading to the production of native gibberellins in maize. One interpretation of the stimulatory action of gibberellic acid and gibberellin XI on the growth of these mutants is t h a t they replace a native gibberellin produced by norinal plants but not by the mutants. I n terms of this hypothesis bean factor I1 might be an intermediate which can be cnnr-erted to an active gibberellin by d-5h u t not by & I . Thus, the genetically controlled eiizytnatic block i n 0-1would be between the production of bean factor I 1 and the active gibberellin. A inore detailed presentation of the biological properties of bean factor I 1 and their biochemical and genetical implications will be presented elsewhere. Experimental Bioassay for Gibberellin-like Substances.-Gibberellin activity was followed throughout t h e purifications by means of biological assays on two genetically different dwarf mutants of Zea ?izeq'sL., d-1 and d-5,according t o the procedure described.'O The material to be assayed was dissolved in 0.1 ml. of water containing 0.05% Tween SOz2and applied t o the first unfolding seedling leaf. After four or five days the lengths of the first and second leaf sheaths were measured. An increase in the length of either of these sheaths of 25'3 or more over the largest untreated dwarf control was interpreted as a positive test for gibberellin-like substances. -1 quantitativc assay was used to determinc t h e relative activities on the different mutants.23 Known weights of each compound were applied t o at least ten plants a t each of three concentration le\-els. After seven days the lengths of the leaf sheaths of treated plants were compared Statistically with non-treated dwarf controls. Isolation of Bean Factor 11. A. Extraction of Bean Seed. --.ipproximately 25.2 kg. (wet weight) of immature bean sced (Phaseolus i'ulgaris I,.) mas soaked in 50 1. of acetonewater ( 1 : l ) for a t least 12 hr. After the extract was removed by filtration, t h e seed was extracted twice more in a similar manner. Tlic ccitnhined filtrates were evaporated to about 40 I . ? n t'aciin. B , Charcoal Adsorption and Elution.--The residual solution from extraction was stirred with 250 g of charcoalz1 and 250 g. of celite25 for two Iir. T h e mixture was filtered and the cliarconl treatment was repeated on the filtrate. The combined charcoal-celite residues were washed 011 the funnel with 2.5 1. of water and were stirred with 5 1. of acetoneewater ( 9 5 : s ) for two hr. After t h e mixture was filtered the elution of the charcoal-celitc residue was repeated in a similar manner. One-millionth of the combined filtrates gave a strongly positive response in both the d-5 and d-1 bioassays. The volume of t h e combined acetone-water filtrates was reduced t o 4.3 1. in Z'UCUO. C. Ethyl Acetate Extraction.-The residual charcoalcclite eluate, buffered with potassium acid phosphate ( 5 7 . 3 g.) and sunicient 3 -11sodium hydroxide to adjust the pH t o 7, \vas extracted wit11 t w o portions of ethyl acetate ( 5 I . ) . This ethyl acetate extr ict, which showed only slight biol(igical activity, was tlisc ~rdctl. .!.fter the pH of the aqueous phase was adjusted t o 2 with hydrochloric acid (concd.), the wlution W : I S cxtrwtrd with four portions of ethyl acetate (22) Sorbitan polyoxyethylcne mimrilauratc. Obtained from Atlas Powder C o . , Wilmington, Delaware. (23) P . hZ. Neely and B. 0 I'hinney, Pliiiil Plrysiol. (Sulipl !, 32, xxxi (1057). (24) Darco G GO Vegetable Charcoal obtained from t h e hlatheson Co., Inc., East R u t h e r f o r d , S e w Jersey (2.5) Hyflo Super-Cel obtnined from Johns hlanville Corp.
THEhf ETIIYL'4TION
May 2U, 1959
(16 1.). Only a small amount of the biologically active material remained in the aqueous phase. The ethyl acetate extract, which was very active in the bioassay, was evaporated t o dryness i n vacuo. D. Silicic Acid Chromatography.-A mixture of 133 g. of silicic acid26 and 67 E[. of celite was tamped d r y into a column 3 X 90 cm. which was washed with successive portions of ethyl ether (75 ml.), ethyl ether-acetone (75 ml.), ethyl ether (50 ml.) and chloroform (100 ml.). T h e residue from the ethyl acetate extract at pH 2 was adsorbed on 15 g. of silicic acid-celite ( 2 : l ) , and the mixture transferred t o the column. T h e column was developed with chloroform (1 l . ) , increasing concentrations of from 5 t o 28y' ethyl acet a t e in chloroform (2.2 l.), 307, ethyl acetate in chloroform (10.4 1.) and ethyl acetate (1 1.). Seventy-eight 200-ml. fractions were collected. Evaporation of fractions 17-24 yielded 0.87 g. of a brown oil which was highly active in the bioassay for d - 5 but only slightly active for d-1. The active material in this fraction is referred t o as bean factor 11. Evaporation of fractions 25-53 yielded 1.14 g. of a brown oil \%hichwas highly active in t h e bioassay for both mutants, d-1 and d-5. The active material in this fraction is referred to as bean factor I. Fractions 54-73 also contained small quantities of bean factor I. E. Charcoal Chromatography.-Sixty-seven g. of charcoal and 133 g. of celite were slurried in water, poured into a column 4 . 5 c m . in diameter, packed under a small pressure head of nitrogen t o a height of 26 cm. and washed with water. T h e bean factor I1 fraction (0.87 g.) from silicic acid Chromatography was added in 15 ml. of acetone-water ( 1 : 1) t o t h e column which was then developed with 50% acetone in water ( I l.), increasing concentrations of from 60 t o 907' acetone in water (0.8 1.) and acetone (1.4 1.). Thirty 100-ml. fractions were collected. Evaporation of fractions 16-23 yielded 0.07 g. of a pale yellow oil containing bean factor 11. F. Countercurrent Distribution.-The fraction from charcoal chromatography containing bean factor I1 (0.07 g.) was distributed between 10 ml. of the upper phase and 10 ml. of the lower phase of a two phase system resulting from the equilibration of equal volumes of n-butyl alcohol and 0.02 JP ammonium hydroxide-ammonium chloride buffer (PH 8). These solutions were introduced into tube 0 of a countercurrent distribution and ninety-nine transfers were effected in this solvent system. The contents of tubes 16-40, which showed activity in the d-5 bioassay, were acidified t o pH 2 with hydrochloric acid (concd.), and the phases were separated. T h e lower phase was reextracted with several portions of n-butyl alcohol. Evapo(20) Merck reagent grade obtained from Rlerck and Co., R a h w a y , New Jersey. (27) hfodel 2-B, H . 0 Post Scientific I n s t r u m e n t C o , Maspeth, N. Y.
ration of the combined n-butyl alcohol extracts yielded 33 nig. of an almost colorless oil. G . Isolation of Crystalline Bean Factor 11.-The bean factor I1 fraction (33 mg.) from countercurrent distribution was dissolved in a c e t o n e w a t e r (95: 5), and the solution was passed over a small charcoal column. The residue (15 mg.) obtained on evaporating t h e column effluent was extracted with warm ethyl acetate, and the solution filtered. The volume of the filtrate was reduced t o 1 ml. in a stream of nitrogen and 1 ml. of petroleum ether was added.28 A flocculent precipitate separated on standing. I t was removed and petroleum ether was again added to incipient turbidity. On standing and cooling a crystalline precipitate separated. Two milligrams of bean factor I1 was recovered as platelets which melted with decomposition from 250255". Methylation.-N-Nitrosomethylurca (26 mg.) dissolved in ethyl ether was treated with 1 ml. of 50'Jo potassium hydroxide, and the diazomethane generated was distilled into ethyl ether according t o a procedure d e s ~ r i b e d . Ap~~ proximately 0.5 mg. of bean factor I1 was triturated with the diazomethane solution. T h e solvent and excess diazomethane were removed by evaporation. The solid residues mixed with a small quantity of Florisi130were slurried in petroleum ether28 and added t o a Florisil column ( 1 X 5 c m . ) . T h e column was developed with 25y0 ethyl ether (10 ml.), 75% ethyl ether in petroleum ether (10 ml.), ethyl ether (60 ml.), and 5%; ethanol in ethyl ether (60 ml.). The ethyl ether eluate and the first 20 ml. of the 57' ethanol in ethyl ether eluate were combined and evaporated t o dryness. The oily residue was dissolved in 10 microliters of chloroform and transferred t o a microcell for the determination of infrared absorption spectrum. T h e methylation of gibberellin AI was carried out in a similar manner.
Acknowledgments.-The authors wish to thank Mrs. Joseph Rogers for technical assistance and also Frank H. Stodola, Northern Regional Research Laboratory, U . S . D . A . , Peoria, Ill. for a supply of gibberellin A1 and gibberellic acid, James Merritt of Merck and Co., Inc., Rahway, New Jersey for supplies of gibberellic acid and methyl gibberellate, and Y . Sumiki, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan for a supply of gibberellin Az. (28) Skellp Solve B, boiling range GO-8O0. (29) "Organic Syntheses," Coll. Vol. 11, John W l e y a n d Sons, Inc., I i e w York, N . Y . , 1963, p. 165. (30) A synthetic adsorbent obtained from Floridin C o . , Tallahassee, Florida.
Los ASGEI.ES, CALIFORSIA
1JOINT COXTRIBUTION FROM THE DEPARTMEST OF CHEWSTRY OF WAYNE STATE UNIVERSITY ASD AGRICOLA,
MINISTERIO DA
2427
O F PYRONONES
THE
INSTITUTO DE QUIMICA
AGRICULTURA, R I O DE JANEIRO]
Naturally Occurring Oxygen Heterocyclics.
1V.I The Methylation of Pyronones*
BY DAVIDHERBST, WALTERB. MORS,OTTORICHARD GOTTLIEBAND CARLDJERASSI RECEIVED NOVEMBER 10, 1958 The contradictory reports in the literature on the diazomethane methylation of pyronones are reviewed and it is shown t h a t such treatment of 6-methyl-2,4-pyronone (triacetic lactone) and 6-phenyl-2,4-pyronone leads in each case to a chromatographically separable mixture of the 2-methoxy-y-pyrone and the 4-methoxy-a-pyrone. These results have a bearing on the structures of several naturally occurring pyrories and attention is again directed to the utility of infrared measurements in settling this point.
Recently, we have reported the isolation from different species of rosewood (Aniba species) of (1) P a p e r 111, P . Crabbe, P. R . Leeining a n d C . Djerassi, THIS J O U R N A L8,0 , 5258 (1958). T h e present paper should also be considered P a r t I V in t h e series "Chemistry of Rosewood." For p a r t 111 see ref. 4. (2) T h e work a t W a y n e S t a t e University was supported b y g r a n t number H-2574 from t h e h'ational H e a r t I n s t i t u t e of t h e National I n stitutes of H e a l t h , U. s. Public Health Service, while t h e investigations
anibine (I), 4-rnethoxyparacotoin (11)3 , 4 and 5,6dehydrokavain (111). 4 The skeletal structures were established rigorously by the course of the alkaline degradation, but the alternate isomeric in R i o d e Janeiro received financial aid from t h e Conselho Nacional de Pesquisas, Brazil. (3) W . B. Mors, 0. R . Gottlieb and C . Djerassi, THISJ O U R N A L , 79, 4507 (1967) ( 4 ) 0. R . Gottlieb and W. B. Mors, J . Oug. Chem., 24, 17 (1959).
