Isolation and Identification of Flavor Components of Burley Tobacco

!I I

ISOLATION AND IDENTIFICATION OF FLAVOR COMPONENTS OF BURLEY TOBACCO

By DONALD L. ROBERTS' and WILMER A. ROHDE' Research Dept., R. J. Reynolds Tobacco Co., Winston-Salem, N.,C. 27102 Tobacco Science, 1972, 16-34, p. 107-112, ISSN.0082-4523.pdf

Silicone DC 710 or 15% Silicone GE XE60 on Chroma Investigations of the constituents of the chloroform extract sorb W (acid ;,,ashed). Liquid cbromato,grap_hic col and the essential oil of good quality burley tobacco were made with an emphasis on the fla-vor components. The chlo,rorform ex umns were run using as adsorbents. Ma.llmckrodt tract gave primarily compounds which had li~t!e or no f!avor; Analytical Reagent Silicic acid o,r Merck Alumina in most of the compounds isol,ated were not sufflc1ently volatile to 20-rnm ID columns or ill some cases 45-mm ID columns. be good flavorants. A second source of burley constituents, the essential oil, which has a rich burley flavor, was investigated by Infrared spectra were run neat, unles,s otherwise distillation and gas chromatography. The compo:unds isolated noted on Perkin-Elmer 21 and 221 infr:ared spectro were identified by comparison of the IR, UV, NMR and mass spectra with those of authentic materials or closely related com photo~eters. NMR spectra were run on Varian HR60 pounds. The isolation and identification of 105 compounds from and AGO spectrometers using deutero,chloroform as the the extract and essential oil of burley tobacco are repo~ed; 56 solvent and tetramethylsilane as the internal standard. of these are reported for the first time as tobacco constituents. Eleven of the compounds are new chemical entities. Flavor The NMR signals are given in tau values with the pro properties of many of the new isolates ar.e gi\/en. ton count in parentheses. Mas,s data were obtained us ing a CEC Model 21-103G Mass Spectrometer with a heated inlet system, The ultraviolet spectra were ob INTRODUCTION tained -with a Beckman DK spectrophotometer. Although extensive work has been done on the con All melting points were determined using a Fisher stituents of various types of tobacco, the compounds Johns melting point apparatus and are uncorrected. responsible for the flavor of these tobaccos have not Elemental analyses were performed ,by Huffman Micro been elucidated. Stedman, in his excellent review of analytical Laboratories, Wheatridge, Colorado. tobacco and tobacco smoke constituents ( 47), lists a large number of compounds which have been isolated EXPERIMENTAL from tobacco-. In this paper we wish to report the com Chloroform Extraction of Burley Tobacco pounds which we have found in burley tobacco, many Preliminary Separations. A total of 187 pounds of of which are new to the tobacco literature and natural aged burley tobacco, USDA grade B4K, was shredded products in general. and packed in a steam-jacketed stainless steel eolu~n Two approaches to if ,olating the constituents of bur in three separate extractions. Hot chloroform was cir ley tob8.cco were taken. The first was extraction of the culated through the tobacco at a column temperature tobacco with chloroform and fractionation of the ex of 50 ° C. The so.Jvent was pumped through the tobacco tract by molecular distillation and liquid chromatog at a rate o,f 2-3 gallons per minute for 2.5 hours and raphy. T'he second was e·xamination of the essential oil was then drained from the column and concentr,ated 0_f burley tobacco by fractional distillation followed by by distillation. The recovered chloroform distillate was liquid and gas chromatography. Some of the results collected, returned to the column, and again conta?ted of the fiTst procedure have been reported previ at 50° with the tobacco for 2.5 hours. The combrned ously (27, 36, 37). extracts were concentrated and worked up as shown in Figure 1. . METHODS AND EQUIPMENT Fraction of Acidic Material 1-D. The molecular d1s u !he gas chromatographic · separations were made ti1late 1-D-. was vacuum distilled thr,ough a 6" micro sing F & M Model 770 Preparative Gas Chromato Vigreaux column and the following compounds were graph and F & M Model 500 Gas Chromatograph. The isolated and identified: a-methylbutyric acid, /l-methyl ~?lunms used were 8' x ¾" OD Stainless Steel prepara butyric acid, n-valeric acid, n-hexanoic acid, phenyl re columns packed with 10% LAC-446 on 60-80 acetic ,add and benzoic acid. f romosorb W (acid washed) and 10' x ¼" OD Stain Frciction 1-R, Isolation of Components. The residue ess Steel columns packed with 10% LAC-446, 10'% from the molecular distillation, 1-R, (41.5 g) was cbro matographed on silicic acid using benzene-ether mix ~~~af~om a,// correspondence should be addressed. 2 Present a-Jdress: ARS tures. Further separations of the subfractions resulted tores Laboratory, Olustee', Fla.. 32078.

(Tobacco Science 107) The 900 g of material 3b was chromatographed on Ilurloy Tobacc.o 187 lb. silicic acid using four fr" colunins. The corre'sponcting fractions from the four columns were then co-mbine Ether Layer solved solid at room temperature. The pentane !Solution j 10% NnOH Extraction was chilled at -20 ° C overnight. The mushy solids that precipitated were identified, according to the IR 1spec. trum, as principally solanesol. 1. Addify Removal of pentane from the filtrate provided ca. 157 2. Extraetwith ether, g of oil. Molecular distillation and chromatography of Acidic Ether Layer this fraction provided suibfractions from which pal }faterial 1 Concentraled mitic acid, stearic acid, unidentified impure long chain Vscooo D'scUko )Acecooe on Rotary ~'.olecular Solution acids, hydrocarbons, so1aneso1 and sterols were isolated. I scill at 70° -w• c Tyrosyl Nonacosanoate (II). Tyrosyl nonacosanoateTobacco Science, 1972, 16-34, p. 107-112, ISSN.0082-4523.pdf was recrystallized from ethanol or acetone as a white, Re,;a~n,ee S,h;;;'::::,,,~. . waxy solid, mp 74-75°. The infrared spectrum (melt) }!at~rial Material - Solids (discarded) 1-R 1-D had hands at 3175, 1718, 1600, 1515, 826 and the dou L Hexane Solutioll (Hot) 2. -20° C blet at 727-716 cm-' characteristic o,f a long-chain meth ylene group. NMR spectrum-r=2.95-3.4(4), 5.91(2), 7.26(2), 7.8(2), 8.75(50) and 9.1(3). Anal. Calcd. for

lkxanc C,,Ho,O,: C, 79.51; H, 11.90. Found: C, 79.6; H, 11.6. SoluLion Ine,olub]~s (Oils) Methanolysis o,f the material gave methyl nonaco tlntcrial 2 lfateriol 3 sanoate and tyrosol. A negative ferric chloride test in Figure 1. Extraction and separation into basic, acidic and neutral dicated that the phenolic hydroxyl is the one esterified materials. by nonacosanoic acid. Isolation of Volatile Burley Tobacco Constituents. The condensate (approx. 330 gal) from the steaming of about 1500 lb of aged burley tobacco (USDA grade C3M) was worked up as shown in Figure 2. The ether in the isolation -of benzoic acid, phenylacetic acid, diluted neutral material (60.59 g) was vacuum dis vanillic acid, azelaic acid, vanillin, and sco,po,letin. tilled through a 36" Nester-Faust spinning band col Separation of Components of Neutral Material 2. umn. Twenty nine fractions plus a cold trap frac The hexane-soluble neutral Material 2 was passed tion were obtained over a boiling range of 30°-130°/ through the rotary molecular still at 105° /0.1 mm for 0.2 mm, including the 14.9 g of pot residue (95% re an initial separation o,f the volatile components. This covery). The acidic material was similarly vacuum distillate (110 g), which had a ~ich, sweet aroma, was distilled and se·parated into four fractions and a pot I dis,solved in hexane and chromatographed on a 7.5 cm 1 residue. ' column (2 kg. of Merck acid-washed alumina). Elution with hexane, hexane-ether mixtures, ether, and 5 % methanol-ether, gave -cuts which were combined accord ing to similarity o,f the- infrared spectra to provide six Table 1. Burley tobacco isolates from chloroform extract fractions. Further fractionation furnished the com Hydrocarbons Reference$ Aliphatic paraffins 47 pounds: paraffinic hydro-carbons (47), neophytadiene Neophytadiene 33 (33), crystalline normal long chain alcohols (mainly Esters Waxy solids of mixed esters (n-long chain) 47 docosanol), a- and /3-3,8,13-duvatriene-l,5-diol (36), a Solanesyl esters 47 Tyrosyl nonacosanoate (II) a.,b and /3-4,8,13-duvatriene-1,3-diol (27), 1,4,8,13-duvate Alcohols traene-3-ol (I), dihydroactinidiolide ( 4), 5-isopropyl- Docosanol 47 o:- and /3-3,8,13-Duvatriene-1,5-diol 36 3-nonene-2,8-dione (10), and a mixture of stigmasterol °'" and /3-4,8, 13-0uvatriene- I ,3-dio,I 21 and /3-sitosterol. 1,4,8,13-0uvatetraene-3-ol (I) n,b So/anesol 47 1,4,8,13-Duvatetraene-3-ol (I). Compound I, mp 126- /3-Sitosterol 47 l Stigmasterol 47 I ~r, was :identified from its spectral prioperties and the Acids structure was confirmed ,by synthe1sis from a-or ,B-4,8,- o:-Methyl butyric 47 Valerie 47 13-duvatriene-1,3-dio,1 by dehydration with magnesium /3-Methy[ valeric 47 Hexanoic 47 sulfate. Spectra.! data: IR-3190, 1654, 1287, 1256, 1212, Azelaic 47 1005, 988, 967, 892, 850, 808 and 768 cm-': UV-1' Myrisfic 41 Palmitic 47 l max. (EtoH) 245 nm (,=14,000); NMR-,=3.92 (1), Stearic 47 Unoleic 47 i 4.5(2), 4.95(2), 8.1(3), 8.26(3), 8.45(3), and 9.12(6, Benzoic 41 split doublet); Mass-M• 288. Phenylacetic 41 Vanillic 41 Solvent Partitioning of Materials 3, 3b and 3c. The Miscellc:ineous Scopoletin 47 hexane-insoluble fraction 3, approximately 2 liters of Vanillin 47 syrup, was partitioned between hexane and 95% Norsolanadione (VI) n IO Oihydroactinidiolide 4,' MeOH-H,O. The hexane layer gave 900 g of Material Nicotine 47 3b while 75 g of the more polar Material 3c was obtained a A new tobacco isolate. from the aqueous methanol layer. b A new compound.

(Tobacco Science 108) II' I :1 on Table 2. Isolates from burley tobacco essential oil .mg Flovor References Flovor References Compound 11ed <-'(;ofllPOUnci LACTONES ,ACIDS • 47 4-Hydroxy-3-methyl valeric smoothing burnt sugar 44 1:on Benzoic Acid weak sweet butter, cream 47 acid ,y-lactone a 41 Heptanoic Acid 47 2-lsopropyl butyrolactone a weak mint waxy, cream, maple sweet, light 41 l,ar Hexanoic Add sweet, winey, fruity, adds body 47 3-lsopropyl butyrolactone" 45 lsovaleric Acid . 3-lsopropyl-ll-valero!actone a coconut, sweet, smoothing Acid a clove, spicy II tra ns-5- Methy I-2- hexeno I c adds body, weak , of fatty, waxy 47 Phthalide a 48 Non,anoic Ac_id • 18 ,y-Valerolactone" light, sweet cis-3-Nonenoic Acid a fatty sweet, waxy, smoothing 47 KETONES ,0% Qctanoic Acid earthy, r.r,ushroom, adds body 31 2-Acetony 1-3- isop ropy 1-6- I 4-Qxonon<1noic Acid a m ethyltetra hydro pyra n sweet, honey, floral, adds body 47 sweet, phenolic, woody 38 !had Phenylacetic Acid fatty (2 is·r::imers) {VIII) a,b i; of Sebacic Acid a 2-Acetony 1-4- isopropyltetra hy- d rofu ran {XVI) a,b ANHYDR!MES sweet ho!ley, hay like, adds body 16 I \dis Dimethylcnaleic !',nhydride, 2-Acetonyl-4-methy ltetra hy- 43 Methylethylmale1c Anhydride burnt sugar, sweet, adds body 16 sweet, fruity ition Ai:f~t! ~o~ e sweet, pungent ketonlc ALDEHYDES almond, cherry 47 , 4-{1 3-Butadienyl)-3,5,5· ' chat Ben:z:aldehvde 2 2, 4- Di meth oxy be n:z:a 1de hyde sweet, hay like tn m ethyl-2-cyc Io hexen• 25 pee- vanilla, sweet 47 I-one (XX!) 11 spicy, peppery, .idds body Vanillin 4- (2- Buteny 1i dene-3,5,5-tri ALCOHOLS weak floral, smoothing 47 methy l-2-cycloh exen- I - 34, 35 157 Benzyl Alc:ohol 47 one (4 isomers) (XX) a spicy, peppery, adds body n-Docosanol Geranylacetone green .4 8 13-Duvatriene-l,3-diol 27 ? of 11 Octahydro-2 5,5,Ba-tetra- ' 13.4' s' 13-Duvatriene-l ,3-dlol 27 28 36 methy\-7-chromanone {XXIV) a camph·oraceous pal (1-3 '3 '13-Duvatriene- l ,5-diol 3,3a ,4,5,7, 7a-Hexa hydro-4,4, fj-3°,a', 13-Duvatri ene-1,5-diol 36 :iain green leaf like 7a-trimethyl-6 {2H) 20 cis-3-Hexenol " ben:wfuranone (XVII) a,b mild 6-Hyd roxy-4, 4, 7a -tri methyl-oda- -ted. 3-Hyd roxy-2,3,4,5,6,7- Tobacco Science, 1972, 16-34, p. 107-112, ISSN.0082-4523.pdf hyd robe niofu ran (XVIII) a,b slight mild 20 floral, citrus h exa hyd ro-3 ,3a, 7, 7-tetra- oate Linalool 0 methyl-ind en-2-one (XXV) a,b sweet green, sweet 43 6-Methyl-5-hepten-2-ol " 2- H yd roxy-4-ketoisophoron e hite, " slight nutty, chocolate, adds body 32 c,-5 8-Oxido-3, 9{ 17), 13- 37 {XIII) a ielt) duvatriene-1-ol 4-Hydroxy-2,2,6-trimethy! dou /3-5,8-Oxido-3, 9{ 17), 13- 37 cyclohexanone (IX) ~ weak, ketonic duvatriene-1-ol lsophorone n earthy, adds body " ·1eth c,-5 8-Oxido-3, 9, 13- 37 3-1 so prop e nyl cycl o pentyl " d'uvatriene- \-ol 47 methyl ketone (XXV!I) a,b sweet pungent (2), Phenylethyl Alcohol flora!, rose floral, smoothing 5-lsopropyl-2-methyl-1,3- Solanol {Vil) a,b nonadien-8-one (Solanone) for smoothing ketonic 9, 10 ESTERS (Ill) sweet-sour, citrus-floral .G. Linalyl Acetate 5-1 sopro py!-3-none n-2 ,8-d i one IO Methyl Tridecanoate {Norsolanadione) [Vl) a sweet ketonic 1.aco 4-Ketodihydroisophorone (XI) 11 weak, sour NITROGEN HETEROCYCLES char.;der :t in- floral, green, winey, adds body 19 4-Ketoisophorone (X) " sweet. ionone " 2-Acetylpyrrole 15 2-Formyl pyrrole sweet, smoothing 4-Keto-2 ,6, 6-trimethyl- " 1·ified 30 cycl o hex-2-en yl id ene lndole adds body, enhances taste 20 sweet, imitation cherry-like a ceta 1de hyd e (XV) a 5- M ethyl-2-Acetyl pyrrol e sweet sweet, cherry, adds body 22 Maltol 11 1-Methyl-2-Formylpyrrole o. " sweet, aromatic, spicy, adds body 42 't:mts. 5-Methyl-2-F-ormylpyrrole cherry, adds body 5-Methyl-2-acetylfuran n popcorn 21 , 4-Methyleneisophorone {XIV} n isophorone-like "Ding 2-Acetyl-6-methylpyrazine a " ketonic, smoothing 43 21 6-Methyl-2,5-heptanedione a " 2-Acetylpyrazine a popcorn :rade 2,5-Diphenylpyridine ~ 4,5, 7, 7a-Tetrahydro-4,4,7a• trimethyl-6 (2H)-benzo pleasant, mild 20 ther- LACTONES 48 furanone (XVI} a,b 1-Caproladone" nutty 4a ,5,6,7-T etrahydro- I ,4a• dis coconut 48 '}'-Capryloladone a 4, 8 d i m ethyl-4-hyd roxy-7-i so- Dihydroactinidiolide slight cooling I col pro p e ny l-2 (BH)-naphthalen• 23 4-Hydroxy-4-methyl hexanoic one { 1-keto-a-cyperone} qreen, sweet 44 :!'rac- acid ,y-ladone a · 4,4a,,5,6-Tetrahydro-4, 4, 7- 5-Hydroxy-4-methyl hexanoic trimethyl-2 (3H}-naphthalen- 30° / acid 6-ladone n one (XXll) a pleasant, weak, green, woody 13, 2't 4. Hydroxy-4-m ethy 1-5-hexen oi c ' t re weak, fruity, mint 44 3,4,7,8-Tetrahydro-4,4,7- acid ,y-ladone a trimethyl-2 {6H)-naphtha!en- ;uum 2-Hydroxy-2,6,6-trimethyl- one (XXIII) n,b weak woody 3-cycl o hex eny 1-a ceti c HYDROCARBONS l pot acid, ·y-ladona (XIX) a spicy, sweet fruity 20, 4 47 Aliphatic paraffins 33 4-Hydroxy-2-methy! valeric Neophytadiene acid ~,-ladone a mint 44

~ New tobacco isolate. Ne-,.v chn11irnl compound,

~rences 47 3l 47 =3.94 (1, q), 4.60 (1, q), 5.11(2), 6.25(1), 8.14(3), 47 The fractions from both the neutral and acidic ma terials were further separated by liquid chromatog 8.83 (3), 9.11 (6) ; Mass-M• 196. 47 raphy and by preparative and analytical gas chro 2-Acetonyl-3 -isopropyl-6 methyl tetra hy dr opyran 36 (VIII). This material consisted of two diastereoisomers 27 n1atography. The materials obtained from the liquid chi:omatography fractions or trapped from the ana of VIII separable by gas chromatography; their struc 47 47 ~Yhcal gas chromatograph were analyzed by obtain tures were determined by synthesis from solano,1 (38). 41 mg their m, NMR, UV and mass spectra. The com Spectral data: (isomer I) IR-1709 and 1081 cm-'; 47 P.0unds isolated are given in Table 2 and were iden NMR-T=6.43(2), 7.39(1), 7.50(1, d), 7.80(3), 8.87(3, 47 47 tified by comparison of their spectra with literature d), 9.08(3, d), and 9.20(3, d); Mass-M• 198. (Isomer 41 II); NMR-T=5.96(2), 7.30(1, d), 7.40(1), 7.82(3), 47 ~pectra or with spectra from synthetic samples. 47 Vhere authentic samples were available comparisons 8.85(3, d), 9.05(3, d), and 9.15(3, d). 47 47 of gas chromatographic retention times were. also 4-Keto-2, 6, 6-trimethylcyclohex-2-enylideneacetalde 47 hyde (XV). Compound XV was identified by comparison 47 lllade to establish identity. 47 with a synthetic sample (20). Spectral data: IR-1660, 47 New Compcnmds 1620, 1578, and 119 cm _,; UV - )c max. (EtOH) 281 47 nm (,=17,200); NMR - T=0.85(1), 3.86(1), 3.97(1), 47 Solano! (VII). Sodium borohydride reduction of so IO 7.60(5), and 8.7G(6); Mass - M• 178. 4, 8 lanone (III) gave solanol (5-isopropyl-2-methyl-1,3- 47 :inadien-8-ol) which was identical with the isolated 4, 5, 7, 7a-Tetrahydro-4, 4, 7a-trimethyl-6(2H)-ben crninr,ound. Spectral data: IR-3280, 1603, 967, 877 zof,uranone (XVI). The structure of XVI was estab , UV-!c max. (EtOH) 230 nm (,=320); NMR-T lished by conversion of XV into 2,4,5,6,7,7a-hexahydro-

(Tobacco Science 109) structure XXVI was assigned to .this isolate. SpectraJ data: IR - 1718, 1160, and 1088 cm-'; Nlv!R _ r=5.97(1, m), 6.35(2, d), 7.45(2, d), 7.85(3, s), 8.32(4, m), and 9.08 ( 6, q) ; Mass - JvP 170. The compound 2-acetonyl-3-isopropyl tetrahydrofuran V was prepared from 1-(3-carboethoxy-2-isopropylpr0• panoyl) aziridine by LiAlHt reduction to 2-hydroxy.3_ I< isopropyl tetrahydrofuran and subsequent base-cata. cnc1 Extract"-bles Aqueous Layer 3 R Discard lyze-d condensation of the hemiacetal with acetone. Spec. l. Hexane 1 ; C 2. Extrac~ed w;ith tra! data: IR - 1710, 1170, and 1080 cm· NJVIR - ,co 5% Na.OH 5.30-5.83(1, m), 6.40(2, t), 7.23(2, m), 7.70(3, s), t, 8.10(4, m), and 9.06(6, q). i1 5 J!e1

(Tobacco Science 110) Lral were totally new compounds and were identified as the suggests that they ,also may be degradation products macrocyclic diterpene 1,4,8,13-duvatetraene-3-ol (I) of solanone. ,, ( 4, and tyrosyl nonacosanoate (II). Very few compounds having flavorant properties --an were found in the chloroform extr:aiet because of their O o, JJ0 c , p l)l"Q ~ 1 loW concentrations relative to the lipid materials present 0 m q //-3- in the extract. In order to reduce the problems attend °'' ;ta. ant with separating the lipids from more flavorful " lJeC- components, an alternative to- solv_ent extracts of 1,~ tobacco was sought. The condensate from the steam ~~ ~ I js), ing of burley tobacco gave the desired result; ex ' 0 ' l traction of acidified steam condensate with chloro " '1) bed form provided an essential oil rich in burley tobacco flavor. ~,( '09, The vrnrk up of the steam condensate is shown m "' in Figure 2. The method of isolation and identification ,n VIII in nearly every case involved trap,ping the pure com me ponent from an analytical gas chromatograph and Yet another series of compounds found in burley ·ras obtaining IR, UV, NMR and mass spectra. The char tobacco is a group which may be considered as caro ire acterizations were then made by comparison of the tenoid degradation products or as isophorone deriva lell oo spectra vvith those of authentic samples, except in a tives. The group provides an interesting study in Tobacco Science, 1972, 16-34, p. 107-112, ISSN.0082-4523.pdf few cases where the compound was not available but the biosynthetic pathways operational in tobacco GO an isomer was synthesized for -comparison purposes. plants during growth and curing. The compounds lUr The compounds which were identified by these tech which have been identified in this series are shown .n- niques are shown in Table 2; the Table also gives an in Figure 3. Several proposals have been made on a 40 indication of the flavor effect associated with many the bi6synthesis of compounds similar to those in of the compounds and whether or not a given com the Figure (5, 8). Recently, a number of the isophor 1ed pound has been reported previously as a tobacco iso one-related compounds were rep-orte-d as constituents ne late. In making the flavor evaluations tobacco was of saffron (49) but the Crn·C,. compounds have not its treated with an ethanolic solution of the selected com been reported previously as natural products. The ne pound to give residual amounts of less than about synthesis of a number of these compounds will be 0.1 percent by weight of the compound with respect discussed in a forthcoming publication. Some C13 ed to tobacco. The tobacco was then smoked by a panel compounds related to rthose in Figure 3 which have us. of experts and compared with a control sample. been reported as burley tobacco constituents are re Several families of compounds were found in bur damascenone, jl-damascone (8), and 4-keto-a-ionol (1). Jm ley tobacco. One of the most important of these in the The compounds XX and XXI were probably formed de by dehydration of 4-keto-a-ionol during the steam g) flavor area is the pyrazines; two pyrazines, 2-acetyl ing of the tobacco; this dehydration may explain yl- pyrazine and 6-methyl-2-acetylpyrazine, were identi fied in the burley condensate. These compounds have why the ionol was not isolated from our steam con densate. of potent aromas reminiscent of buttered popcorn. Neu ,yl rath, Dunger, and Kustermann reported in 1971 the One of the eleven new chemical ·entities described 1g', identification of a large number of alkyl and fury! in this paper is 2-acetonyl-4-isopropyltetrahydrofuran 2d pyrazines in tobacco smoke (17). Another family of n nitrogen heterocycles found was the pyrroles; six o pyrroles, including indole, were identified. Acetyl e- pyrrole has been known as a tobacco constituent for 3. some time (19), and recently Luttich, in a patent, has ,o J::t· ,ca,¢ ~ndicated that other 2-acyl pyrroles are also present a: d- o, 1~ ~obacco (15). The acyl pyrroles possess aromas similar to the aromatic aldehy,des such as benzalde X xn XIII hyde, but ·sweeter and heavier. Another heterocycle which has been isolated and which has useful flavor OJ n Properties is 2-acetyl-5-methylfuran. In addition to d these compounds, the lactones and ketones are im portant contributors to the overall flavor of burley .e tobacco. ;5 XVII A series of the- anomalous terpenoids related to t s~lanone (III) (10) have been found in tobacco and f b present the compounds appear toi be unique to 0 '0 acco. Previously, the duvane (cembrane) diter benes and their open chain analogs (IV, V) have /:n reported as tobacco ,constituents (7, 12). These XVIII m 1 erpenes, and particularly the open -chain com = ;ounds, have been proposed as solanone precursors. b our additional derivatives of solanone have now n e~~ isolated from burley tobacco; these are norsola i a tone (VI) (10), solano! (VII) and two diastereo Psomers of 2-,acetonyl-3-isopropyl-6-methyltetrahydro /ran (VIII). Compound VIII is the norketo deriva- 1:ve of VII which exists in the cyclic form. Examina Figure 3. Corotenoid degrodotion products from burley tobocco. Ion of the structure of some of the other isolates

(Tobacco Science 111) (XXVI). Compound XXVI is somewhat anomalous Pheitooxygenation senslbilisee de l'homosafranate de methyle Helv. Chim. Acta 51: 481-494. 1968. , · since the 2-acetonyl-3-isopropyltetrahydrofuran struc 5. Burden, R. S., and H. F. Taylor, The Structure and Chernicai Transformations of Xanthoxin. Tetrahedron Letters: 4071-41}74 ture would be the one expected if the compound were 1970. . derived from solanone. The 4-isopropyl substitution 6. Colonge, J., M. Costantini, and M. Ducloux, Sur la P:repa:ra- tion des Alcoyltetraehydropyrannois-2. Bull. Soc, Chim, France suggests that XXVI may be derived from 3-isopropyl 1966: 2005-2011. 7. Courtney, J. L., and S. McDonald, A New C20 a,/3-Unsatur butyrolactone and that the 3-isopropyltetrahydrofuran ated Aldehyde (3, 7, 13-Trimethyl-10-isopropyl-2, 6, 11, 12-tetraae: analog may yet be found since both isopropyl butyro catetraen~l-al) (I) from Tobacco. Tetrahedron Letters, 495-466 1967. ' lactones were isolated. 8. Demole, E., and D. Berthet, Identifi.cation de la :qamascenone et de la /3-Danmscon dans le Tabac Burley_ Helv. Chim. Acta 54: The monoterpene 3-isopropenylcyclo,pentyl methyl 681-682. 1971. ketone (XXVII), although previously prepared by Set 9. Griffith, R. B., R. R. Johnson, and A. D. Quinn, Org-anolep tically Improved Tobacco Product U, S. Patent 3,174,485 .Mar. 23 tine, Parks and Hunter by Lewis acid treatment of 19C5). ' 10. Johnson, R. R., and J. A. Nicholson, The Structure, Chemis limonene oxide ( 46) and by Leffingwell and Shackel try and Synthesis of Solanone. A New Anomalous Terpenoid Ke ford from pyrolysis of 1-hydroxyneodihydrocarvyl tone from Tobacco. J. ,Org. Chem. 30: 2918-2921. 1965. 11. Kim, Y. C., and G. H. Cocolas, Glutarnic Acid Analogs, 'rhe Synthesis of 3-Alkylglutamic Acids and 4-Alkylpyroglutamic Acids. J. Med. Chem. 8: 509-513. 1965. 12. Kinzer, G, \V., T. F. Page, Jr., and R. R. Johnson, Structure of Two Solanone Precursors from Tobacco. J. Org. Chem, 31: 1797-1800. 1966, 13. Leffingwell, J. C., A New Procedure for Alkylation of Iso phorone at the 4-Position. Tetrahedron Letters: 1653-165~. 1970. 14. Leffingwell, J. C., and R. E. Shackelford, A Novel Rmg Con 0 traction during the Vapor Phase Pyrolysis of Substituted Trans- 1-Hydroxy-2-Acetoxycyohexanes. Tetrahedron Letters: 2003-2006, 1970, 15. Luttich, W. R. G., Tobacco Flavoring. U. S. Patent 3,580,259 acetate (14), has not been reported previously as a (May 25, 1971). Tobacco Science, 1972, 16-34, p. 107-112, ISSN.0082-4523.pdf natural product. 16. Neurath, G., Treating Tobacco. U. S. Patent 3,610,253 (Oct. 5, 1971). Examination o,f the list of components isolated from 17. Neurath, G., M. Dunger, and I. Kustermann, Untersuchung der "Semi-Volatiles'' des Cigarettenrauches. Beitr. Tabak Forsch, burley tobacco (Tables 1 and 2) shows a large number 6; 12-20. 1971. of compounds which do not fit into any known pattern 18. Morton, A. A. et al, Polymeriza;tion, XII. The Metalation of Olefins and Dienes and Their Use in a-Polymerization of Buta of biogenesis. This result should not be entirely un dienes. J. Am. Chem. Soc. 72: 3785-3792. ]950. 19. Onishi, I., and K. Yamamoto, Studios on the Essential Oils expected inasmuch as tobacco is subjected to various of Tobacco Leaves: Part XI. Phenol Fraction (III). Bull. Agr, curing, heating, aging and fermentation conditions in Chem. Soc. 21: 90-94. 1957. 20. (a). Rice, VV. Y., and D. L. Roberts, Synthesis of 4.-Keto- the course of its preparation for manufacture into 2, 6, 6-trimethyl-cyclohex-2-enylidene-acetaldehyde and Related Compounds. In preparation. (b). Isler, 0., M. Montavon, R. tobacco producits. Thus, processing techniques may Ruegg, G. Saucy, and P. Zeller, Polyene Aldehydes, U. S. Patent contribute to the formation of certain constituents. No. 2,827,481 (March 18, 1958). 21. Roberts, D. L., Process of Imparting a Popcorn-like Flavor While many of the compounds may appear to be spe and Aroma to Foodstuffs and Tobacco by Incorporating l'yrazine Delivatives Therein and the Resulting Products. U. S. Patent Ko. cific to tobacco, more thorough investigations of other 3,402,051 (Sept. 17. 1968). plant materials, made possible by the development of 22. Roberts, D. L., Smoking Product Having Flavorant Additive, U. S. Patent No. 3,334.637 (Aug. 8, 1967). sophisticated gas chromatography-mass spectrom 23. Roberts, D. L., The Structure of a New Sesquiterpene-1- Keto-a-cyperone--Isolated from Tobacco. Phytochemistry. 11 :2077- eter -combinations, have shown or will show that many 2080. 1972, of these compounds are present in other natural 24. Roberts, D. L., Tobacco. U S, Patent No, 3,217,717 (Nov, 16, 1965). products. 25. Roberts, D. L., '.robacco. U. S. Patent No. 3,217,71S (:[\Tov. 16, 1965), 26. Roberts, D. L., Tobacco. U, S, Patent No. 3,280,824 (Oc,i. 25, SUMMARY J 966), 27. Roberts, D. L .. and R. L. Rowland, Macrocyclic Ditcrpenes. a- and /3-4, 8, 13-Duvatriene-1,3-diols from Tobacco. J. Org. Chem, As a result of the investigation of constituents of 27: 3989-3995, 1962. burley tobacco, 105 compounds have been identified; 28. Roberts, D. L., and J. N. Schumacher, Tobacco Composition, U.S. Patent 3,217,716 (Nov. 16, 1965). 56 of them are reported for the first time as tobacco 29. Roberts, D. L., and J. N. Schumacher, Tobacco Product. U. S, Patent 3,380,456 (Apr. 30, 1968). constituents. Eleven of these are new to the chemical 30. Rodgman, A'., and L. C. Cook, The Composition of Cigarette literature. Although many of the compounds identified Smoke. XI. Heterocyclic Nitrogen Compounds from 'I'urkish To bacco Smoke. Tob. Sci. 7: 174-177. 1962. contributed to the flavor of burley tobacco, none of 31. Rohde, Vl. A., Tobacco. U. S. Patent 3,313,307 (Apr, 11, them alone possesses the flavor characteristic of bur 1967). 32. Rohde, "\V. A., 'I'obacco Product. U. S. Pa.tent Pending. ley tobacco. The flavor is presumed to be derived from 33. Rowland, R. L., Flue-cured Tobacco. II. Neophyladicne. J. Am. Chem. Soc. 79: 5007-5010. 1957. the sum of many of these components and many more 34. Rowland, R. L., Process for Preparing 4-(2-Butenylidene)- \Vhich have not yet been identified. 3,5,5-trimethyl-2-cyclohexen-1-one. U. S. Patent 3,268,589 (Aug. 23, 1966). 35. Rowland, R. L., Tobacco, U. S. Patent 3,211,157 (Oct. 12, 1965). ACKNOWLEDGEMENTS 36. Rowland, R. L., and D. L. Roberts, Macrocvclic Diterpenes Isolated froim Tobacco. a- and /3-3,8,13-Duvatriine-1,5-diols. J. The authors express their grartitude to Drs. J. N. Org. Chem. 28: 1165-1169. 1963. L 37. Rowland, R. L., A. Rodgma.n, J. N. Schumacher, D, , Schumacher and R. L. Rowland for their valuable Roberts, L. C . .Cook, and VV. E. ·walker, Jr., Macrocyclic Diter· assistance in the identification work. We thank the pene Hydroxy Ethers from Tobacco and Cigarette Smoke. J. Org. Chem, 29; 16-21. 1964. . Analytical Division for providing the spectra used 38. Rowland, R. L., and J. N. Schumacher, Private Communica tion. throughout this work. We appreciate the assistance 39. Sabeitay, S., L. Traband, and F. Emmanuel, Constituents of Mr. J. A. Giles gave us in the preparation of this the -Concrete of Tobacco Leaves (Nicotiana Tabacum). Compt, Rend. 213: 321-323. 1941. manuscript, and for aid in the flavor evaluations, we 4.0, Schumacher, J. N., Private Communication. 41. Schumacher, J. N., Tobacco. U. S. Patent 3,372,699 (]Jar. thank Dr. J. C. Leffingwell. 1 Il 9 \l ~8dh.umacher, J. N., Tobacco Product. U. S, Paten,t 3,381,690 (May 7, 1968). LITERATURE CITED 43. Schumacher, J. N., ;and D. L. Roberts, Tobacco Product, U.S. 1-,atent 3,381,691 (May 7, 1968). 1. A'asen, A. J., B. Kimland, and -C. R. Enzell, Tobacco Chemis 44. Sohumacher, J. N., D. L. Rob'erts, and W. A. Rohde, To- try 7. Structure and Synthesis of 3-Oxo-a-ionol, A New Tobacco bacco. U. S. Patent 3,372,700 (Mar. 12, 1968). To- Constituent. Acta Chem. Scand. 25: 1481-1482. 1971. 45. Schumacher, .T. N., W. A, Rohde, and D. L. Roberts, 2.Adler, R., R. L, Peck, and L. Thompson, Chemistry of Cigar bacco Product. U. S. Patent 3,380,457 (Apr. 30, 1968). Butt Odor. II. Further Investigations on the Dis1tillable Portions. 46, Settine, R. L., G. L. Parks, and G. L. K. Hunter, The Re· Tob. Sci: 15: 121-123. 1971. arrangement of Limonene and Carvomenthene Epoxides. J. Qrg, 3. Bailey, P. S,, Uber die Ozonisierung von' Camphen. Ber. 88; Chem. 29: 616-618. 1964. 795-801, 1955. 47. Stedman, R. L., The Chemlcal Composition of Tobacco and 4, (a), Bailey, W. C., Jr., A. K. Bose, R. M. Ikeda, R. H. New Tobacco Smoke. Chem, Rev. 68: 153-207. 1968. man, H, V. Secor, and C. Varsel, The Isolation of 2-Hydroxy-2,- 48. Teague, C. E., J:r., J, N. Schumacher, and W. A. Rohde, 6,6-trimethylcyclohe:xylidene-acetic Acid -y-Lac;tone and Its Syn Tobacco. U. S. Patent 3,372,701 (Mar. 12, 1968). thesis. J. Org. Chem. 33: 2819-2822. 1968. (b). Demole, E. and P. 49. ZarghMD.i, N. S., and D. E .. Heinz, Monoterpene AldehydeS Enggist, Synthese des (±)-loliolide, (±)-actinidiolide, (±)-·di and Jsophorone-Related Compounds of Saffron. Phytochemistr)' hydroactinidiolide et derives a partir d' acide homosafranique. 10: 2755-2761. 1971.

(Tobacco Science 112)