Chemistry and Biochemistry
of Green Tobacco The chemical composition and properties of tobacco at the time of processing are determined in advance by inheritance and the cultural history of the crop. Environmental influences, especially moisture and mineral nutrition, may result in extensive modification in the relative proportions of nitrogenous and carbohydrate constituents with attendant changes in smoking properties of the manufactured product. A unifying concept built around the Krebs tricarboxylic acid cycle may be developed to account for inherited and culturally induced modifications in tobacco composition. Available evidence appears to be in reasonable agreement with this concept. Rather large gaps in basic information revealed by these considerations point to the need for extensive investigation of the constituents and the chemical processes of living tobacco.
R.
F. DAWSON
Columbia University, N e w York, N . Y.
as tissue constituents with succinic reportedly a third. All evidence ( 1 7 , 22) points to the ready interconversion of these acids in the tobacco leaf with malic and citric acids particularly active in this respect. It should be noted (Figure 1) that malic acid represents an oxidation product of citric. Studies of the citric acid cycle catalysts in tobacco have been strangely few. It is therefore impossible a t the present time to state whether the species contains the complement of enzymes that would be required if such a cycle were operative. Kempner ( 1 2 )showed several years ago that the terminal oxidase of tobacco leaves is undoubtedly cytochrome oxidase. According to some authors, acetic acid or its equivalent-for example, acetyl phosphate-occupies a pivotal position through which the readily reversible pathways of carbohydrate syntheeis and breakdown are coupled to the citric acid cycle. It is known that acetate is rapidly and completely oxidized in tobacco leaves (19,17). Vickery and Abrahams ( 1 7 ) fed excised tobacco leaves with various organic acids of the cycle and of the fermentative pathways. Feeding with malate, citrate, and isocitrate led to appreciable accumulation of citric acid. Acetate fed in the dark was utilized but did not appear as a net increase in citrate content of the leaf tissues as might have been expected. This observation could be used as an argument against the possibility of occurrence of such a cycle as outlined here but for the current report of Zbinovsky ( 2 2 ) that isotopic carbon from acetic acid infiltrated into tobacco leaves is found predominantly in citric acid when the leaves are kept in the dark. Culture on acetic acid containing C1*in the light led to an accumulation of the isotope in malic acid, The dark conversion of malic to citric, so frequently reported for tobacco leaves, was also encountered by these authors. It would thus seem that the failure of the New Haven group to detect a net conversion of acetate to citrate by quantitative analysis may have been complicated by the presence of back reactions involving citrate. I n this connection i t should also be noted that of the acids employed by Vickery and Abrahams, only acetate would have required initial phosphorylation with a free energy input of considerably more than 2 to 3 kcal. per mole. Also, acetate would require a stimulated output of oxaloacetate in order to undergo extensive oxidation through the cycle (Figure 2). Acetate is thought to be the precursor of the fatty acids and the end product of their oxidation in plants as well as in bacteria and animals. If acetate actually occupies the pivotal position assigned to it in the cycle, then i t is easily seen how fatty acids
OBACCO is outstanding among plant species largely because of its enormous leaf area and its ability to produce nicotine. Otherwise, the problems of tobacco production center about control of characteristics that are common to many species and, in general, sensitive to the same genetical and environmental influences. The objectives of such control differ widely according to the type of tobacco concerned and manufacture intended. Frankenburg (8, 9) has summarized available information regarding the composition of cigar and cigarette tobaccos in relation t o manufacturing processes Consequently, emphasis will be placed here on the nature of the chemical pathways by which variations in composition of the growing crop may be understood. It is convenient to consider the chemistry of green tobacco in terms of three relatively recent biochemical concepts. First, the different constituents of the plant are thought to be produced by specific, enzymically catalyzed reaction pathways, all of which are linked directly or indirectly to one central cyclic pathway for the combuption of organic acids. In its principal forms, this cycle has been termed the Krebs cycle or the citric acid cycle. Secondly, the detailed sequential nature of these pathways as well as points of linkage and maximum rates at which the pathways are traversed are determined by the array of hereditary units possessed by the individual or variety. These units are generally termed genes, although their nature is not yet fully understood. Thirdly, environmental factors control the relative rates with which different biochemical pathways are actually traversed and hence account for extensive variations in tissue composition. Among such factors are temperature, light intensity, and the environmental concentrations of liquid and gaseous water, of oxygen and carbon dioxide gases, of nitrate, sulfate, phosphate, calcium, potassium, ammonium and magnesium salts, and of the trace elements iron, manganese, copper, zinc, molybdenum, and boron. In what follows, the chemistry of tobacco will be discussed from the standpoint of these three concepts.
T
Biochemical Pathways in Green Tobacco
Evidence for the occurrence of a cyclical system for integrating all pathways of metabolism into a single unified scheme is derived chiefly from experiments with other species and hence is still largely inferential for tobacco. At least five of the nine known organic acids of the Krebs cycle have been isolated from tobacco. Of these malic and citric are quantitatively the most important
266
INDUSTRIAL A N D ENGINEERING CHEMISTRY
February 1952
could not only be respired to carbon dioxide and water but also how fruits and seeds can convert fat to carbohydrates and vice versa during their development and germination. In this connection, it is suggestive that the tobacco seed is rich in fat but poor in starch and other storage polysaccharides. During the development of the young plant from the germinating seed, i t is likely that acetate from fat breakdown is utilized to reverse the normal pathways of fermentative carbohydrate breakdown with the result that sugars and starch are produced as well as the structural polysaccharides cellulose, the hemicelluloses, the pectins, and perhaps also lignin. These structural substances would be available for the formation of new cell walls and tissues in the growing regions of the plant body. Bonner and Arreguin ( 8 ) have recently discovered another interesting series of reactions involving acetate. Tissues of the Guayule plant (Parthenium) are apparently able to transform acetate into acetone which with more acetate yields 3-methyl2-butenoic acid. Biosynthesis of rubber according to Bonner and Areguin:
2CHaCOOH (Acetate)
--
CAQBOHYDRATES 2NH3
+
CHa C:O COOH
c
it
C DYRUVIC
ALANINE
/rfc /rf
Photosynthesis ? Photosynthesis ? I1 +cos
C C
FATTY ACIDS
/r/
Y L A C E T I K TERPENES
Or a n k &d
cude
C C
C-C C C I
ACONITIC ISOCITQIC CITRIC
OXALOSUCClNlC
acid)
The latter is readily converted by the plant into tfK polyterpene rubber. Tobacco yields, on ether extraction, a complex mixture of Clo-CII and ClrCll paraffin hydrocarbons, waxes, alcohols, mono- or polyhydric esters of the C1-CL4fatty acids involving aliphatic, terpene, and furfuryl alcohols, eugenol, isoeugenol, and furfuraldehyde. These substances are largely responsible for the aromatic properties of fresh tobacco leaves and, together with the phenolic substances, catechol, rutin, caffeic, and chlorogenic acids, and the tannins, are thought to determine the aroma of burning tobacco. It is interesting to speculate that acetate might be the precursor of many if not most of the ether-soluble substances in the green leaf. I n a general way, they appear to be quantitatively related to the metabolism of the carbohydrates and may therefore represent end products of specialized reaction pathways originating with some relatively simple product of carbohydrate metabolism such as acetate. Undoubtedly the most important reaction sequence linked to the citric acid cycle in green plants is that through which carbon skeletons originate-namely, the photosynthetic assimilation of carbon dioxide. The studies of Calvin and Benson (1) have shown that the earliest detectable products of photosynthesis are either identical with or very closely related to the intermediates of the fermentative pathways and of the citric acid cycle. It has long been known that hydrogen from the photochemical splitting of wrtter is used to reduce carbon dioxide in the green cell. But BjIorless cells also fix carbon dioxide on a small scale; Vishniac and Ochoa ($0)have recently reported the astonishing fact that an enzyme from pigeon liver, washed chloroplasts, and triphosphopyridine nucleotide, when irradiated in the presence of pyruvic acid and carbon dioxide, produces malic acid. Ochoa’s in vitro photosynthesis: Enzyme preparation from pigeon breast muscle, triphosphopyridine, manganese, isolated chloroplasts, pyruvate, carbon dioxide, and light: COa
reduce oxaloacetic acid, which is an intermediate between pyruvate and carbon dioxide on the one hand and malate on the other. Triphosphopyridine nucleotide normally removes hydrogen from fermentative and Krebs cycle intermediates during carbohydrate breakdown and passes it on to other enzymes which, in air, eventually catalyze the union of hydrogen with oxygen to form water. It thus seems that enzymes, such as were used by Ochoa, act to rexrerse some of the reaction sequences of the cycle or of closely linked pathways with the result that hydrogen is added to the system rather than abstracted. There is nothing to indicate that this process would possess any unusual features in tobacco.
CHa CH3 (+ Acetate) CHI CO --f CO C-CHI +Rubber CHe CH3 II COOH (Acetone) CH COOH (Acetoacetic (3- Methy I-2-butenoic acid)
267
e
(Pyruvate)
COOH C:O CH2 COOH
COOH
e CHOH
(Oxaloacetate)
CHe COOH
(Malate)
I n these reactions, hydrogen is apparently extracted from water by the action of chlorophyll and light. This hydrogen can then be taken up by triphosphopyridine nucleotide and used to
A WART IC
ACIDS e GLUTAMIC Outline of Krebs Tricarboxylic Acid Cycle and Probable linked Reaction Sequences
DQOTEIN
Figure 1.
OTHER AMlNO
An extremely important linked series of reaction pathways is that by which inorganic nitrogen is assimilated in the plant. Here nitrate from the soil is reduced with hydrogen obtained from the dehydrogenation of members of the citric acid cycle. The resulting ammonia is taken up by two components of the cyclc, oxaloacetic and a-ketoglutaric acids, and also by pyruvio acid, the latter an intermediate between glucose or starch and acetate. The products, aspartic and glutamic acids and alanine, respectively, -ne thought to provide the nitrogen for synthesis of the remaining natural amino acids by transamination reactions with suitable a-ketoacids. The extensive studies of Vickery and Pucher (19) on starving and curing tobacco leaves have shown that during protein breakdown the carbon skeletons of the amino acids are oxidized almost completely to carbon dioxide and water and that the remaining nitrogen is utilized in the synthesis of asparagine and glutamine. This probably involves the citric acid cycle according to the conclusions of these authors. Vickery, Pucher, Schoenheimer, and Rittenberg (18)used “6 to prove that the processes of nitrogen assimilation and dissimilation in the tobacco plant exist in dynamic equilibrium: continuous degrada tion is balanced by continuous resynthesis. Whereas the tobacco leaf is the seat of photosynthetic activity, the root system is apparently the locus for much of the nitrogen assimilation. Schmid (16)has analyzed the stream of nitrogenous compounds that flows away from the root toward the stem and leaves. Of the total nitrogen present in this stream, 8.5% occurred as amino acid-N, an equal amount as amide-N, 2.7% as ammonia-N, 20% as nicotine-N, and the remainder as unreduced nitrate. Much of the latter is ordinarily stored in the stalk of the tobacco plant. The net result of nitrogen assimilation is the withdrawal of a portion of newly photosynthesized carbon chains from the carbohydrate system into the general pool
INDUSTRIAL AND ENGINEERING CHEMISTRY
268
of nitrogenous compounds. The extent of such diversion can vary over a wide range in response to fluctuations in form and quantity of nitrogen available from the environment. Since the end products of nitrogen assimilation in tobacco are principally protein and nicotine, it obviously becomes possible to direct the composition of a given crop toward the carbohydrate-aromatic side or alternatively toward the protein-nicotine side merely by regulating the nitrogen supply of the crop. It is of considerable significance, therefore, that the nitrogenous constituents of cigar tobaccos form about 24% of the total Solids while only about 15%
CH,COOH (ACETIC ACID)
'YOOH HOOCCH(0y) HCH,COOH
HOOCCOCH,COOH
(OXALOACETIC
41
ACID)
HOOCCH(OH)CH,COOH (MALIC
ACID)
Figure 2
(*NH3)
eCHBCH(NH2)COOH (Alanine)
-
11. COOH COOH C:O (=kNH3) HCNH, (*NHs) CH2 , -x CHI COOH COOH (Oxaloacetate) (Aspartic acid) 111. COOH
C:O
(*NHj)
COOH HCNH, CHZ CONHz (Asparagine)
COOH COOH HCNHz ( z ~ N H : ) HCNHz CHz CHz CH, CH2 COOH CONHz (Glutamic acid) (Glutamine)
e
CHz CH, COOH ( a-Ketoglutarate)
virus protein undergoes its mtocatalytic multiplication. Pirie ( 1 4 ) has obtained a similar protein preparation by different procedures but has criticized the conclusion that it is related as a precursor to the virus protein. According to Camus, Eggman, and Wildman (3) night temperatures during the growing period exercise a profound effect on the quantitative relationships among the different proteins of the cell. Thus, aa night temperature increased from 6" to 26" C., the amount of particulate protein in the leaves of Cuba white tobacco increased, while the amount of soluble cytoplasmic protein reached a minimum b e tween 14"and 20" C. Nicotine is the one product of nitrogen assimilation that clearly distinguishes tobacco from virtually, but not quite, all other plants. This alkaloid can largely be removed from tobacco by chemical processing, by breeding and selection, or by grafting the plants to a nonalkaloidal root-stock such aa tomato or eggplant. Nicotine is primarily a product of root metabolism (6, 6). The weight of accumulated evidence suggests that it is formed during nitrogen assimilation and is definitely not a product of amino acid metabolism as postulated by various authors. During the growth of the plant, nicotine is transported to the leaves where it continues to accumulate in ever-increasing amounts until the onset of senescence. I n some varieties of tobacco, there is pesent in the leaf a mechanism for the removal of a methyl group from pyrrolidine nitrogen of nicotine. The product of this reaction, nornicotine, is less volatile than nicotine. The reaction is not reversible and its specificity is low: N-methyl anabasine as well as the N-ethyl homologs of the latter and of nicotine are readily dealkylated (7'). Genetical Control of Chemical Composition
of the solids of cigarette tobaccos contain nitrogen according to Frankenburg (8)(Table I). The reasons for this difference lie in part in the fact that cigar tobaccos receive heavier fertilization with nitrogen and that they are harvested before senescent changes bring about a migration of nitrogen from the leaf as happens in many cigarette tobaccos. The proteins of the tobacco leaf have been examined by Wildman, Bonner, and Cheo (21). Pathways of nitrogen assimilation via the Krebs cycle intermediates:
I. CHsCOCOOH (Pyruvate)
Vol. 44, No. 2
There seem to be three generally distinguishable categories, the proteins of the particulate structures in the cell, the nucleus, plastids, and mitochondria; a heterogeneous fraction of soluble cytoplasmic protein; and finally an electrophoretically homogeneous nucleoprotein which constitutes about 75% of the actual leaf cytoplasmic protein. This last fraction has the interesting property of containing in a bound form all the plant growth hormone of the leaf. Furthermore, it has the activity of a phosphatase and in addition seems to be the specific nitrogenous fraction of the leaf a t the expense of which the tobacco mosaic
Perhaps the major problem in tobacco research is the interpretation in chemical terms and the practical control of what is a t present almost entirely an intuitive notion, "quality in tobacco." Information regarding the control of chemical composition of the green leaf is of critical importance in this connection, since the green leaf is the starting point of the manufacturing processes aimed a t bringing out the smoking qualities of the tobacco. Control of chemical composition in the living plant is accomplished as much through genetical means as by environmental manipulations. This is due to the fact that, as a consequence of its long history of cultivation and selection by man, the tobacco plant has come to possess a more or less fixed type of heredity. This simply means that the variational degrees of freedom for any specific inherited characteristic are severely limited in the species Nicotiana tabacum. According to Clayton ( 4 ) the Indians had selected tobacco long and painstakingly for leaf size and for high nicotine content. I n this process the particulate hereditary units (genes) for milder characters originally present have been irretrievably lost. There seems no doubt that quality can be improved by going back to the wild parents of N. tabacum, which are known, and to their relatives and repeating some of the process of breeding and selection which nature and the Indians contrived without benefit of science centuries ago. Modern plant breeding methods can vastly accelerate the process, but before such a program is feasible, the chemist must come up with qualitative characterization of quality and with techniques for its quantitative estimation. Among the chemical aspects of tobacco known to be genetically controlled are nicotine content, the semichlorotic condition of the stem and leaf of white burley tobacco, the tendency of burley and Maryland tobaccos to produce relatively more framework substances including cellulose and relatively less soluble nitrogen compounds and organic acids than cigar types although grown on the same soil types, and perhaps the tendency of cigarette tobaccos generally to produce higher contents of carbohydrates and lower contents of nitrogen compounds than cigar tobaccos.
February 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
Environmental Control of Chemical Composition in Tobacco Extensive study has been given to identifying and evaluating those environmental factors which are of greatest importance in producing tobacco ef good quality. Since tobacco is used for diverse purposes in manufacturing, it is evident that several specialized crops must be grown each with its own particular cultural treatment in order to bring out the more or less unique properties conditioned by different combinations of environmental circumstances. Frankenburg (8) has correctly emphasized that plants of different strains tend to become more alike under identical cultural conditions. According to Garner (IO) the major factors influencing the composition and properties of tobacco can be reduced to a few common denominators, most of them originating in the root environment-that is, the soil. These are soil and air moisture, soil gases, and inorganic soil nutrients, particularly nitrogen. The effects of these variables individually and collectively are so complex that no fully satisfactory analysis has ever been achieved. Consequently, it is impossible to derive a polyfunctional equation to express either growth or chemical composition of a single variety grown under different circumstances. Certain limited generalizations can be made, however. First the impact of a given environmental factor at different levels of intensity can be expected to influence the chemistry of the plant by altering the relative proportions of root and shoot. This is equivalent to altering the relative capacities of the individual for carbon and nitrogen assimilation. Secondly, the effects uf continued inc’reases or decreases in intensity of the factor may exceed the limits of growth response and continue to influence chemical composition although growth be little or not affected.
Table 1.
Average Composition of Green Tobacco leaves
(8) Class of Compound Carbon only Nitrogenous
Per Cent of Dry Weight of Leaves Cigar tobacco Cigarette tobacco 62.0 72.5 24.0 15.5
An example of this may be seen in the array of leaves on the single stalk of almost any variety of tobacco. The lowermost have easiest access to the ascending stream of water, minerals, and nitrogen. Consequently, they grow progressively larger and contain relatively more nitrogenous compounds. Upward, the leaves are smaller with lower absolute contents of water and nitrogen. This picture can be altered to permit more nearly equal growth and composition at all levels by topping. If topping is not too high nor too late, the excess photosynthate from the leaves descends to nourish the roots which respond with renewed growth. If sufficient soil nitrogen and moisture are present, assimilated nitrogen and nicotine are sent back to the shoot which results in higher than normal contents of protein and alkaloid in the leaves a t a11 levels. By increasing soil nitrogen supplies still more, the contents of protein and nicotine, for instance, can be considerably increased even after leaf enlargement (growth) has ceased. Steinberg (16)has recently discovered an interesting relation‘ship between an environmental factor and the synthesis of amino acids in tobacco. Deficiencies in the soil of certain of the minor elements lead to a higher than normal tissue concentration of amino acids. Different elements have different effects both qualitatively and quantitatively, but in certain cases one of the principal amino acids to accumulate is isoleucine. When this amino acid accumulates or when it is fed to tobacco seedlings in sterile culture through the roots, certain rather striking morphological changes in the leaf occur which are similar if not
Table II.
2 69
Average Composition of Green Tobacco leaves
(8) Class of Compound Carbon only Cellulose and lignin Pentosans Pectins Ether-soluble8 (volatile oils, waxes, resins, paraffins) Tannins Oxali~acid Carbohydrates Ether-soluble organic acids (citric, malio, suocinio, eto.) Unidentified compounds Containing nitrogen Proteins Alkrtloids Sol. compounds
Per Cent of Dry Weight of Leaves Cigar Ci arette tOb8000 t&aOCO 9.6 3.0
7.0
10.0 2.0 7.0
7.0 2.5 2.0 3.0
7.5 2.0 2.0 23.0
11.0 17.0
11.0 8.0
17.3
12.2 1.3
3.0
a. 7
4.0
identical to those that characterize the very interesting disease of cultivated tobacco known as “frenching.” This observation would seem to provide an interesting and perhaps profitable entering wedge into the extremely little understood field of the coupling between chemical activity and morphological expressions in living plants. The classical example of control of chemical composition by manipulation of cultural conditions is the case of cigar-filler tobaccos contrasted with flue-cured cigarette types (Table 11). The latter are grown in extremely light soils where root aeration and water supply are good. However, fertilization, particularly with nitrogen, is limited. The consequence is a plant which exhibits mild but not severe growth restriction. The leaves are light and thin and develop light colors on curing. The sugar content is high, but varietal characteristics do not favor a high content of aromatics. On the other hand, cigar-filler types are usually grown on heavier ground with considerably heavier fertilization. While water supply is adequate, leaf development is relatively greater than in the case of most cigarette tobaccos. Topping leads to the production of a heavy, ash-rich leaf which contains much nitrogen including nicotine, Desirable aromatic properties are developed during curing and fermentation, although these are probably not completely identical with the substances responsible for the aromatic properties of cigarette tobaccos. From a table presented by Garner (10) it may be calculated that cured cigar-type tobaccos contain on the average about 40% of carbon compounds and 24% of nitrogen-containing compounds, while the, corresponding figures for Type 13 fluecured tobacco were 71oJ,and 70/,, respectively. It would appear that luxury nitrogen metabolism (in excess of that required for reasonably normal growth) may be a characteristic feature of cigar tobacco culture. Many of these interrelationships can be understood in terms of the citric acid cycle. Thus. the aromatic Turkish tobaccos are grown under circumstances which drastically limit the availability of inorganic nutrients and of water. I n this case, carbon from photosynthesis backs up in the cycle leading to an excess production of acetate or its equivalent. Acetate, as has been shown, can go to carbohydrate, fat, and probably also to volatile oils and resins of the ether-soluble class. The result is a leaf poor in protein and rich in the products of carbohydrate assimile tion including the aromatics. The lighter cigarette tobaccos receive a better nutrition, but the balance between nitrogen and carbon assimilation is still on the side of carbon. There is enough nitrogen to enable a substantial amount of protein synthesis and enough accompanying nutrients of soil origin to permit nearly normal cell expansion and growth, but the composition and metabolism of the leaf are still predominantly carbohydrate in nature. Genetical lack of easily traversed pathways for the synthesis of
270
INDUSTRIAL AND ENGINEERING CHEMISTRY
aromatics constitutes one distinguishing feature between this and the Turkish or Oriental types. I n cigar tobacco a near excess of all nutrients combined with topping practices leads to an abundance of carbon compounds for the production of framework substances and of nitrogenous compounds necessary for the formation of protoplasm. This may mean that there is little competition between the different reaction pathways leading away from the cycle for carbon skeletons. The presence of abundant nitrogen does, however, serve to divert more of the acids of the cycle into amino acid and nicotine synthesis leaving less available for the synthesis of starch and free sugars. The extent of such diversion increases as nitrogen supply is increased with all other factors held constant (11). Vickery et al. (18)noted that tobacco which had absorbed NlsH,CI through the roots contained 8.2% isotopic nitrogen in the leaf proteins in 72 hours. Hydrolysis of the leaf protein and isolation of individual amino acids revealed that higher concentrations of NL5had gone into glutamic and aspartic acids than any others. It was concluded that these two amino acids had exchanged nitrogen with the ammonia of the cell most rapidly. In the light of earlier discussions, t’iis result would seem to support the idea that citric acid cycle intermediates provide the gateway aa well as the rate controlling medium for the entry of nitrogen into the metabolic processes of the growing plant. literature Cited (1) Benson, A. A., Brookhaven Conference Rept. 70 (C-13), 119-38 (1950). (2) Bonner, J., and Arreguin, B., Arch. Biochem., 21, 109-24 (1949).
Vol. 44, No. 2
(3) Camus, G. C., Eggman, Id., and Wildman, S. G., Abstract of paper presented at 1950 meeting of the Botanical Society of
America, Columbus, Ohio.
(4) Clayton, E. E.,personal communication. (5) Dawson, R. F., Advances in Enzymol., 8 , 203-51 (1948). (6) Dawson, R. F.,Am. J . Botany, 32,416-23 (1945). (7) Dawson, R. F.,J . Am. Chem. SOC., 73, 4218 (1951). (8) Frankexiburg, W. G., Advances in Enzymol., 6, 309-87 (1946). (9)Ibid., 10, 33-41 (1950). (10) Garner, W. W., “Production of Tobacco,” Philadelphia, Blakiston, 1946. (11) Garner, W. W., Bacon, C. W., Bowling, .J. D., and Brown, D. E., U. S. Dept. Agr., Tech. B,uZZ. 414 (1934). (12) Kempner, W., Plant PhysioZ., 11, 605-13 (1936). (13) Krotkov, G., and Barker, H. A , , Am. J . Botany, 35, 12-15 (1948). (14) Pirie, N. W., Biochem. J., 47, 614-25 (1950). (15) Schmid, H., Ber. Schweiz. Botan. Ges., 58, 6-44 (1948). (16) Steinberg, R. A., Abstract of paper presented at 1950 Research Conference on Chemistry of Tobacco, State College, Pa. (17) Vickery, H. B.,and Abrahams, Marjorie D., J. Biol. Chem., 180,3745 (1949). (18) Vickery, H.B., Pucher, G. W., Schoenheimer, R., and Rittenberg, D., Ibid., 129,791 (1939); 136,531 (1940). (19) Vickery, H.B., Pucher, G. W., Wakeman, A. J., and Leavenworth, C. s., Conn. Agric. Expt. Sta., Bull. 399, 757-829 (1937). (20) Vishniac, W., and Ochoa, S., Nature, 167, 768-9 (1951). (21)Wildman, S. G.,Cheo, C. C., and Bonner, J., J . BioZ. Chem., 180, 985-1001 (1949). (22) Zbinovsky, V.,Abstract of paper presented at the 1951 meeting of the American Society of Plant Physiologists, Minneapolis,
Minn. RECEIVED August 8, 1951.
ALKALOIDS OF TOBACCO Identification and Determination The value of some newer techniques in studying the chemistry of tobacco is exemplified by their application to tobacco alkaloids and their derivatives. The newer techniques mentioned are ultraviolet and infrared spectrophotometry, chromatograph, countercurrent distribution, photochemical oxidation, reciprocal grafts with Nicotiana, radioactive tracers, statistical analysis, and improved pyrolysis, distillation, and colorimetric procedures.
J. J. WILLAMAN Eastern Regionul Research Lcborufory, Philadelphia 18, Pa.
OR LL long time researchers on tobacco chemistry had to be content with studying empirical groups of organic substances -fiber, pectin, protein, alkaloids, amino and amide nitrogen, carbohydrates, polyphenols, and pectins. A wealth of information has resulted, not only on the fresh plant of various types and grades (36, @, 44),but on curing, fermentation, and aging (18, 19). It has always been realized, however, that such studies could well be supplemented by determining the individual constituents, if only methods were available for doing so. Recently chemists have been given many new instruments and procedures for the examination of plant material. Some of these have been applied to all groups of the constituents of tobacco, but it is proposed to limit this discussion largely to the tobacco alkaloids. There are at least three reasons for this choice: First, most of the recent detailed work on tobacco concerns the alkaloids; secondly, they are a complicated group and hence well illustrate what can be done with the newer techniques; thirdly, these substances are of vital interest in tobacco processing and in studies of the physiological effects of smoking. The chemistry of the tobacco alkaloids has been studied for many years. The names of Splith, Wenusch, Pictet, Pinner,
F
and many others bring to mind the important invest,igations on tobacco carried on without the aid of the newer analytical techniques. Nicotine received the most attention in these earlier works. It was not until Woodward, Eisner, and Haines (64) studied the pyrolysis of nicotine that large quantities of homologs became available for study and a new phase in tobacco alkaloids began. Nicotine is converted to myosmine by pyrolysis over quartz at 570“ C. The conversion is about 18% on the basis of the weight of the original nicotine and 33y0 on the basis of nicotine consumed in the pyrolysis. Myosmine had previously been obtained from tobacco smoke by Spath, Wenusch, and Zajic (40). However, its ready availability from the pyrolysis of nicotine afforded the opportunity to study its chemical reactivity. The study by Haines, Eisner, and Woodward (97) showed that the ring structure normally applied to myosmine was easily hydrolysed in water to the open chain derivative, 3-pyridyl-waminopropyl ketone. The presence of a primary amino group made possible a new and easy determination of myosmine by the Van Slyke method and has been employed most successfully by
