Tank for =-traction of rice mliib
Elution of the vitamin irom fuller's earth
Final orystallirstion of the batch of vitamin
FIGGRE 1. DESCENDXNO SIZEOF SCALE OPERATIONS IN EXPERIMENTAL ISOLATION PEOCEDERE
THE BERIBERI VITAMIN R. R. WILLIAMS Bell Telephone Laboratories, 463 West Street, New York, N. Y.
11
The story is told of a 26-year research on the characterization of the beriberi vitamin, its isolation, the determination of its structure, and its synthesis. This vitamin is required by all living things, both plant and animal. The uses of the synthetic vitamin are discussed in the light of this fact and of the probability of a widespread insufficiency of it in human diets.
N
OTEWORTEY bioehemicsl studies have come to fruition during the past two or three decades. We have but to mention adrenalin, thyroxin, insulin, cortin, and the more vaguely known active const,ituents of the pituitary, adrenals, and thymus, and immediately there comes to mind the sex hormones, the carcinogenic hydrocarbons, the bile acids, the sterols, the fat-soluble vitamins, the antianemic constituents of the liver, ascorbic and pantothenic acids, glutathione, and the mongrel family of the vitamins B. One reason for this trend to the biological is that here we find ourselves measurably nearer to the ancient mysteries 01 life and consciousness and even that of personality. Far fronr disavowing an interest in thesc specnlative and perhaps faneiful realms, biochemistry is today contributing significantly to the prohienis of lrnman society and human behavior.
in 1910 by one of its earlier students, Colonel E. B. Vedder, M.C., IION of the George Washington University Medical School, then Captain on the Army's Board of TropicalMedicine in Manila. Only a few months previously Fraser and Stanton had reported to the Congress of Tropical Medicine their epoch-making findings in the Malay States as to t.he association of beriberi with polished rice and recalled attention to the almost ignored work of Eijkniann in Java on dietary polyneuritis in fowls. Fraser and Stanton liad proved their ease by the issuance of the customary nrhite rice to a camp of 150 laborers and brown rice-that is, partly decorticated-to anotlier equal group. Epidemics of beriberi occurred arnong the white-rice eaters; the brown-rice eaters were free. Later, on exchanging the diets of the two camps, beriberi showed a reversed ineidence \\-hich left no doubt of a casual relationship. Beriberi was very p r e d e n t in Xanila. There, as is usual elsewhere, it takes two forms of similar character except that the so-called wet type shows extensive edematous swelling of tlre extremit.ies; in the dry typo muscular atrophy is externally conspicuous. Lameness, ataxia, nunibness of the extremities, and loss or exaggeration of patellar reflexes reveal a disturbance of the motor and sensory nerves. In its more acute stages there is labored breat.hing and cyanotic dis-
Human Beriberi Another often concealed motive of bioclreniistfy is humanitarian. Arising from an association wit,li the practice of medicine, this field of chemistry not only whets our curiosity but by its promise to relieve sufferinggrat.ifiesour humanistic impulses. The story of beriberi and the antineuritic vitamin as it has been unfolded to me over a period of more than 25 years has had such an aspect. I was indrodueed to the subject 980
SEPTEMBER, 1937
INDUSTRIAL AND ENGINEERING CHEMISTRY
981
coloration about the mouth and nose which suggests a disturbance of tlie respiratory function; this suggestion has been confirmed in recent years by the finding of pyruvic (i. e., acetoformic) acid in beriberic blood analogous to acetoacetic acid in diabebes. Hypertrophy of the right heart occurs, and deat.h ensues froin heart failure. Especially destructive is the infantile form of the disease. About 25 years ago this form of the disease wan first reproduced in puppies and thus identified by Andrews in Manila. For this purpose Andrews suckled puppies at the breasts of human mothers who had just lost their babies of the then mysterious disease laon. The enlistment. of these bereaved, poor, and ignorant mothers in the cause of science required some diplomacy; Andrews says one mother refused to nurse a puppy until its eyes were open. Infant mortality in Manila regularly reached the staggering figure of 50 or even 55 per cent. In contrast to western cities, mortality was much greater among breast-fed than among bottle-fed babies in spite of the unsanitary tropical milk supply. No more elquent testimony t,o the deficiencies of the prevailing human dietaries (!odd be foiind. Mothers who lacked an unknown element secreted a deficient milk. Later evidence suggesis that it is also poimunos. Undertaking the work under such circumstances, it was natural that tlie chemist should be drawn into personal contact with the disease and, in company with his medical associates, tent the progress of his chemical fractionations of rice polish not only upon experimental animals but upon human patients as well. The most prevalent form of beriberi is of a chronic or rudimentary type, most conspicuous aniong women of childbearing age. Treatment of such cases brings about a gradual improvement over periods of weeks or months. However, in acute beriberi, especially in infants, response is usiially dramatic. On several occasions in tho absence of a medical associate, I went a t the call of a public health inspctor to the Toiido slum district of Manila to nttend without benefit, of medical certificate reported cases of beriberi. Often I dosed the baby myself to he sure to get as much of nry precious preparation inside and as little outside the baby as possible and sometimes sat down beside the ansious mother on the split bainboo floor of the little nip" shack to await results. Within as little as 3 hours I have seen the cessation of the weird, almost soundless crying which, due probably to paralysis of the larynx, is characteristic of the last. stage of the malady. Easing of the gasping breathing so011 followed, and then the smoothing of the wild-pulse, the Pading of blue lips, a hungry nursing, and peaceful sleep.
process. For years the sole objective of our work was increased efficiency of process so t,hat we should at the end o b tain enough vitamin to study it. Such work requires some patience and skill; it requires vastly more faith and courage, and a rigorous adherence in spite of disaster to one's ideal and objective. To the intestinal fortitude of those who joined me in this enterprise I pay just tribute. With these I include Seidell in America, Peters in England, Suzuki and Ohdake in Japan, and Jansen's group in Java, all of whoin persistently hammered at this all-important objective through long and difficult years. At last we had in sight a gram or two of vitamin. We had to he sure i t was pure; otherwise the analysis for carbon, hydrogen, nitrogen, and sulfur would only be misleading. Wrong information is worse than none at all. Its composition turned out to be C,JI,nS~SOCI?.
Isolation of the Vitamin
User a period of 2 or 3 years we had worked up several tons of rice polish in 300-pound lots. hferck &- Company then tindertook the same process on a much larger scale and were soon providing not only for our experinrents but also for others. We had the good fortune at t,lie outset of the striictural work to hit upon a useful cleavage reactioii of the vitamin picked from oiir past experience in its isolation. In the presence of sodium sulfite at a pII of 5 tlie vitamin splits neatly and quantitatively without the loss of an atom according t o Equation 1:
Observing such profound effects, the young and impressionable chemist inevitably conceived the desire to know what was responsible for them. The crude rice polish preparations were variable aiid uncertain in their action, and their use wold throw little light upon tlie function in the body of the beriberi-preveiiting substance which later came to be called vitamin. We now know that crude rice polish extracts cout,ain only 1 part of vitamin in 50,000. The search was destined to last 25 years and to enlist the talents of biochemists in every civilized country of the world. Fortunately we did not know that tlien. .Tansen in Java was the first to isolate the subst.ance in pure form. Windaus in Germany 6 years later showed the presence of sulfur in the molecule. However, an efficient means of obtaining t.he substance io quantity for chemical study was still lacking. This we accomplished in 1933 using the methods of Funk, Seidell, and Jansen; these methods were reinforced by our own expedients, notably the use of quinine for the elution of the vitamin from fuller's earth which is used as a n adsorbent in an early stage of the
Sulfite Cleavage of the Vitamin In order to determine the structure-that is, how these various atomn are arranged with respect to one another in the molecule--it was necessary to split the molecule into progressively snialler pieces until t,hey could Ire recognized a8 previously k n m n substances. Knowing tlie identit,y of each fragment, we might, hope to learn how to put them together again to form artificially the original siibst,ance. In these phases of tlie pr0jec.t we enlisted first H. T. Clarke and later E. R. Bricliman and J. K. Cline. Each conbribiited important ideas alii1 skill in the study of striictnre nnd synthesis. This work is, t,hPreEoro,tile aacoiriplisliiiieiitof 8. groiip r:rtilcr than of an individod.
FIGI.BE 2. MODELOF VITA MI^ M~LZCWLE Kitrope", oxygen, sulfur. and chlorine stoms are lettered: the others are oaibon (black) snd hydrogen (rbite).
CI?RI~NIOSCI~ i KaBOa
-
CoH9N$Oa i CoHJWI I I1
+ ZNaCl (1)
Prodnet I of the sulfite cleavage proved difficult to identify. It is insoluble in organic solvents, very sparingly soliible in water, readily soluble in alkali, and infusible tip to 140" C. As shown in Equation 2, HCI C~H~N,SO~--C~H~N*SO, ( =C~HAXO~H.OII) +ma (2) 1
111
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INDUSTRIAL AXD ENGINEERING CHEMISTRY
982
on reaction with aqueous hydrochloric acid, product I splits out ammonia and yields product 111,also an infusible crystalline substance. Product I or I11 yields sulfuric acid on heating with water a t 200’ C. and sulfurous acid on fusing with alkali, indicating the presence of a sulfonic group. Significant organic residues could not be isolated.
the compound corresponded in melting point and composition to 4-methylthiazole-5-carboxy acid, synthetically prepared by Wohman in 1890: CHI I
N
kH-k IV. 4-Methyl-thiazole-5;carboxy acid
The Pyrimidine Portion We had long suspected from its ultraviolet absorption spectrum that product I, which we will call an aminosulfonic acid, contains the pyrimidine nucleus and that the amino group is in position 6. This conclusion was based not only on the absorption but also on the difficulty of splitting out ammonia, and the fact that this amino group, although primary, reacts with nitrous acid or with benzoyl chloride only under vigorous conditions. The task we were now facing was to find where the sulfonic group is located and where the extra two carbon atoms are in the aminosulfonic acid which has the composition CeHeNsSOs. The spectrum of this aminosulfonic acid is single-banded in acid solution; it shows vestiges of a second band in water solution and becomes definitely double-banded in alkali. A similar behavior with acid and alkali is observed in 6-aminopyrimidine and all its alkylated derivatives. This series of compounds, several of them new to chemistry, were synthesized for the purpose. For brevity we must omit many details and pass on to later experiments that supplied conclusive evidence. Equation 3 shows a reduction of the aminosulfonic acid with sodium in liquid ammonia, CeHoNaSOs 2Na + CeHgNa NalS03 (3)
+
+
,b=CCOOH
The thiazole nucleus had never before been found in nature. It is probably quite important to the’physiological action of the vitamin. These findings enabled us to conclude that the original sulfite cleavage product must have one of two structures: ~
CHa
I
C===CCH243H20H (or -*CHOHCH3)
N/ \CH--$
The latter could be excluded by the optical inactivity of our product. Its identity was confirmed by the following synthesis:
CHI
d S \ Thio-
~ C C H ~ C H Z O H + &=CBr.CH2CH@H-c N/ (6) AH %H-S
I
Bromoaoetopropyl formamide alcohol (enol)
yielding a base, CsHsN3,whose picrate proved on mixed melting point t o be identical with that of 2,5-dirnethyl-B-aminopyrimidine, the synthesis of which is indicated in Equation 4:
V. 4-Methyl-B-(phydroxy) ethylthiazole
Linkage of the T w o Nuclei
Having established the structure of the two fragments produced by sulfite cleavage, we shall N=COH N=CC1 N=CNH2 consider -the relationship-of the two HNBR ~ C O nuclei to one another. Obviously alcoholic NH3 ACHs * CH3 CHa (4) the sulfonic group serves as a flag t o CHa& ‘ c A C H 3 + CH3A ACH3 p0c13 I I1 mark on the pyrimidine portion the & !%I ?;% H J-JH &-AH A-CH original point a t which the thiazole nucleus was attached t o it. But which atom of the thiazole was involved in this linkage? This valuable piece of evidence settled at once all question We had noted that the vitamin whose habits suggest it to of the 6-aminopyrimidine grouping and also fixed the number be a strong base is split by sulfite into a slightly acidic and a and position of the side chains. Equation 5 indicates a slightly basic substance. This loss of basicity by cleavage with synthesis which yielded 2-methyl-6-oxy-5-pyrimidine methyla neutral reagent suggested the disruption of a quaternary sulfonic acid : ammonium base or a sulfonium base. Methyl iodide was N=COH N=COH found to add to the thiazole half of the vitamin molecule to NazS03 produce a quaternary base in the familiar way. This methCH3& dCH2-OEt CHsb LCH8OsH (5) iodide (product VI) betrayed a peculiar and striking kinship 4-CH I II J-AH to the vitamin: CH3 This was proved by absorption spectrum and otherwise to be identical with the oxysulfonic acid (product 111)derived from the vitamin. This settled the structure of the pyrimidine half of the vitamin.
Ab
-
Thiazole Portion We now return to the sulfite cleavage reaction and consider the other half of the molecule, although chronologically the work was done in the reverse order. Product I1 of the sulfite cleavage (Equation 1) is a weakly basic oil and is chloroform soluble. Nitric acid oxidation yields product IV. This product is identical with one previously obtained by Windaus by oxidizing the vitamin itself with nitric acid. Although Windaus regarded it as a pyrrole derivative, such an oxidation without loss of sulfur, as well as other facts, suggested to H. T. Clarke a thiazole nucleus. A literature search showed that
VI
Potentiometric titration of the quaternary methiodide; product VI, with alkali showed no sharp break when 1 mole was reached, and only after 2 moles had been added did the pH move upward, indicating the presence of an excess. In the case of the vitamin chloride, we are dealing with the salt of a diacid base, since there are two ionic chlorines in the molecule as we have seen in the empirical formula. On titrating this chloride with alkali, we found a sharp rise when 1 mole was reached, corresponding to the formation of neutral
SEPTEMBER, 1Y3i
INDUSTRIAL AND ENGINEEKING CHEMISTRY
or monoacid salt, but from t.hat point onward it behaved like the metbiodide of the thiazole with no further break until a total of 3 moles had been added. The parallelism of the two compounds in respect to this extraordinary behavior left no doubt that the vitamin is a quaternary thiazole.
Complete Structure of the Vitamin
983
shaking with silver chloride into the more familiar chloride hydrochloride, identical in every way with the natural product. Needless to say, the product can be produced much more cheaply by synthetic means. It is now commercially available in quantities corresponding to thousands of tons of rice polish. Its cost is a fraction of that of the natural suhstance in the pure state.
With this piece of information, we can now write the complete formula of the ritamiii : CHI K=CNHI-HCI
1
1
CH,& CCH?-----------N
!
C-CCHZCH20H
'c:
&>CH VII.
Vitamin chloride hydrochloride
The action of alkali may be explained as follows: One mole of alkali reacts with the diacid salt and liberates the monochloride; further addition of alkali begins to liberate the strong quaternary base, but this is not stable; the hydroxyl group goes over rapidly but not instantaneously to the scarbon atom to form the neutral pseudo base or carbinol. Such a process of migration of hydroxyl is well known in cyclic quaternary bases. In effect it uses up hydroxyl ions and prevents the p H from rising. Nor does the pH rise even when 2 moles have been added because quaternary thiazoles such as this undergo ring opening in alkaline solution, forming an acidic sulfhydryl group. Only when this group is neutralized with a third mole of alkali does the alkalinity rise sharply. On adding an acid, the whole series of reactions undergoes reversal and the vitamin is regenerated.
Synthesis of Vitamin The conIirmation of the structure by synthesis of the vitamin proceeds according to the following series of reactions: HCOOLt
i
Ef0COCWIC*2aOEt
-
LIOCOC*C*tOEt
Coiiilea", .Yirrck and Cornpony. Ino.
Parslyred condition
Recovery one day after treatment
FIGVRE3. TREATMENT OF
RERlBERlC PIQEoN WIl'H SYNTHETIC YlTAMlS
The first trial of this synthetic product on polyneuritic rats represented the most tense and dramatic moment of 26 years of research. It was with much elation t.hat I translated the conservative report of the pharmacologist into the message, "The rats say yes." The structure which we proposed last summer has since been confirmed by both German and British workers. We learn further that the German I. C . Farbenindustrie has applied for patents on another synthesis which has not yet been published. The facts indicate that had we disdained commercial methods and neglected patent protection at each stage, the world would now be facing a monopolistic control of the synthetic product in purely commercial hands. The experience illustrates most forcibly that, mnch as one may admire the motive, it is sometimes impossible to dedicate inventions to free public use by publication unless the scientific worker is prepared to withold all publication until his achievement i s complete. If he attempts, without patent proteetion, to promote the cause of science by publishing the evidence as it develops, he may only he paving the way for exploitation by another of the invention which he thinks to lay freely in the lap of humanity. A patent on a single step is usually, or at least often, sufficient to afford commercial control. vi'e have assigned our patents to Research Corporation, founded many years ago by F. G. Cottrell to exploit inventions and to apply the profits to support research and kindred public ohjeets.
Uses of the Product
The resulting bromide hydrobromide has the typical physio:logical properties of the vitamin and can be converted by
Now that the vitamin is abundantly available, what uses will it serve? Fimt, we should mention the knowledge it will bring of the physiological function of this substance which is required for the growth and well being of all living things, both plant and animal. Yeast, bacteria, mushrooms, peas, tomatoes, beetles, birds, goats, monkeys, rabbits, rats, mice, and men have all been shown experimentally to use it as part of their normal processes. According to recent work of Lohmann and Scliuster the pyrophosphoric ester of tbe vitamin is the prosthetic ~ O U D of an enzyme which decarboxylates pyruvic acid. Without it pyruvic acid accumnlates and poisons the tissues. A fuller
984
INDUSTRIAL AND ENGINEERING CHEMISTRY
FIGURE 4. TON8 O F RICE POLISH WERE REQUIRED TO ISOLATE ENOUGH NATURAL VITAMINFOR STUDYOF CHEMICAL CONSTITUTION
understanding of these matters underlies fundamental phases of physiology. A better knowledge of its behavior will broaden the new science and in many ways assist in the mastery of constitutional diseases, notably neuritis, arthritis, and gout. Certain practical and immediate uses are already evident. Although we have known for more than 20 years the cause of and means of preventing beriberi, relatively little has been done in the Orient to curb it except perhaps in the Dutch East Indies. Until the sale of polished rice i R prohibited or restricted in the East, as has often been proposed by sanitarians, the disease will flourish and there will be need for treating it. The food habits of a people change but slowly, and the meager available statistics show no general falling off in the incidence of beriberi either in Japan or in the Philippines. I n the outlying provinces of the Philippines the recorded mortality is on the rise partly because of its increasing recognition by practitioners, partly because of the spreading displacement of hand-hulled rice by machine-milled rice consequent to industrialization of the East. The number of people who are partly incapacitated by beriberi is much larger than the number who die; one authority estimates the total number of current cases in the Philippines a t 150,000.
Supply of the Vitamin i n Occidental Diets What of the need for it in the supposedly well-fed western world? Here also we practice decortication of our staple grains and although we protect ourselves largely from the ravages of forthright beriberi by variety in diet, the average intake of this vitamin is suboptimal. There are few foods in which it is superabundant; many staples are almost devoid of it. The rich sources, aside from the brans of grains, are ripe peas, beans, and lean pork. White bread, macaroni, corn meal, peeled potatoes, and beef muscle contain very
VOL. 29, NO. 9
little. Even cow’s milk, according to good authoyity, offers a rather scant supply to nursing infants. Many fruits and vegetables contain moderate amounts on a dry basis, but as they are composed of 80 or 90 per cent water we eat very little of them net. Moreover this vitamin is very soluble in water and is sensitive to heat so that it may largely be lost in cooking, especially if liquids are drained off either in the kitchen or at table. Certainly many individuals suffer from lack of the vitamin a t times. This is especially true of women during pregnancy and lactation when the daily requirement rises to three to five times the normal. It is reasonable to suppose that modern trends in daily habits may have much to do with modern trends in disease and physical defect. I am thinking particularly of the rise or relatively greater persistence of constitutional disease-e. g., diabetes, cancer, and nephritis. One of the hygienic factors of greatest potential significance is the food supply. I n some ways we have materially bettered our diets in recent years. The rising use of dairy products provides more vitamin A; the vogue for sun tan and fortified foods means more v i t a m i n D; the g r e a t l y increased p r a c t i c e of distributing fresh f r u i t s a n d vegetatables throughout the year by the aid of refrigeration supplies more vitamin C; but we look in vain for any such significant upturn of our intake of the beriberi vitamin. Courtesy, Mer& and Company, Inc. Our knowledge FIGURE5. SYNTHETIC VITAMININ QUANTITY is yet m e a g e r , but I venture to Train loads of rice polish would be needed to produce an equivalent amount. call a t t e n t i o n again to the fact that, although grain eating is older than history, the practice of decorticating and degerminating grains for human food is primarily a product of the machine age. We do not yet know whether to attach weight to such an experiment as the recently reported development of cancer in experimental animals through deprivation of vitamin E, which happens also to be abundantly present in wheat germ. But we shall be stupid if we do not inquire further and diligently. More knowledge will erase many apparent inconsistencies. In the meantime, we will do well to keep an open mind and work industriously to learn the ultimate significance of each of the ever-increasing number of recognizable essential food factors of which none is more important than the beriberi vitamin. RECEIVED June 12, 1937. Presented before the General Meeting at the 93rd Meeting of ihe American Chemical Sooiety. Chapel Hill, N. C..April 12 to 15, 1937.
.
