H a r r i s o n H o w e Lecture . . .
WSMitoiiiiil Reserch on K A R L FOLKERS Director, Organic and Biochemical Research Merck and Co., Inc., Rahway, N. J.
Department
The story of vitamin B12 is not com plete; it has just started to unfold . .
. . . Nevertheless, the chemical, biological, and therapeutic develor^ments are already extensive enough that only certain significant features of this combined research may be reviewed in this lecture. The discovery by Minot and Murphy in 1926 that pernicious anemia could be treated therapeutically with whole liver opened a new chapter of research in nutrition. Soon after this epoch-making discovery, various groups of investigators began to fractionate liver extracts for the purification of the principle which is active in the therapy of pernicious anemia. Cohn and Minot and their associates described their notable work during the period of about 1927 to 1930. West and Dakin and their associates apparently studied fractionation over the longest period of time. Randolph West was a clinician with a fine appreciation of the chemistry of the problem, and it was the good fortune of us in the Merck research laboratories to become associated with him when we became interested in this problem. We all regret that Dr. West passed away recently; we are glad that he lived to participate in the completion of the isolation of the vitamin. Other groups of investigators were Laland, Klem, Strandell, and associates (1935-36), Wilkinson (1932-40), and Karrer and his associates (1937-38). SubbaRow and Jacobson reported their first studies during 1935-38. Presumably, Dr. SubbaRow continued the work until his recent untimely death; he is known for a multiple factor hypothesis. Castle and his associates were concerned more with the factor in beef muscle and the effect of gastric juice on the activity of this factor, or the extrinsic factor and the intrinsic factor. Other investigators published studies which cannot be mentioned in the interest of brevity. Several years ago, we started work in the Merck laboratories on the fractiona1634
tion of liver extract to purify the "antipernicious anemia" principle; we collaborated with Randolph West of Columbia University for clivi^r:1 tests. Later, w e collaborated with Mary Shorb and George Briggs of die University of Maryland concerning the development of a microbiological assay. This research led to the isolation of a red crystalline compound which was designated vitamin Bi2 [Rickes, Brink, Koniuszy, Wood, and Folkers (1948)]. This crystalline compound shows a microbiological activity of 11 million L L D units/mg„ or 1.3 X 10 ° of one microgram per milliliter supports half-maximal growth. Its refractive indexes were found to be a, 1.616; £, 1.652; 7, 1.664. The crystals darken on a hotstage at 210 to 220° and do not melt below 300°. The new vitamin was found to b e clinically active when a single dose of 3 to 6 fMg. was injected intramuscularly in patients with pernicious anemia. One can imagine the excitement and amazement on die occasion of testing a dose of a substance which is almost too small to b e seen -with the unaided eye. The approximate vitamin Bi- contents of four samples of commercial liver ex-
tracts which are on the market for parenteral use were determined microbiologically, and it was found that t h e extracts obtained from four companies varied considerably in their Bi2 contents and none of these contained more than 0.02^ of the vitamin on a dry-weight basis. Initial Characterization Studies With the crystalline vitamin available, but in minute quantities, the characterization of the substance was initiated. Emission spectrographic analysis showed the presence of cobalt and phosphorus in the molecule; thus, vitamin Bi» may be thought of as a cobalt coordination complex [Rickes, Brink, Koniuszy, Wood, and Folkers (1948)]. Nitrogen is present, and sulfur is absent. Aqueous solutions of vitamin Bi- may be autoclaved at 120° for 15 minutes without loss of microbiological activity. However, microbiological inactivation in 0.015 N sodium hydroxide takes place at a rate of 20% in 0.67 hours, 45% in 6 hours, and 90% in 23 hours. Similarly, inactivation in 0.01 N hydrochloric acid takes place at a rate of 18% in 3 hours, 75% in 23 hours, and 89% in 95 hours.
FOLKERS, director of organic and bioKARL chemical research at Merck & Co., Inc., Rail-
way, »N. J., never lets a year go by without doing something of extraordinary interest in the chemical world. Last year h e gave a paper on vitamin B12 before the International Congress of Biochemistry in Europe, and later delivered the Harrison Howe Lecture. I n 1948 h e was elected to the National Academy of Sciences. I n 1947 he collaborated^ in work which led to the isolation of crystalline vitamin B12. One keeps on going back, and finds him the recipient of the ACS Award in Pure Chemistry in 1941, the co recipient of the Mead Johnson and Co. Award in 1940, and in 1935 beginning a series of experiments on curare.
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Because of the presence of cobalt in ' t h e vitamin, it was of interest t o test cobalt ion u n d e r conditions w h e r e vitamin B12 is active. However, 1 /*g./ml. of cobaltous ion is inactive for t h e growth of L. lactis, a n d cobaltous ion in q u a n t i ties of 150 fxg. and 500 fig. w e r e inactive clinically. T h e negative clinical response w a s expected in view of t h e amount of cobalt ion in daily food intake. As p a r t of o u r program o n t h e distribution of this vitamin, numerous source materials other t h a n liver h a v e been investigated. T h e properties of a red crystalline c o m p o u n d which has been isolated from t h e culture filtrate of a griseinp r o d u c i n g strain of Streptomyces griseus have b e e n c o m p a r e d with those of vitamin B12, a n d it was concluded that t h e red c o m p o u n d from S. griseus a n d vitamin Bi 2 a r e identical [Rickes, Brink, Koniuszy, W o o d , a n d Folkers ( 1 9 4 8 ) ] . T h e color, melting points or decomposition t e m p e r a t u r e s , a n d t h e refractive indexes of both c o m p o u n d s are t h e same. Both c o m p o u n d s show t h e presence of t h e elements cobalt a n d phosphorus. Both comp o u n d s show t h e same biological activities. T h e activity of each for t h e growth of Lactobacillus lactis is about 11 million units per milligram. Animal protein factor activity for chicks is evidenced at a level of 30 /u.g. p e r kg. of diet. Both compounds show c o m p a r a b l e activity in h u m a n s w i t h pernicious anemia. At t h e Glaxo Laboratories in E n g l a n d , t h e r e was also research on t h e isolation of t h e anti-pernicious anemia factor. T h e r e was a report on two amorphous r e d fractions from liver, which w e r e not homogeneous a n d which showed only general absorption w i t h slight steps at 2,750 A. a n d 5,050 A. T h e clinical activity of such a purified concentrate was in t h e r a n g e of 300 to 600 fig. for patients with pernicious anemia. T h e molecular weight of t h e active substance was believed to b e a b o u t 3,000 [ S m i t h ( 1 9 4 8 ) ] . S o m e w h a t later, it was a n n o u n c e d that final purification a n d crystallization of the factor h a d also b e e n achieved [Smith and Parker ( 1 9 4 8 ) ] . In a later paper, t h e presence of a b o u t 4.0% of cobalt in t h e c o m p o u n d indicating a molecular weight of about 1,600 was r e p o r t e d , and it was found that t h e c o m p o u n d possesses not three atoms of phosphorus as originally believed, b u t one a t o m of phosphorus [Smith ( 1 9 4 9 ) ] . A comparison of t h e red crystalline c o m p o u n d isolated in t h e Glaxo Laboratories with vitamin Bi 2 has shown that they are identical. A g r o u p of investigators at t h e British D r u g Houses in London h a v e also isolated a crystalline anti-pernicious anemia factor from liver [Ellis, Petrow, a n d Snook ( 1 9 4 9 ) ] . This crystalline factor is red, possesses 4 . 0 % of cobalt, a n d exhibits an absorption spectrum w i t h maxima at 2,780 A., 3,610 A., and 5,500 A. Hydrolysis of t h e factor yielded one substance reacting w i t h ninhydrin; n o purines could b e d e t e c t e d in t h e hydrolyzates. It was reported t h a t hydrolysis b y 2 0 % h y d r o V O L U M E
2 8,
NO.
20
» =• »
chloric acid gave a n acidic black amorphous solid containing cobalt. An exchange of samples has shown that this crystalline factor a n d vitamin B12 are identical. Some additional properties of vitamin B12 have been determined [Brink, Wolf, Kaczka, Rickes, Koniuszy, Wood, a n d Folkers ( 1 9 4 9 ) ] . Vitamin Bu> has 4 . 5 % cobalt, showing a minimum molecular weight of 1,300. An ebullioscopic molecular weight determination g a v e a value of 1,490 ± 1 5 0 . Vitamin Bm is optically active. It is a polyacidic base as revealed by titration i n glacial acetic acid. T h e absorption spectrum of the substance in water show maxima at 2,780 A., 3,610 A., a n d 5,500 A., w h i c h d o not shift markedly with a change in p H . Numerous microanalyses show that vitamin B l 2 has a composition typified by C8i _^Hso_ KSNMOISPCo. T h e formulas G^ILe- wNuOiaPCo a n d CeaHss- M N U O I S P C O agree very well w7ith t h e analytical results. Acid hydrolysis of vitamin Bi» does not result in the liberation of amino acids; thus, vitamin B12 is not a polypeptide. Alkali fusion of vitamin B12 yields products w h i c h give the positive test with Ehrlich's reagent characteristic of certain cyclic five-membered nitrogen-containing compounds. Crystalline vitamin B12 has been converted into crystalline vitamin BJ2a by t r e a t m e n t with a platinum catalyst and h y d r o g e n [Kaczka, Wolf, a n d Folkers ( 1 9 4 9 ) ] . D u r i n g this reaction the color of the aqueous solution changes from red t o b r o w n a n d , on exposure to air, the color changes back to red. T h e red crysstalline vitamin B12.1 has refractive indexes which are slightly different from those of vitamin B12. A cobalt content of 4 . 5 8 % a n d a phosphorus content of 2 . 4 3 % reveal that the molecule of vitamin Bi2a is n o t grossly different from t h a t of vitamin B12. T h e absorption spectrum of vitamin Bi 2a shows maxima at 2,700-2,770 A., 3,150 A., 3,525 A., 4,150 A., a n d 5,300 A. which are similar to, but different from, those of vitamin Bt2. Vitamin Bi 2a is highly active biologically as is vitamin Bio, a n d has shown its activity in tests with microorganisms, rats, chicks, and m a n . It is to b e recalled that Smith had reported in 1948 on two amorphous red fractions from liver which w e r e not homogeneous, but which indicated two chemically related compounds. A red crystalline compound has been isolated from Streptomyces aureofaciens w h i c h has been designated vitamin Bi2b [Pierce, Page, Stokstad, a n d Jukes ( 1 9 4 9 ) ] . In this case, it was observed that after charcoal adsorption, chromatography over silicic acid resulted in t h e formation of t w o red b a n d s or fractions. On further purification, these two red fractions gave two ^ed crystalline compounds. O n e of these compounds contained cobalt and phosphorus, and s h o w e d absorption maxima at 2,730 A., 3,510 A., and 5,250 A. It was biologically active for chicks and for the growth of L. leichmanii 3 1 3 . T h e crystals of t h e second red crystalline compound » M A Y
15,
1950
w e r e said to appear similar to those of vitamin B12. Biological Characterization of Vitamin B12 T h e biological aspects of vitamin B12 m a y he separated into at least two categories; (a) therapeutic treatment of h u m a n diseases, ( b ) nutrition of poultry a n d animals. T h e minimum effective dose for therapeutic treatment of pernicious anemia is thought to b e about one microgram daily, or multiples ot this dose at longer intervals. T h e dose for a proper remission has b e e n estimated a t 100 /ig. [Spies, Suarez, Lopez, Milanes, Stone, Toca, Aramburu, a n d Kartus ( 1 9 4 9 ) ] . Alternatively, 3 fig. daily for six weeks, and t h e n 1 fig. daily for a maintenance dose has been suggested [Jones, Darby, and Totter ( 1 9 4 9 ) ] . No evidence of toxicity from vitamin B12 has been reported. Vitamin B12 has hematopoietic activity for the following diseases which are associated with anemia: pernicious anemia (uncomplicated and with neurologic inv o l v e m e n t ) , nutritional macrocytic anemia, certain cases of macrocytic anemia of infancy, sprue (tropical and nontropical). It is being studied in certain diseases which a r e unassociated w i t h anemia. In five cases of amyotrophic lateral sclerosis, injections of vitamin B12 resulted in great subjective improvement. It was not curative [Spies a n d Stone ( 1 9 4 9 ) ] . T h e Intrinsic F a c t o r and Vitamin B12 T h e studies of Castle a n d his associates over a period of years h a v e shown that foods such as beef muscle contain a heatstable substance, designated the extrinsic factor, which produces little or no response w h e n ingested t>y a pernicious anemia patient unless normal gastric juice is also taken with it. T h e activity of gastric juice has been ascribed to the so-called intrinsic factor. The relationship b e t w e e n t h e extrinsic and intrinsic factors has been newly investigated, particularly in regard t o t h e clinical activity of vitamin Bio. Recent results are as follows. Vitamin Bi2, active parenterally at t h e microgram level, has been found to be essentially inactive when administered orally at a dosage level of 5 /xg. daily, b u t active at this dosage when administered orally together with normal human gastric juice [Berk, Castle, Welch, Heinle, Anker, a n d Epstein ( 1 9 4 9 ) a n d Hall, Morgan, and Campbell ( 1 9 4 9 ) ] . Much of the early work was based u p o n the possibility of a chemical reaction between t h e extrinsic a n d intrinsic factors to give another substance which was the effective one [Castle and Ham ( 1 9 3 6 ) ] . Donald Wolf, Thomas W o o d , John Valiant, a n d t h e author have carried out certain experiments on the treatment of vitamin Bu» with gastric juice, a n d on t h e purification of the extrinsic factor from beef muscle. First, w e assayed the gastric juice of six apparently normal individuals for vita1635
min B^-like activity. T h e average value for the " L L D activity" of gastric juice was approximately 100 units per ml. Daily oral doses of gastric juice in quantities u p to 300 ml. were known to produce no hematopoietic response; it is evident that this volume of gastric juice contained only a b o u t 3 fig. of vitamin B12 or its equivalent. Such an amount of vitamin Bw is ineffective orally, but effective parenteraily. It was known years ago [Morris, Schiff, Burger, and Sherman (1932) ] that a vacuum-concentrate of 450 ml. of gastric juice produced a hematopoietic effect when administered intramuscularly; this result can b e interpreted today as d u e to the probable presence of an a d e q u a t e amount of vitamin Bia-like activity in the concentrate. Thus, normal human gastric juice appears to contain vitamin B12 or its equivalent. Next, w e tested the stability of vitamin B12 w h e n it w a s dissolved in gastric juice. T h e solutions were examined for changes in absorption spectrum a n d microbiological activity. T h e absorption spectra of the solutions showed no changes during incubation at p H 2. There was no evidence of a change in the microbiological activity of the solutions a t pH 2, 3, and 7 for the growth of L. lactis. In another experiment, vitamin B 12 was recovered in crystalline form from the gastric juice. It was concluded that there was no evidence for a chemical reaction between vitamin B12 and the intrinsic factor which resulted in a gross structural change in vitamin B12. A possible reaction between vitamin B12 and the intrinsic factor in which there was a very small structural change in the large vitamin B12 molecule, and which could produce as little as 5 micrograms of hematopoietically orally-active product is being studied further. A concentrate with vitamin Buj-like activity from beef muscle was prepared which was active hematopoietically when given parenteraily to three patients with pernicious anemia. The concentrate showed 5 0 0 L L D units per mg., and was given parenteraily in a daily dose of about 20 mg.; this dose had a microbiological activity which was equivalent to about o n e microgram of vitamin B12. Thus, the extrinsic factor in this concentrate elicited a hematopoietic response on a microgram-basis very similar to that of vitamin B12, and it seems highly probable that t h e vitamin Bis-like activity of this concentrate is identical with the extrinsic factor. W e are indebted to Byron Hall of the Mayo Clinic in Rochester, Minnesota for t h e clinical aspects of this experiment. Chemistry of Vitamin B12 The phosphorus in vitamin Bt2 has been liberated a s phosphate by acid hydrolysis [Ellis, Petrow, a n d Snook ( 1 9 4 9 ) ] . Although n o phosphate is liberated at 25° for 17 d a y s in 2 0 % hydrochloric acid, phosphate is liberated at 100°. T h e phosphorus content of 2 % as found b y phos-
1636
phate liberation has been compared with a phosphorus content of 1.9-2.3% as d e termined by another method. Degradation of vitamin Bu b y acid hydrolysis gave a crystalline product having the composition C8HioN2, which has been identified [Brink and Folkers ( 1 9 4 9 ) ] . This reaction is expressed by the conversion of Crc-tKjHw.MNuOisPCo to CeHioNa. Thus, the crystalline degradation product is only about one tenth of vitamin B12, but this part is very interesting. The C P substance melted at 205 to 206° it was basic a n d formed a crystalline picrate melting at 273 to 275°. It was optically inactive, and had a characteristic absorption spectrum which changed reversibly with p H . T h e degradation product, C9H10N2, was suspected of being a substituted benzimidazole. Its absorption spectrum resembled that of 2,5-dimethylbenzimidazole. In the chemical Literature, there is described t h e reaction of benzimidazole with benzoyl chloride in alkaline solution to give the dibenzoyl derivative of 1,2-diaminobenzene. This reaction was applied to t h e degradation product,
CBHION 2 J a n d
a
new
product,
C--
H20N2O2, was obtained which melts at 262 to 2 6 3 ° . When synthetic 1,2-diamino4,5-dimethylbenzene ( I ) was treated with benzoyl chloride in alkali, the dibenzoyl derivative ( I I ) was obtained and found to melt at 262 to 262.5°. When synthetic
CH,/NN-HCOC 6 H 6 CHJ^XHCOC»H6 II 1,2 - diamino - 4,5 - dimcthylbenzene was treated with formic acid, 5,6-dimethylbenzimidazole ( I I I ) was obtained; it melted at 204 to 205°. There were no de-
?CU
pressions of melting points when the degradation product, C0H10N2, a n d the synthetic 5,6-dimethylbenzimidazole were mixed, or w h e n their respective dibenzoyl derivatives were mixed. Thus, vitamin
HCOH HCOH H0CH
I
CH 2 OH VI CHEMICAL
B12 has been degraded to 5,6-dimethyIbenzimidazole ( I I I ) , which is a new benzimidazole. The l,2-diamino-4,5-dimethylbenzene moiety ( I V ) is not without some familiarity in the vitamin field. T h u s , it is a moiety of riboflavin (vitamin B2) ( V ) . T h e isolation and characterization of Llyxoflavin ( V I ) from t h e h u m a n heart myocardium was recently reported [Pellares and Garza ( 1 9 4 9 ) ] . L-Lyxoflavin also has this 1,2 - diamino - 4,5 - dimethylbenzene moiety. It differs from vitamin B2 only in that the carbohydrate moiety is related to L-lyxose, whereas this moiety in vitamin B 2 is related to D-ribose. T h e partial formula ( V I I ) may now be written provisionally for vitamin Bm.
CH
VII Future Vitamin Research Let us consider t h e future of vitamin research. There is obviously much more to learn about the chemistry of vitamin B12. T h e molecular size of vitamin But is several times that of some well-known vitamins and this factor in conjunction with the complexity of vitamin B12 indicated that a substantial amount of research is required to elucidate its chemistry. Biochemical advances on vitamin B12 are expected, and perhaps future medical research on vitamin B12 will disclose entirely new therapeutic applications. There is more to learn yet about the intrinsic factor and h o w it enhances the absorption of microgram-quantities of vitamin B15. Other new vitamins w h i c h are unrelated chemically to vitamin B12 will be elucidated. A highly purified concentrate of "vitamin B12," which is a growth factor for rats, has been reported [Novak and Hauge ( 1 9 4 8 ) ] . "Vitamin Bu" has been described as a brown crystalline compound isolated from urine which has very high actiyity on cell proliferation in vitro and on hemopoiesis in vivo [Norris and Majnarich ( 1 9 4 9 ) ] . T h e r e are numerous publications on microbial factors. T h e r e is more to learn yet about the chemistry of compounds for the anemias. For example, it has b e e n recognized that some patients have macrocytic anemia with megaloblastic arrest of t h e bone marrow which does not respond to liver extract, folic acid, or vitamin B12 [Spies, Stone, Toca, and A r a m b u r u ( 1 9 4 9 ) ] . Again, a patient has b e e n observed [Mueller, Hawkins, a n d Vilter ( 1 9 4 9 ) ] who had macrocytic anemia with megaloblastic maturation arrest of t h e bone marrow and other symptoms which were not completely improved b y liver extract, B12, or, folic acid, and a deficiency of an unknown factor was suggested. DELIVERED as fifth annual Harrison Howe lecture, sponsored by the Rochester Section of the ACS, at the University of Rochester, Nov. 7, 1949.
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