Jan., 1951
ADDITIONALDATAON VITAMIN
with hydrobromic acid. The hydrogenation of apo- and isoapo-&erythroidine yielded the same compound, octahydro-apo-@-erythroidine. Oxidation of apo-&erythroidine gave formic acid.
[CONTRIBUTION FROM THE RESEARCH
335
Interpretation of these reactions and degradation products of @-erythroidine allows one to formulate tentative structures for these products. RAHWAY,N. J.
RECEIVED JULY 18, 1950
LABORATORIES OF MERCK& CO.,INC.]
Vitamin B12. XIII. Additional Data on Vitamin B12a BY EDWARD A. KACZKA, ROBERT G. DENKEWALTER, ARNOLDHOLLAND AND KARLFOLKERS ABSORPTION SPECTRA In a recent communication,' crystalline vitamin IN WATER OF: B*a was described as a reaction product of vitamin -VITAMIN B,* 200BU and hydrogen over a platinum catalyst. The VlTPMlN 8 4 p o ----VITAMIN Blz0 described physical properties of vitamin B12a (FROM ~ . G R I S E ~ ~ ) showed that it could be differentiated from vitamin Bls although these two compounds are obviously very closely related in composition and properties. The results of the lirst preliminary biological tests which were reported,' were not intended as a critical and final evaluation, but to show that vitamin B'Za possesses high vitamin B12 activity. Vitamin Bun has been examined further, and samples, including the one described,' have been 260 300 340 380 4 2 0 460 500 540 580 WAVELENGTH IN Mp. found to be about 98% pure by solubility analyses. Fig. 1. Vitamin B'Za separates from aqueous acetone in needle-like or bladed crystals which belong to the orthorhombic systems. The color of the crystals slight variations in the fiH of the solutions and is somewhat darker red than those of vitamin this influence is demonstrated in Fig. 2. During BIZ. On the micro-block, the crystals start to the early work, the PH variations of water soluwere not deterdarken at about 200°, with more decomposition tions of samples of vitamin appearing to take place than with vitamin B12, mined, It is evident that vitamin B12a and the but do not melt below 300'. The refractive in- crystals from S. griseus cannot be differentiated by dices of two samples (dried in vacuo a t room tem- absorption spectrum (Fig. 1) or refractive indices perature) which resulted from two separate hydro- (Table I). genation experiments are given in Table I. When PBSORPTION SPECTRA the crystals of sample 2 were heated for two hours OF VITAMIN B,,o: a t 100' in vacuo, the refractive indices were found -IN WATER to be: CY, 1.604 * 0.004; P, 1.640 * 0.004; y, -PHOSPHATE MIXTURE PH 5 0 ' ---_PHOSPHATE MIXTURE PH 88' 1.654 * 0.004. This heating of crystals of vitamin Blza does not seem to alter very much the @ and y indices, but does seem to cause a progressive increase in the CY index. The refractive indices may be used as a criterion for identity and reproducibility of vitamin BIZa.
I\
TABLE I REFRACTIVE INDICES OF VITAMIN BIZ. Sample 1 2 Vitamin Blr (from s. griseus)
B
a
1.580 * 0.002 1.640 A 0.002 1.657 1.580 0.002 1.640 * 0.002 1.656 f
1.581 t 0 . 0 0 2
L Y
0.002
Amorphous and crystalline concentrates from culture broths of Strefitomyces griseus, which showed vitamin BIZ activity in microbiological assays, yielded a red crystalline compound after further fractional crystallization. The absorption spectrum of this red crystalline product is like that of vitamin BIZ., but differs from that of vitamin B11 as shown in Fig. l. The slight variations in the absorption spectra of vitamin BIZ. and the crystals from S. griscUs in t h e regions 265-275 w, 300-320 m p and 350-358 m p are influenced by (1) k h ,Well amd Fotkw. Tma
JOURNAL,
V l , 1614 (1949).
,
0
300
E
*
340
I
"
e
I
"
380 420 460 WAVELENGTH IN M,I.
I
500
"
540
"
' 500
Fig. 2.
* 0.002
1.640h 0 . 0 0 2 1.657 a0.002
'
260
--
A sample (NP-92-58-4)z of vitamin B12b,* has been compared with vitamin B12a obtained from the hydrogenation reaction with the following results: The two samples cannot be differentiated by absorption spectrum as illustrated by Fig. 3. The sample of vitamin B1za used for this comparison was found to be 98.5% pure. When powdered samples of vitamin B12a were dried a t 100' for two hours in vacuo and then dissolved in water, the spectrum was found to be, on immediate (S) Through the cowtony of Dr. T. E. Jukes of the Lederle Laboratories, Division of tho Amerimn Cyanamid Company. (8) Piorco, Page, S t o w and Jukr, TEN Jonnwu, 71, 2959 (1949).
336
EDWARD A. KACZKA, ROBERTG. DENKEWALTER, ARNOLDHOLLAND AND KARLFOLKERS Vol. 73
examination, as shown in Fig. 3 ; however, the absorption a t 3150 A. started to change within minutes, and after 24 hours was as shown in Fig. 3 . Another sample of vitamin B l a b (Lederle NP119-17-6)4 was dried similarly and dissolved in water; the absorption was determined within two minutes, particularly a t 3150 B.,and after time intervals of one and five hours. A decrease in the absorption of this solution of Vitamin B12b was observed within this period similar to that seen for vitamin BlZn.
E:
i
240
i .---
.~. ..,-.
I
I
I 280
I
320
I 360
I 400
WAVE
. .q
.,
I 440
I WO
I
10
I
560
TABLEI11 ACTIVITYOF VITAMINBlZaIN RATS 00
Substance
, mp.
LENGTH
Fig. 3.-Spectra in water of vitamin BI?, after drying in vacuo a t 100" for two hours: -, solution a t t = ca. 1 min.; -.-, solution a t t = ca. 15 min.; ---,solution a t t = 24 hr.; * * . , spectrum of vitamin BEb (Lederle NP-9258-4) after drying a t 100" for two hours in vacuo; water solution a t t = over 30 minutes.
A sample of vitamin B12al vitamin B12b (NP92-58-4)) and a mixture of the two, were examined by paper-strip chromatography. The two samples and the mixture showed a single spot of comparable RF values of about 0.14, and there was no evidence of a separation of the mixture, whereas mixtures of vitamins Blz and B12a, and B I and ~ B12b did separate. The solvent system used was 10% acetic acid40% butanol-50yo water. This sample of vitamin B12b was found to be insoluble in 85% acetone solution previously saturated with vitamin B12a. Vitamin B12b is readily soluble in 85% acetone. These data indicate not only that the sample of vitamin B12b was substantially pure, TABLEI1 h~ICRORIOI.OGICAI, DATA
L. leichmannii' Sample auto L. locfis Aseptic claved (Titriaddn. with metric)S CUP)^ of sample. medium, units/@g. units/@g. units/pg. units/pg.
1 Vitamin Biza 7,700 2 Vitamin Btza (from S. griseus) 6,600 3 Vitamin B12b (Lederle NP-92-58-4) 6,800 4 Vitamin B I ~ 11,000
11,000
but that the major component was identical with vitamin B l 2 a . An infrared absorption bands a t 2140 em.-' exhibited by vitamin B12, but which is absent in the spectrum of vitamin B12b, is also absent in the spectrum of vitamin BEa. Vitamin Biza, the crystals from S. griseus and vitamin BlZb were assayed microbiologically using Lactobacillus lactis and Lactobacillus leichmunnii. The results which are summarized in Table I1 show that those sampIes cannot be differentiated I)y these assays. Vitamin B l 2 has shown "animal protein factor" activityg when fed to rats on a diet devoid of animal protein and containing o.25Y0 of thyroid powder. Vitamin B E a was first assayed' at a level of 0.125 pg. in this test and, as seen in Table 111, the response was equivalent to that elicited by 0.063 pg. of vitamin Bl2; when one-half the dose of vitamin B12a was fed, the gain in weight was also comparable to that elicited by an equal weight of vitamin BIZ. Thus, vitamins B12 and B12a have comparable activities in this test.
9,500
10,800 11,000 10,000 11,000 11,000
2,100 2,350
1,900 11,000
(4) Kindly sent by Dr. R. Stokstad. (5) Caswell, Koditschik and Hendlin, J. Bid. Chem., 180, 125 (1949). (6) Foster, L d l y and Woodruff, Science, 110, 507 (1949). (7) Hendin arid Soars, Z -Rid. Chcm. in press.
Controls (undosed) Vitamin Btz Vitamin BIZ Vitamin Blza Vitamin Blza
Dose fed daily, pg.
... 0,063 ,125 .063 .125
Wt. No. of increment, male rats g . , 15 days
10 10 10 10 10
27 56 61 60 59
It was stated' that vitamin BlZa has 30 * 15% of vitamin B12 activity in chicks. Additional tests with vitamin B12a1 as well as a preliminary comparison of vitamin B U a and vitamin BlZb (NP92-58-4)) indicate that both the same order of vitamin B 1 2 activity, approximately 50%, in the chick assay, under the conditions employed. These comparisons of vitamin BIZa and the sample (NP-92-58-4) of vitamin BIZb have not revealed a difference between them, but have provided results which can be interpreted only on the basis that vitamins B 1 2 a and B12b are identical. Dr. Randolph West made the first clinical test of vitamin B12a on a pernicious anemia patient. He reported that it possessed activity like vitamin Blz and in one patient about 30% of a maximal hematological response was observed with a dose of 25 pg.l Dr. Tom SpieslO has further examined the activity of vitamin B l z a and has found that it is effective in promoting clinical improvement and in producing positive hemopoietic responses in people with Addison's pernicious anemia, tropical sprue, non-tropical sprue, nutritional macrocytic anemia and in one case of megaloblastic anemia of infancy. Because of the extreme variability from patient to patient, comparative studies on the effect per unit of weight of vitamin B12 and vitamin B12a will take some time to evaluate. However, it has been established that vitamin B12al like vitamin BIZ,produces a rise in reticulocytes, (8) Brockman, Pierce. Stokstad, Broquist and Jukes,THISJOURNAL, 72, 1042 (1950). (9) Emerson, Proc. SOC.Ezp. Bid. and M c d , TB,392 (1949). 110) Personal communication.
Jan., 1951
ISOLATION O F
VITAMIN BlZb FROM NEOMYCIN FERMENTATIONS
red blood cells, white blood cells, platelets and hemoglobin, and promotes a return of the bone marrow to normal. Experimental Vitamin Blh from the Reaction of Vitamin BIZ$th Hydrogen in Presence of a Platinum Catalyst.-A solution containing 349 mg. of vitamin B1s in 70 ml. of water was shaken with approximately 300 mg. of platinum oxide catalyst and hydrogen gas under substantially atmospheric pressure a t cu. 25” for 40 minutes. During the reaction, the color of the solution changed from red t o brown. The solution was then diluted with acetone t o a volume of cu. 400 ml. and, after a short time, the catalyst settled. After removal of the catalyst by centrifugation, the solution was further diluted with acetone t o a volume of ca. 900 ml. and after a short time vitamin Blh began t o crystallize in the form of darkred slender needles. After allowing the solution to stand overnight, 200 mg. of vitamin Bl%,was obtained. Recrystallization of this product was accomplished by dissolving the compound in ca. 30 ml. of water and diluting the solution with CU. 300 ml. of acetone; yield 150 mg. A solubility analysis showed that the product had a purity of 98.5%.
Acknowledgment.-We are indebted to Dr. Charles Rosenblum for measurements on the [CONTRIBUTION FROM THE
337
crystallographic properties of vitamin Baa, Mr. Fred Bacher for solubility determinations, Dr. N. R. Trenner for infrared absorption spectrum determination, Dr. David Hendlin and Miss M. Soars for microbiological assays and to Drs. Gladys Emerson and Walther Ott for animal assays. Summary A new crystalline product, designated vitamin B12a, has been produced from vitamin Bl2 by utilizing a catalytic hydrogenation technique and has been characterized by several criteria. Using the described criteria, it is concluded that vitamin Bus, the red crystalline product isolated from concentrates from s. griscus, and vitamin Bltb are identical. Vitamin Blza has a biological activity like that of vitamin Blt in assays using L. lactis, L. leichmannii, rats, chicks and humans. RAHWAY, NEWJERSEY
RESRARCH LABORATORIES OF THE UPJOHN
RECEIVED JULY 31, 1950 COMPANY]
The Isolation of Vitamin BIZbfrom Neomycin Fermentations BY WILLIAMG.
JACKSON,
GEORGEB. WHITFIELD,WILLIAMH, DEVRIES, HARRISON A. NELSONAND JOHN S. EVANS
The isolation from liver of the anti-pernicious fermentations.120*a It was differentiated from B12 anemia factor, vitamin Blt, has been reported by by the location of its absorption peaks a t 352 and workers in this countryla and in England2; its 525 mp.” The corresponding Bl%peaks are a t 361 isolation from streptomycin fermentations has been and 548 mp.l*314 The relation of vitamin B12a to announced.’b**a The “animal protein factor” is ap- B18bhasnot been completelyclarified.16a~bBoth were parently identical with, or owes its principal activ- obtained from BE by supposedly similar hydrogenaity to vitamin Bls or B12b.4J Vitamin Bl%has been tion conditions and their absorption spectra are converted to B12a by hydrogenation,6and to B12b by nearly identical, save in the 315 mp region where hydrogenation’ and by “prolonged mild acid hy- B12a has a density 80,” vs. 43 for B12b. drolysis.”ab Vitamin Blzb has been shown8to be the Methods of Assay.-Fermentation harvests and full equivalent of B12 against Addisonian pernicious brown extracts were assayed microbiologically anemia. We wish to report the isolation of vitamin against crystalline BIZ in a Lactobacillus lactis B12b from neomycin fermentations, and to provide Dorner turbidimetric assay, adapted from that of the working details of an extraction process by Shorb.’” The medium is a modification of that which one may start with a neomycin fermentations used by Guirard, et aL1’ and obtain the vitamin in pure crystalline form. Extracts having a purity of 5 mcg. per mg. or Vitamin B12b has been isolated from liver,l0 from better usually had a pink or yellowish-pink color aureomycin fermentations” and from streptomycin and showed optical peaks a t 526 mp and at 352 mp, (1) (a) Rickes, Brink, Koniuszy, Wood and Folkers, Science, 107, strongly suggestive of the Bllb spectrum and With 396 (1948); (b) ibid., 108,634 (1948). intensity at 526 mp corresponding roughly to the (2) Smith and Parker, ibid., 48, viii (proceedings) (1948). microbiological potency. At this time Dr. Fricke (3) (a) Smith, Fantes and Ball, Abstracts of Papers, A. C. S. Meeting, Spring 1950, p. 10A; (b) Brockman, Pierce, Stokstad, Broquist of the Abbott Laboratories kindly supplied us with and Jukes, ibid., p. 11A; (c) Folkers, ibid.. p. 11A. the complete quantitative ultraviolet and visible (4) Stokstad, Page, Pierce, Franklin, Jukes, Heinle, Epstein and spectrum of crystalline B12bisolated from streptoWelch, J. Lab. Clin. Mcd., 88, 860 (1948). (5) An excellent review article has just appeared summarizing the mycin fermentations, and comparison showed our history of the anemia problem, animal protein factor, folic acid and the semi-purified extracts to be similar if not identical Ba vitamins: Smith, J. Phurm. Pharmacal., 9, 409 (1950). (6) Kaczka, Wolf and Folkers, THISJOURNAL, 71, 1514 (1949). (7) Brockman, Pierce, Stokstad, Broquist and Jukes, ibid., 73, 1042
(1950). (8) Lichtman, Watson, Ginsberg, Pierce, Stohtad and Jukea, Proc. SOC.E x g f l . Bioi. M i d , TS, 643 (1949). (9) Fermentation data are covered in a separate paper by Nelson, Calhoun and Colingrworth, presented st the September, 1QS0,A. C. S. meeting, Chicago, Ill. (10) Stokstad, Jukes, Pierce, Page and FranMin, J . E M . Chnn., 180, 647 (1949). (11) Pierce, Page, Stokstad and Jukes, THISJOURNAL, 71, 2962 (1949).
(12) Fricke, Lanius, DeRose, Lapidus and Frost, Fcderalion Proc., 9, 173 (1950). (13) Ellis, Petrow and Snook, J . Phurm. Pkavmacol., 1, 60 (1949). (14) U. S.Pbarmacopoedia XIII, Third Sheet Supplcment, p. 8. (15) (a) Stokitad, Jukes, Brockman, Pierce and Broqdst, Fcdrratio?? The identity of Birs and PYOC., 9, 122 (1950). (b) ADDEDIN PROOR: Bub has now been claimed (Brink, Kuehl and Folkers, Science, 11% 354 (1950)) and the name “hydroxo-cobalamin” has been proposed (Kaczka. Wolf, Kuehl and Folkers, i b i d . , p. 354). Vitamin Bu, by this nomenclature. is “cyano-cobalamin.” (16) Shorb, J . Blot. Ckcm., 169,455 (1947); J. Eoct., 63,669 (1947). (17) Guirard, Snell and Williams, Avcb. Eiochcrn.)9, 861 (1846).
