alkylatcd iiiniiio nitro coiiipouii(ls tleerc;i:+. Summary nicirkctlly with increasing size of the alkyl groups. 1. ~ , - l , ~ ~ - ' ~ r i i o ~ l ~ ~ ~ l11:)s i r iI)WII ~ ~ t sl iy ~- ~ l ~ ~ ~ ~ i l i 'I'he iiicltiiig points of the trilialogeno derivatives thesizeti. of ;iiiilinc, iiiononietliyl:iniliiie and diinetli~~lntiiliiic 2. p - I odoari i li 11 e , p - iotlc )di me t 1i y 1:I i i i 1i 1ic , ?,-I,( ishow tlic same decrease; thus, tlic iiieltiiig points triiodoaniline and 2 , I ,(j-triiodotliii:etli~.lniiilirie for the tricliloro derivatives arc icS.5, : 2 n i i c l have been examiiicd froiii the staritlpoiiit o f halo< 2 3 " ; for the tribromo, 122, ;{!I and < 2 h 0 , re- gen reactivity, in reactions with acid stannous spectively ; and for the triiodoaniline and di- cliloride, bromine and nitrous acid. ;{. X s predicted from the theory of damped resoIiiethylaniline, 1% and (i9-iOo, rcspectivcly. The authors believe t h a t these decreases are an- nance, the halogen atoms i n 2,~,O-triiorlodimethylother example of the same phenomenon recorded aniline show a relatively low ortlcr of reactivity. by Arnold, Pierce and Barnes. EDMONTON, ALBERTA, CANADAKECEIVEIIAPHII.28, 1947
[ C O N TR1iiUTIO.V FROM THE RESEAKCIi r2ABORATORIES O F
MERCKA N D
CO., ISC.]
Isolation of Rhizopterin, A New Growth Factor for Streptococcus Lactis R BY EDWARD L. RICKES,LOUISCHAIETA N D Experiments performed in this Laboratory very early demonstrated the presence, in some natural innlterials, of more than one factor capable of supportiiig the growth of Streptococcus lack^ R' on a "folic acid" deficient m e d i ~ n i . ~ Differential assays of concentrates from several natural sources indicated t h a t some possessed a much higher activity for S. lachis R than for Lactobacillus This property was shown to a striking degree by a charcoal adsorbate6 derived from the purification t)t' Rhizopus nigricans fumaric acid fermentatioii liquors. The fractionation of ai1 eluate of this adsorbate by means of chromatographic adsorption procedures led to' the isolation, in crystalline form, of a compound highly active for S. I n c h R and substantially inactive for L . casei.ti Subsequent investigations have shown that this new growth factor is a Because of the nature and source of the factor, the name rhizopterin has been adopted for this compound, formerly referred to as the "S.L.R. f a ~ t o r . " ~ It was found t h a t rhizopterin was relatively weakly adsorbed on alumina and appeared in the initial eluates. It could be distinguished frorn succeeding more strongly adsorbed fractions, which were active for both organisms. The more strongly adsorbed material which appeared iii the later eluate fractions corresponded closely in chromatographic adsorption behavior and biologi(1) Present address: National Institute of Health, Bethesda, Md. ( 2 ) This organism iri also known as Sfreplococcus faccalis R . (3) Assay method of Mitchell, Snell and Williams, T H I SJOURNAJ., 63, 2284 (1941). (4) Norit eluate factor assays using
L.casci: Hutchings, Bohontm and Peterson, J. B i d . Chcm., 141, 521 (1941). (5) This adsorbate was obtained from Chas. Pfizer and C o . ( f . J. H. Kane. A. Finlay, P . F . Amann, U. S. Patent 2,327,191 < l!>+3). ( t i ) Keresztesy, Rickes and Stokes, Science, 91, 465 (1943). (7) Wolf, Anderson, Kaczka, Harris, Arth, Southwick. Mozingo and Folkers, THISJ O U R N A L , 69, 2753 (1947). ( 8 ) Rickes, Trenner, Conn and Keresztesy, ibid., 2751 (1947). (9) Stokes, Keresztesy and Faster, Scirncr, 100, 522 (1944).
JOHN
C. KERESZTESY'
cal activity t o other concentrates of vitamin Bc (pteroylglutamic acid). T o obtaiii rhizopterin in crystalline form, it was necessary to effect a 200,000-fold purificatioii from the initial charcoal eluate. This was accomplished through the following sequelice oi steps : rlutioii of the charcoal adsorbate, readsorptiori by norit A arid elution, adsorption by fuller's earth and elution, precipitation a t pH 7 , chroniatographic adsorption on alumina, and crystdlization as the free acid or ainnionium snit. The microanalytical data for rhizopterin are iii best agreement with the formula C15Hil'N&4. The pterin-like nature of the compound is iiidicated by a comparison of the absorption spectra of rhizopterin and xarithoptcriii (Figs. 1 a i d 2 ) . The pterin nature is further dcmonst.rated by the potentiometric titration (lata for these two corn-p u i i d s (Table I ) . ,4n equivalent weight.of I(i7.3 TABLE I Initial pH hIidpoint Neutralization point
Rhizopterin
Xanthopterin
4.3 7.2 10.2
5.0 9 0 10.8
was calculated for rhizopterin. Since the poteiitiometric titration indicated t h a t it is a dibasic acid, one may assume a molecular weight or :{:$.?, which is in agreement with the value of 340 calcuO ~ . physical prolwrties lated for C I ~ H I ~ N ~Other of rhizopterin, such as its insolubility i l i water and common organic solvents, its solubility i i i acid and alkali, and the failure of the corr,puuiid to melt up t o 300' fprther indicate the correlation of rhizopterin with the pterins. Later chemical and physico-chemical studies confirnied the pterin-like nature of the c o m p o ~ n d . ~ . ~ It was found t h a t 0.000034 y of rhizopterin per ml. of culture medium is necessary t o produce half maximum growth of S. lactis R. I t is essentially inactive for L.casei.
2730
n--
i l
---
T
-__ I aiitl tlic p l l inaiiititiiietl a t 2.5 I)y thc .~tltlitionof 50%
I
2300
2700 3100 3500 3900 Wave length in A. Fig. 1.-Absorption spectrum of rhizopteriii: (1) PH 12.6, (2) PH 7.0 and (3) PH 1.3.
r '
-
"
"
"
I
'
20
e.
0
X
15
q" 10 5
2300 Fig. 2.-A
2700 3100 Wave length in
3500
3900
A.
bsorption spectrum of xanthopterin : (1) p H 12.6, (2) pH 7.0 and (3) pH 1.3.
Rhizopterin was found t o be inactive for hernoglobin formation and growth promotion in chicks on a diet containing all known nutrients except members of the folic acid group. When the compound wgs fed a t the rate of 1 mg. per r a t per day, the granulocytopenia which was induced i n rats on a folic acid deficient diet containing succinylsuiiathiazole was not cured. Vitamin B, (pteroylglutamic acid) was effective a t 20 y per day. Experimental Elution of Adsorbate.-Moist charcoal adsorbate6 (180 lb.) was stirred for one hour with 150 gal. of 40'% ethanol containing 0.25% sodium hydroxide. The eluate showed an activity for S. Eactis R corresponding to 0.004 to 0.007 y of rhizopterin per mg. of solids, or a total of 140 t o 200 mg. of rhizopterin, and a n activity for L.cash corresponding t o a total of 20 mg. of pteroylglutamic acid. Readsorption on Norit.-The eluate was acidified with dilute sulfuric acid t o PH 2 t o 3. Norit A (5 lb.) was added and the mixture stirred for one hour and then filtered. Elution of Readsorbate.-The combined readsorbates from five accumulated batches were suspended in 150 gal. of 30% ethanol containing 0.5% sodium hydroxide. The suspension was stirred for one hour and filtered. This eluate contained the equivalent of approximately 1.O g. of rhizopterin and 100 mg. of pteroylglutamic acid. Fuller's Earth Adsorption and Elution.-The eluate (150 gal.) from the norit readsorption was diluted t o 300 gal. with water and promptly adjusted to p H 2.5 with sulfuric acid. Fuller's earth (6.5 kg.) was added slowly
sitlftiric liltcrctl.
The inistiire w:is stirrer1 for one hour :tiirI 'The filtrate w:ic; trratctl w i t h :L srcoiirl cqii;il ;tiiiouiiL of fuller's earth ant1 then tliw:trc!ctl. ?'he coinhined fuller's earth :i(Isorl):tte>were suspcndcri i i i :L inistiire of 15 gal. of ethmol (!)5yo), 5 gal. of concciltr:itetl (2Xyo,) ;iinnioiiiiiin hyriroxirlc :tiid 3 )g i l . of w;tter a n d stirretl for one hour. The suspension wits filtered m r l the filtratc concentrated in zlacz~o to 3 to 5 liters. O n standing i n the cold this solution, the p H of which wit\ approximatcly 7, deposited a precipitate containing the Active mxterial. Additional active material was recovered from thc filtrate in the form of a precipitate which separated on :tci-iificatioii to PI3 2.5. pH 7-Precipitate .-The precipitatc was separated from the liquor by centrifugation, washed with 6% acetic acid followed by alcohol and acctone, and dried in wactdo. This precipitate (90 t o 1.50 9.) contained 600 mg. of rhizopteriii of approximately 0.5% parity. Six batches of this mitcrinl totaling 886 g. were combined for further purification. Chromatographic Adsorption.-A portion of the combined pH 7-insoluble fractions (126 9.) was extracted three times by stirring for one-half hour with 1 liter of 0.5 S hydrochloric acid. The insoluble residue was removed by centrifugation and discarded. The extract was stirred with 5 volumes of 9.5% ethanol, the PH was adjusted to 4.5 with 30% sodium hydroxide and the precipitate which formed was removed by filtration and discarded. The clear filtrate (i8 liters) was stirred with aluminum oxideln (100 to 150 g . ) for one hour. The alumina was removed and the filtrate chromatographed under pressure on a column 72 inm. in diameter containing 200 g. of alumina. The column was washed with 2 liters of 95% ethanol and the filtrate d'iscarded. Elution was then carried out with a mixture containing 20 volumes of methanol, 10 volumes of concentrated (28%) ammonium hydroxide and 70 volumes of water. The PH of the effluent was carefully followed after the addition of the ammoniacal eluant. Immediately following a rise in the PH of the eluate, fractions were taken as indicated in Table 11. :tritl.
TABLE 11 Fraction
1 2 3 4 5 6
Volume, ml.
Rhizopterin.
Solids,
mg.
mg.
100 100 100
2.2 100.0 50.0 13.6 7.3 4.2
100 250 2 50
70 840 710 470 320 250
Purity,
%
3.0 11.9 7.0 2.9 2.3 0.68
A total of 7 columns similar to that described above was required for processing the combined batch of PH 7411soluble material. The first four or five fractions from each of the seven chromatograms were combined. The resulting solution contained approximately 60 y of rhizopterin per mg. of solids and represented a total of 1.2 g. of rhizopterin. This solution also showed an activity for L.casei corresponding t o approximately 20 mg. of pteroylglutamic acid. The combined fractions were concentrated i n vacuo to 2 liters. The solution was made alkaline (PH 12) with ammonium hydroxide and treated with five volumes of 95% ethanol. The precipitate which formed was removed by filtration and discarded, and after removal of ixcess ammonia by distillation in vacuo, the filtrate was acidified to PH 4.5 with glacial acetic acid. This solution was then chromatographed on a column 72 mm. in diameter which contained 400 g. of alumina. The column was washed thoroughly with 80% ethanol and eluted with 50% methanol containing 3% concentrated ammonium hydroxide. Fractions (Table 111) of 100 ml. each were taken in the manner described for the first chromatogram. The material showing L . cas& activity was eluted from the column in later fractions not illustrated in the table. (10) Merck-Brockman alumina was used throughout this work.
?’Aill.E
I;~.LCLIOII
1 2 3 4 5
Itliizo~itrrin, mK.
1x
111 I
