JR., R.P. GRABER AND KARLFOLKERS Vol. 68 1460 1’. A . KuEIiI,, JK., R. L. P E C K , C. E. HOFFHINE,
[ CONTRIBUTION F R O M THE IZESEARCH I~AKOKATORIESO F h f E R C K 8r CO., I K C . ]
Streptomyces Antibiotics. VIII. Isolation of Streptomycin BY FREDERICK -4. KGEHL,JR., ROBERT L. PECK,CHARLES E. HOFFHINE,JR., ROBERTP. GRABER AND KARLFOLKERS Some chemical and biological properties of streptomycin helianthate, hydrochloride, sulfate and p- (2-hydroxy-1-naphthylazo) -benzenesulfonate have been described.’ The preliminary steps in the purification of the streptomycin concentrates and the preparation of these salts of streptomycin are described herein. The first concentrates of streptomycin, which were prepared for use in microbiological experiments, were obtained from the culture broths of Streptomyces griseus by the following steps : adsorption on Norite-A, elution with dilute acid, neutralization and concentration in vucuo to a residue. The crystalline reineckate of streptomycin, which has been d e ~ c r i b e d ,was ~ obtained by a sequexe of steps similar to that used for streptothricin r e i n e ~ k a t e . ~These steps were : charcoal adsorption, elution with mineral acid, precipitation with phosphotungstic acid, conversion of regenerated bases to crude picrate, chromatography of picrate and finally preparation of the reineckate. Another method for the purification and isolation of streptomycin has been describe~l.~The steps were as follows: clarification of broth with charcoal, adsorption on charcoal, elution with inethanolic hydrogen chloride, and chromatography with alumilium oxide. A three-step charcoal adsorption process for the purification of streptomycin also has been used.6 Our sequence of steps for the isolation of streptomycin, which differs in several respects froin those described above, is as follows: charcoal adsorption, elution with methariolic formic acid, precipitation with picric acid, conversion to the hydrochloride, chromatography, and finally conversion to the heliant.hate. The steps consisting of charcoal adsorption, elution with methanolic formic acid, precipitation with picric acid, and direct conversion to the hydrochloride were car(1) K u e h l , P e c k , WCValti a n d Folkers, Science, 102, 34 (1945). ( 2 ) Schatz, ?3n:ie and W a k s m a n , Proc. SOL.E x p . B i d . M e d . , 66,
00 (1!144). ( 3 ) ITricd :ind Winterstriner. S,.irnce, 101, 013 (1945). ( 4 ) C:rrter, C l ~ r r k IJickinan, , I , w , Skell a n d Strong, J. I3iol. Chcin., 160, 337 (1043). ( 5 ) 1.t. Puge a n d Campbell. i b i d . , 162, 163 (1946).
ried out essentially as described for streptothricim6 The concentrates of streptomycin hydrochloride showed an average activity of about 100-200 units/mg. The chromatographic step was carried out either with columns of aluminum oxide or Darco G-GO. By these chromatographic procedures, fractions showing activities up to about 7.50 units/mg. were obtained. Treatment of samples of streptomycin hydrochloride, which showed an activity of about 400 units/nig., or higher, with the sodium salt of helianthine (methyl orange) or of p-(2-hydroxy1-naphthy1azo)-benzenesulfonicacid (orange 11), yielded the corresponding crystalline salts of streptomycin. The helianthate was utilized for preparative purposes After recrystallization the helianthate was converted into the hydrochloride or other salts such as the hydrobromide or sulfate. The sulfate was obtained crystalline, but it was difficult to reproduce and i t was frequently contaminated with streptidine sulfate.? The helianthate also was converted directly into the crystalline streptomycin trihydrochloride-calciunl chloride double salt .8 The desirable precautions in the conversion of the helianthate into other salts have already been noted.? Potentiometric titrations were carried out on three salts of streptomycin and the data obtained were as follows: helianthate, eq. wt. 1600, PK‘a 7.50; calcd. for 1495.6; hydrochloride, eq. wt. 740, pK’a’’ 7.66; calcd. for C21H37N7012.3 HC1, 6S9; hydrobromide, eq. wt. 869; calcd. for C21Ha7N7012.3 HBr, 823. As already stated,8 a cryoscopic molecular weight determination on streptomycin hydrochloride in water gave a value of about 800 for the free base (calcd., 580). A colorimetric determination of the helianthine in streptomycin helianthate gave a value of 205 for the combining weight (calcd. combining weight, 193). The results of these determinations and the microanalytical ( 6 ) P e c k , Walti, G r a b e r , Flynn, Hoffhine, Alllrey a n d Folkers, THISJ O U R N A L , 68, 772 (1946) ( 7 ) P e c k , G r a b e r , WaItl, Peel, IT(tRhtne a n d Iqolkers, r b ~ d, 68, 20 (1946) ( 8 ) P e c k , Brlnk, K u e h l , Flyun, Waltt a n d Folkers, rbrd , 61, 1866 (1948).
ISOLATION OF STREPTOMYCIN
Aug., 1946
1461
was adsorbed on charcoal from the culture broths of Streptomyces griseus and eluted from the adsorbate with methyl alcohol-water solutions containing formic acid. The eluate was concentrated into a crude amorphous streptomycin formate. The streptomycin was purified further by precipitation with picric acid followed by conExperimental version t o the hydrochloride. The details of the manipuWe are indebted t o Dr. H. B. Woodruff and Mr. D. lations of. these steps are essentially the same as those Hendlin of the Microbiological Department for the assays. described for streptothricin.6 The preparations of strepB . subtilis was employed as a test organism in a cup assay tomycin hydrochloride obtained by these procedures method which they developed and which was similar t o the showed 100-200 units/mg. Chromatographic Purification of Streptomycin with paper-disc plate method.9 of acid-washed aluminum Preparation of Crude Concentrates of Streptomycin Aluminum Oxide.-Columns Hydrochloride froin Culture Broths.-The streptomycin oxide were prepared and used for the chromatographic purification of streptomycin in the same manner as has TABLE I been described for streptothrich6 The data obtained on representative concentrates of streptomycin hydrochloride CHROMATOGRAPIIIC PURIFICATION O F STREPTOMYCIN RYare summarized in Table I . DROCHLORIDE WITH ALUMINUM OXIDE Aluminum oxide was also suspended in methanol soluProducts-tions of crude streptomycin hydrochloride for the removal % of gross impurities. From a given sample of concentrate, ActivAdsorbate the fractions obtained by chromatography were of higher ActivActivity" ity Elureactivity than those obtained by treatment with a suspenity units/ covunits/ Ad,rorbent ates sion of aluminum oxide. g. Type ml. ered g. mg. mg. g. Chromatographic Purification of Streptomycin with Darco 800 Alumina 200 2.42 28 45 78 372 G-60.-The columns of Darco G-60 and filter paper mix600 9.67 46 154 ture were used for the purification of streptomycin ac113 2000 8.67 35 cording to the procedure which has been described for -streptothricin.0 A summary of typical results is given in 10.1 Table 11.
data are in agreement with the molecular formula C21H57-39N701~.3 H X for these streptomycin salts. Only one of the three salt groups of streptomycin can be titrated with sodium hydroxide solution.
7 -
30.0
110
600
illumina
500 500
1500
4.122 2.687 3,145
380 150 160
46 12 1 :j
--
TABLE I1 CHROMATOGRAPHIC PURIFICATION O F STREPTOMYCIN DROCHLORIDE WITH
73 20
150
400
Alumina
100 500 500 1000
1.040 4,795 1.851 1.651
370 330 220 240
12 53 1:I 13
-.
9 1. 11.4
170
243
4:1 Alumina !mpercel
50 100 100 200 630
0.434 1.275 0.847 0.987 1.333
690 400 210 180 200
50 100 100 1000
0.237 3.678 1.711 2,786
0 410 190 160
250 150 250 330
0.698 1.031 0.729 0.4132
500 490 290 160
Adsorbate Units/ G. mg.
-4dsorbent Filt er Darco paper G-60 pulp, Eluates ml. g. g.
!I
200
333
1:l .4lumina !iupercel
55
15
25 25 150
0.232 .419 ,730
350 330 220
Cb 17 28 34
2.04
250
144
16
25 27 25 50 75
.lo0 .148 .111 . 130 .115
700 540 360 270 2FO
14 15 8 7 6
2.75
350
145
15
25 25 50 50 250
.202 .355 .820 .367 .208
780 1100 400 220 210
16 21 33
40 25 25 25 500
041 332 .503 502 1 150
880 870 530 410
3 26
375
38
25 26 50 50 500
0.046 092 369 584 1 090
650 510 540 500 300
4 6 25 36 40
-.
... 4 ti 1 (I 19
-
88 6.0
225
160
Alumina
23 34 14 4-.
3.20
350
136
14
I it
3 54
40
400
Alumina
100 100 200
10.6 7.2 6.0
720 538 433
54: 27 18
9C
1 0
500
30
.Alumina
15 15 50 100 35b
0.147 0.147 0.146 0.141 0.317
640 700 620 540 420
18 21 19 15 27
101
The accuracy of the assay method was estimated t o be 15%. Water. (9) Loo, Shell, T h o r n b e r r y , Ehrllch, McGuire, Savage a n d SylJ B a d , SO, 701 714 ~I(i.t.7)
ve5ter
Art i vity
Activity recrivunits/ ercn.
250
74: 16.6
-----Products-
1.90
1!i 2Li 10 14
HE.-
DARCOG4O
2.7
300
120
20
g.
mg.
8
4
24
18
Streptomycin He1ianthate.-A 26.3-g. sample of streptomycin hydrochloride (500 units/mg.) was dissolved in 390 ml. of methanol and the solution was heated t o approximately 5.5'. A solution containing 20.8 g. of methyl orange in 1456 ml. of water was heated t o 75" and added :t the methanol solution. After standing overnight a t 10 , the mixture was centrifuged and t h e rryctalline product
1462 F. A. KUEHL,JR., R. L. PECK,C. E. HOFFHINE, JR., R. P. GRABER AND KARLFOLKERS Vol. 68 was washed with approximately 200 ml. of water. Re- hot aqueous methanol, The crystals were then collected crystallization of the helianthate was accomplished by dis- by filtration and washed with water, isopropanol, acetone solving it in 3 liters of hot 33% aqueous methanol and and ether, and dried a t 25" in vacuo over phosphorus allowing the solution t o stand overnight at 10". The pentoxide. The yield was 6.3 g., activity about 300 units/ helianthate was then removed by centrifuging and washed mg. with 200 ml. of water and with 200 ml. each of isopropanol, Anal. Calcd. for CnlH3,N7012(ClsH12S~04S)a.5H?O: C, acetone and ether. The weight of the dried product was 50.09; H, 5.06; N, 11.01; H:O, 5.4. Calcd. for CZIHW 32 g. (87y0 yield). S ~ O ~ ~ ( C ~ ~ F I I ~ N ~C,O 50 ~ S03; ) ~I€, . ~5.17; N ~ OX,: 10.99; Anal. Calcd. for C Z ~ H ~ ~ N ~ O ~ Z ( C ~ ~C, H 50.59; I I N ~ OHzO, ~ S )5.4. ~ : Found: C , 50.01; H, 5.34; N, 11.03; weight H , 5.53; N, 14.99; S, 6.43. Calcd. for C2,H39N7OI2- loss on drying to constant weight a t 100" in vacuo, 5.3. (CliHlbS303S)3: C , 50.52; H , 5.65; N, 14.97; S, 6.42. Conversion of Streptomycin Helianthate to Streptomycin Found: C, 50.38; H , 5.86; N, 15.02; S,5.76. Sulfate.-A sample of streptomycin helianthate weighing I n the melting point determination on the micro-block, 32.6 g. was suspended in 150 rnl. of water by stirring methe helianthate decomposed a t 220-226 '. The crystals chanically. Thirty milliliters of 2 N sulfuric acid was were dark red in color, but under the microscope they added slowly and the stirring was continued for forty-five appeared as flat yellow plates. Upon drying, the crystals minutes. A t the end of this period, the helianthine was in lost solvent of crystallization and changed into an amor- crystalline form and was removed by filtration through a layer of Darco G-60. The colorless filtrate was lycphilphous copper-like powder. ized, yielding a voluminous fluBy white powder. After Anal. Found: C , 50.53; H , 5.83; N, 14.34. for two hours a t 100' in vacuo, the product weighed After one more recrystallization, the following data were drying ~ 12.2g. ltshowed [ a ]-79.5°(c,170inwater)andhadan obtained. activity cf about 700 units/mg. This preparation conA n d . Found: C, 50.45; H, 5.83; N, 14.81. tained 0.99% of ash and the values for carbon, hydrogen After three more recrystallizations, the following data and nitrogen given below are corrected to an ash-free basis. were obtained. Anal. Calcd. for (CzlH37;'u'7012)~-3Hk30i: C, 34.75; H, 5.54; N, 13.47; Sod, 19.80. Calcd. for (C21H&TO12)2. Anal. Found: C, 50.64; H, 5.90; K,14.13. Conversion of Streptomycin Helianthate to Streptomycin 3&SO(: C, 34.61; H, 5.80; N, 13.45; SOa, 19.76. Hydrochloride.-A 29-g. sample of streptomycin helian- Found: C,34.45; H , 6.16; N, 13.45; S04,20.2. Conversion of Streptomycin Sulfate to the Free Base.thate was added to 465 ml. of concentrated hydrochloric A sample of amorphous streptomycin sulfate weighing acid-methanol solution (ratio 1:26 by volume). After the mixture was thoroughly stirred, the suspension was 126 mg. was dissolved in about 5 ml. of m-ater and the filtered through a thin layer of Darco G-60 and the filter solution was treated with the calculated amount of barium cake was washed with 85 ml. of methanol. The filtrate hydroxide solution. After removal of barium sulfate, the and washings were poured into 4500 ml. of acetone and the filtrate was concentrated to a residue. After washing with mixture was centrifuged. The precipitated streptomycin acetone and drying, streptomycin base was obtained as an hydrochloride was washed with acetone and dried; yield amorphous light yellow powder; 520 units/mg. 13 g. (97%), activity about 785 units/mg., [or]= -86.7" Anal. Calcd. for C2&7x@12: C, 43.49; H, 6.45; S , (c, 1% in water). A sample was dried a t 100" in vacuo to 16.91; calcd. for Cz1H39?j,Ols: C , 43.37; IT, 6.76; X, constant weight before analytical determinations were 16.85. Found: C, 43.03; 11, 6.57; N, 15.87. made. Acknowledgment.-The authors wish t o thank Anai'. Calcd. for C21H37N7012.3HC1: C, 36.61; H, Dr. N. R. Trenner, Mr. Walter Bastedo, Jr., 5.85; 1, 14.23; C1, 15.44. Calcd. for C Z I H ~ ~ N ~ O W ~ H C ~ : C, 36.50; H , 6.13; N, 14.19; C1, 15.39. Found: C , and Mr. R. N. Boos and their associates for 36.80; H, 6.09; X, 14.39; C1, 15.59. physical chemical measurements and microConversion of Streptomycin Helianthate to Streptomycin analytical determinations. Hydrobromide.-When a 219-mg. sample of streptomycin Summary helianthate was treated with methanol-hydrobromic acid in the manner described for the hydrochloride, streptomyStreptomycin has been isolated from the culture cin hydrobromide was obtained as an amorphous white powder, yield !38 mg., activity abobt 680 units/mg. The broths of Streptomyces griseus by the following sequence of steps : charcoal adsorption, elution product was difficult to purify. Anai. Calcd. for C21H3,N701~,3HBr:C , 30.67; H , with methanolic formic acid, precipitation with 4.91; N, 11.92; Br, 29.1; eq. wt., 822. Calcd. for CZIHS- picric acid, conversion to the hydrochloride, N7Ol2.3HBr: C, 30.60; H, 5.13; N, 11.88; Br, 29.1. chromatography with aluminum oxide or Darco Found: C, 31.25; H , 5.58; N, 11.61; Br, 28.0; eq. wt., G-60, and finally conversion to the crystalline 869 (potentiometric titration). helianthate. Streptomycin p(2-Hydroxy-I-naphthy1azo)-benzeneThe results of various determinations and sulfonate.-Five grams of streptomycin hydrochloride, activity about 700 units/mg., [ G ] D -86.5" (c, 0.9% in analyses are in agreement with the formula water), was dissolved in 75 ml. of methanol and a solution C ~ I H ~ , - - ~ ~ N for ,OL streptomycin ~ and show that of 5 g. of Orange I1 dissolved in 280 ml. of hot water was i t has three basic functional groups. added. On cooling, the bulky crystalline precipitate was RAFIWAY, S . 5. RECEIVED APRIL 10, 1946 collectrd by centrifugation and recrystallized twice from
