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F.H. STODOLA, 0. L. SHOTWELL, A. 31. HORUD, R.G. BENEDICT AND A. C. RILEY,JR. !CONTRIBUTION FROM
THE
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NORTHERN REGIONAL RESEARCH LABORATORY1]
Hydroxystreptomycin, a New Antibiotic from SfvepfornycesGriseocarneus2 BY FRANK H. STODOLA, ODETTEI,. SHOTWELL, ANNE MARIE BORUD,ROBERTG. BENEDICTAND .ARTHUR C. RILEY,JR. A new member of the streptomycin series has been degraded to streptidine, N-methyl-L-glucosamine and 2-hydroxymethyl3-hydroxy-1,4-pyrone. These cleavage products indicate that the new antibiotic differs from streptomycin only in having a hydroxymethyl group instead of a methyl in the streptose portion of the molecule.
In a preliminary note3 we reported the isolation
On the basis of these studies the formula (V) is indicated for hydroxystreptomycin. composition C21H39N7013 produced by a new species -4s paper chromatography proved to be so useof Streptomyces, S. griseocarneus, obtained from a ful in detecting the new streptomycin we show in Japanese soil. Since the new antibiotic differed Figs. 1 and 2 some separations of the various strepfrom streptomycin by only one hydroxyl group, it tomycins to assist future workers in the characteriwas given the name “hydroxystreptomycin.’ ’ zation of the members of this growing group. In In the present paper are given the details of that the figures streptomycin is designated as A, manwork along with some data on the paper chroma- nosidostreptomycin as B and hydroxystreptomycin tography of the streptomycins. as C. The cleavage of dihydrohydroxystreptomycin In a recent note, Grundy and co-workers4 have with methanolic hydrogen chloride gave streptidine described the isolation of a streptomycin which and a disaccharide isolated as the hexaacetate (I). appears to be identical with hydroxystreptomycin. Under similar conditions dihydrostreptomycin Their antibiotic was produced by a Streptomyces yields streptidine and a pentaacetate. The extra isolated from a soil sample collected a t North Chihydroxyl group indicated by the acetyl determina- cago, Illinois. of a new member of the streptomycin series of the
Experimental Isolation of Crude Hydroxystreptomycin.-The method of 0 purification was essentially that il developed by earlier workers on c streptomycin.6 To each liter of i \ filtered culture liquor was added HC c-oIr 11 g. of Nuchar (2-250 N, the CH~OCOCH~ o H-COCOCHa 11 /I mixture stirred for 30 minutes, €IC C-CHOH I1 CHaCOO-CH the carbon removed by filtra\ tion, and washed in succession 0 with one liter of water, 100 cc. C -H , of 50% ethanol and 100 cc. of absolute methanol. The airI CHPOCOCII~ I1 dried carbon was stirred for five streptidine-0 -CI$ minutes with sufficient meth0 0 anol-hydrochloric acid mixture /CIi -0-X-methyl-L(0.5 cc. concd. HC1 per liter of ll I/ 0 I1 , glucosamine methanol) to permit stirring. C C ,‘ After filtration the filtrate was /\ H--C C--OH 0.-c--&-orr €10-c CH concentrated in vacuo to onefourth volume and added to ten volumes of acetone. The precipitate was separated by centrifugation and washed twice with acetone. Drying in vucuo I11 IY gave a somewhat hygroscopic tion on the hexaacetate was shown not to be in the tan powder assaying about 30-40v0 hydroxystreptomycin. crude product (25 g.) was dissolved in 50 cc. of water, glucosamine portion of the molecule by the isola- theThis $H adjusted to 5.8, the Insoluble material removed by tion of a pentaacetyl N-methyl-a-L-glucosamine centrifugation and the solution placed on a column (6.5 X identical with the pentaacetyl derivative from 45 cm.) of acid-washed Harshaw alumina (adjusted to pI-1 streptomycin. That the extra hydroxyl is located 4.7). For development 80% methanol was used. One cubic centimeter portions of the eluate gave about in the streptose moiety was demonstrated by alka- hundred 75% of the activity in fractious 9 through 16. The methline degradation of hydroxystreptomycin to the anol in these fractions was removed in vacuo and the resultpyrone (11) which is an isomer of kojic acid (111). ing aqueous solutions lyophilized to white powders the puriThe corresponding product from streptomycin is ties of which ranged from SO-SO%. Preparation of the He1ianthate.-Crude hydroxystreptomaltol (TV). mycin (1.54 9 . ) of about 80% Imrity ryas dissolved in 44 cc. of 50% methanol. ‘To this solutioii w i b added 2.2 g. of (1) One of the laboratories of the Bureau of Agricultural and IndusCH
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trial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. Article not copyrighted. (2) Presented before the Division of Agricultural and Food Chemist r y at the 118th Meeting of the American Chemical Society, Chicago, Illinois, September, 1950. (3) R. G. Benedict, F. H. Stodola, 0. L. Shotwell, A. M Borud and :. A l.incleIIfzlsrr, .5l:l,;,,,?, 112, 77 ! I X , l J ) .
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(4) W. E. Grundy, J. K Schenck, 11. R. Clark, Jr., M. P. Hargie, K.K. Richards atid J. C . Sylvester, A r c h . Biochcm., 28, 150 (1950). ( 5 ) A. Schatz, el al., PYOC.SOL. E r p . B i d . Mcd., 66, G6 (1944); H. E. Carter, et ai., J. B i d . Cliem., 160, 337 (1945); F. A. Kuehl, Jr,, et’al., Science, 102, 3-1 ( 1 ~ 4 s )I
