[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
263
Condition B. Condition B is the same as condition A except that the column is 0.6 cm by 75 cm of Chromobeads type C2, and the column effluent is monitored by an automated ninhydrin-hydrazine procedure. 2:~ The prepared reagent for this modified detection system is more stable than that used in condition A. Column flow rate is 0.9 ml/min. The retention times and color yields for various hydrolysis products and related compounds are given in Table I, and a comparison of quantitative results for different streptothricin-type antibiotics is given in Table II. 18 :~Technicon Corporation, Res. Bull. No. 20, Technicon Chromatography Corp., Chauncey, New York, 1968.
[11] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Aminoglycoside Antibiotics
By HAMAO UMEZAWA and SHINICHI KO•DO I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . II. P r e p a r a t i o n of Resin Columns . . . . . . . . . . . . . III. Extraction and Purification b y Using Carboxylic Acid Resins . . . . A. Isolation of K a n a m y c i n . . . . . . . . . . . . . . B. Isolation of 4-Amino-4-deoxy-a,a-trehalose . . . . . . . . C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a . D. Separation of N e b r a m y c i n s on a Large Scale . . . . . . . . E. Separation of Derivatives of 3 ' , ¥ - D i d e o x y k a n a m y c i n B . . . . . IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins . . . . . . . . . . . . . . . . . . A. Isolation of K a s u g a m y c i n by Amberlite IR-120 . . . . . . . B. Separation of Validamycins by Dowex 50 . . . . . . . . . V. Extraction a n d Purification Using Cellulose and Sephadex Exchangers A. Separation of Gentamicin C Complex b y CM-Sephadex . . . . . B. Separation of Lividomycins b y CM-Sephadex . . . . . . . . VI. Nonionic Adsorption C h r o m a t o g r a p h y b y Anion Exchange Resins. A. P r e p a r a t i o n of Nonionic Columns . . . . . . . . . . . B. C h r o m a t o g r a p h i c Procedure . . . . . . . . . . . . . C. Application to Separation of K a n a m y c i n s . . . . . . . . . 1). Application to Separation of Neomycins . . . . . . . . . E. Application to Separation of Destomyeins . . . . . . . . . F. High-Pressure Liquid C h r o m a t o g r a p h y . . . . . . . . . .
263 267 268 268 269 270 271 271 272 273 273 273 274 275 275 276 276 276 277 277 278
I. I n t r o d u c t i o n A m i n o g l y c o s i d e a n t i b i o t i c s ( T a b l e I ) p r o d u c e d b y Streptomyces, Micromonospora, a n d Bacillus h a v e b e e n e x t r a c t e d a n d p u r i f i e d b y a p p l i c a -
264
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
TABLE I AMINOGLYCOSIDE ANTIBIOTICSa
1. Monosaccharide antibiotics: nojirimycin, 3-amino-3-deoxy-D-glucose, b streptozotocin, prumycinc 2. Simple disaccharide antibiotics: trehalosamine, mannosyl glucosaminide,d 4-amino4-deoxy-a,a-trehalose" 3. Inositol-containing antibiotics: kasugamycin, myomycin/ 4. Inosamine-inosadiamine-containingantibiotics: (1) Inosamine group: minosaminomycing (2) Streptomycin group: streptomycin, mannosidostreptomycin (streptomycin B), hydroxystreptomycin (reticulin), dihydrostreptomycin, glebomycin (bluensomycin) (3) Actinospectacin (spectinomycin) (4) Hybrimycin group: hybrimycin A1, A2, Aa, BI, B~, Bah (5) Neamine group: neamine (neomycin A), paromamine (6) Kanamycin group; kanamycin (A), B, C, NK-1001, NK-1012-1, NK-1012-2, NK-1013-1, NK-1013-2, NK-10O3,i tobramycinJ (nebramycin factor 6), apramycink (factor 2), 6"-0-carbamoylkanamycin Bk (factor 4), 6"-0carbamoyltobramycin~ (factor 5'), gentamicin A, C~, C~, C2, sisomicin,t verdamicin,~ G-418~ (7) Neomycin group: neomycin B, C (streptothricin BII, BI), neomycin Lps, Lpc, paromomycin I, II, lividomycin A,° B,p mannosyl paromomycin,q ribostamycinr (SF-733), butirosin A, B,* Bu-17O9 E~, E.~t (8) Destomycin group: hygromycin B, destomycin A, B, A-396-I,~ SS-56C ~ 5. Other cyclitol-containing antibiotics: v'alidamycin A,~ B, • C, D, E, F~ Most antibiotics were described in H. Umezawa, "Index of Antibiotics from Actinomycetes." University of Tokyo Press, Tokyo, 1967. b S. Umezawa, K. Umino, S. Shibahara, M. Hamada, and S. Omoto, J. Antibiot. Ser. A 20, 355 (1967). c S. Omura, M. Tishler, M. Katagiri, and T. Hata, Chem. Commun., p. 633 (1972). d M. Uramoto, N. Otake, and H. Yonehar~, J. Antibiot. Ser. A 20, 236 (1967). • It. Naganawa, N, Usui, T. Takita, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 145 (1974). I j. C. French, Q. R. Bartz, and H. W. Dion, J. Antibiot. 26, 272 (1973). a M. Hamada, S. Kondo, T. Yokoyama, K. Miura, K. Iinuma, H. Yamamoto, K. Maeda, T. Takeuchi, and H. Umezawa, J . Antibiot. 27, 81 (1974). h W. T. Shier, K. L. Rinehart, Jr., and D. Gottlieb, J. Antibiot. 23, 51 (1970). i M. Murase, T. Ito, S. Fukatsu, and H. Umezawa, "Progress in Antimicrobial and Anticancer Chemotherapy," Vol. II, p. 1098. Univ. of Tokyo Press, Tokyo, 1970. K. F. Koch and J. A. Rhoades, Antimicrob. Ag. Chemother. 1970 309 (1971). k K. F. Koch, F. A. Davis, and J. A. Rhoades, J. Antibiot. 26, 745 (1973). z D. J. Cooper, R. S. Jaret, and H. Reimann, Chem. Commun., p. 285 (1971). " P. J. L. Daniels and A. S. Yehaskel, 13th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., September, 1973. n p. j. L. Daniels, A. S. Yehaskel, and J. Morton, 13th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., September, 1973. o T. Oda, T. Mori, Y. Kyotani, and M. Nakayama, J. Antibiot. 24, 511 (1971).
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
265
P T. Mori, Y. Kyotani, I. Wutanabe, and T. Oda, J. Antibiot. 25, 149 (1972). q T. Mori, Y. Kyotani, I. Watanabe, and T. Oda, J. Antibiot. 25, 317 (1972). E. Akita, T. Tsuruoka, N. Ezaki, and T. Niida, J. Antibiot. 28, 175 (1970). P. W. K. Woo, H. W. Dion, and Q. R. Bartz, Tetrahedron Left., p. 2125 (1971). t H. Tsukiura, K. Saito, S. Kobaru, M. Konishi, and H. Kawaguchi, J. Antibiot. 26, 386 (1973). J. Shoji ~nd Y. Nakaguwa, J. Antibiot. 23, 569 (1970). " S. Inouye, T. Shomura, H. Watanabe, K. Totsugawa, and T. Niida, J. Antibiot. 26, 374 (1973). S. Horii and Y. Kameda, Chem. Commun., p. 747 (1972). T. Iwasa, Y. K~meda, M. Asai, S. Horii, and K. Mizuno, J. Antibiot. 24, 119 (1971). YS. Horii, Y. Kameda, and K. Kawahara, J. Antibiot. 25, 48 (1972). tion of an ion-exchange chromatography. Among these antibiotics, streptomycin, dihydrostreptomycin, k a n a m y c i n (which can be also called k a n a m y c i n A), k a n a m y c i n B, a mixture of gentamicins C1, C2 and Cla, a mixture of neomycins B and C, a mixture of p a r o m o m y c i n s I and I I , and ribostamycin are commercially available as chemotherapeutic agents useful in treating infections. H y g r o m y c i n B and destomycin A are used as animal anthelminties. K a s u g a m y c i n and validamycin are used for prevention of plant diseases. More than one aminoglycoside antibiotic is generally produced by the same strain as in cases of k a n a m y c i n s A, B, and C by S t r e p t o m y c e s k a n a m y c e t i c u s , 1 gentamicins C1, C,_,, and C ~ by M i c r o m o n o s p o r a purpurea"- and butirosins A and B by Bacillus circulans, a Therefore, complete separation of the analogous antibiotics which are produced in a same culture filtrate has been studied. These antibiotics are adsorbed by cation exchangers such as resin, cellulose, and Sephadex ion-exchangers, and separated. Commercially available cation exchangers used for their separation are shown in T a b l e II. A weak cation exchange resin possessing carboxylic acid as the functional group is most useful not only in extraction of these antibiotics from culture liquids, but also in separation of a mixture of these antibiotics into each components. For the first time in order to extract streptomycin ill about 1949, tile resin process was introduced into industrial extraction of natural products. This resin was a polyacrylic acid resin, Amberlite IRC-50, manufactured by R o h m and H a a s Co., Philadelphia. 1K. Maeda, M. Ueda, K. Yagishita, S. Kawaji, S. Kondo, M. Murase, T. Takeuchi, Y. Okami, and H. Umezawa, J. Antibiot. Set. A 10, 228 (1957). 2j. p. Rosselet, J. Marquez, E. Meseck, A. Murawski, A. Hamdan, C. Joyner, R. Schmidt, D. Migliore, and H. L. Herzog, Anlimicrob. Ag. Chemother. 1963, 14 (1964). H. W. Dion, P. W. K. Woo, N. E. Willmer, D. L. Kern, J. Onaga, and S. A. Fusari, Antimicrob. Ag. Chemother. 2, 84 (1972).
266
[11]
METHODS FOR T H E STUDY OF ANTIBIOTICS
TABLE II COMMERCIAL CATION EXCHANGERS FOR SEPARATION OF AMINOGLYCOSIDE ANTIBIOTICS
Functional group Matrix Resin
Sephadex Cellulose
Carboxylic acid
Phosphonic acid
Sulfonic acid
Amberlite IRC-50" Amberlite CG-50~ Amberlite IRC-84" Duolite CC-3b Lewatit CNP ~ Bio-Rex 70d CM-Sephadex C-25I CM-celluloseg
Duolite ES-63b Bio-Rex 63d
Amberlite IR-120~ Dowex 50~ Duolite C-20b Lewatit SP-120° SE-Sephadex C-25s
P-cellulosea
Manufactured by Rohm and Haas Co., Philadelphia, Pennsylvania. b Diamond Shamrock Chemical Co., Resinous Products Div., Cleveland, Ohio. c Naftone, Inc., Park Ave., New York. d Bio-Rad Laboratories, Richmond, California. DoT Chemical Co., Midland, Michigan. I Pharmacia Fine Chemicals, Division of AB Pharmacia, Uppsala, Sweden, CM-: carboxymethyl group, SE-: sulfoethyl group. g Carboxymethyl celluloses and cellulose phosphates are supplied by many different manufacturers. Even now it is widely used in antibiotic industries. Streptomycin adsorbed on a column of this resin in the Na* form was eluted with aqueous mineral acid in a good yield. Extraction of neomycins and kanamycins which are stable in alkaline solution is more efficiently accomplished by adsorption of NH4 * form of this resin and by elution with aqueous ammonia. A mixture of m a n y kinds of aminoglycoside antibiotics are efficiently separated into each components by a linear gradient elution of ammonia from a column of Amberlite CG-50 (chromatographic grade, a polyacrylic acid resin). 4 Processes using strong cation exchange resins possessing sulfonic acid as the functional group were introduced into extraction of weakly basic aminoglycoside antibiotics. In this case also, Amberlite CG-50 resin was utilized for further purification, eluting the weakly adsorbed antibiotics with water. Adsorption of aminoglycoside antibiotics on a resin is often interfered with by various kinds of cations in culture filtrates. Multivalent inorganic cations such as Ca 2÷ and Fe 3÷ are tightly bound to cation exchange resins. Therefore, these cations are first removed from culture liquids before application of the resin processes. 4H. Yamamoto, Y. Ikeda, S. Kondo, and H. Umezawa, unpublished data, 1973.
[lll
C H R O M A T O G R A P HOF Y AMINOGLYCOSIDES
267
For separation and purification of aminoglycoside antibiotics, cellulose and Sephadex ion-exchangers are also useful. The most efficient chromatography for separation of analogous aminoglycoside antibiotics can be accomplished by the use of anion exchange resin, Dowex l-X2 (Dow Chemical Co., Midland, Michigan). This technique by nonionic adsorption chromatography was first employed for separation of kanamycins (Rothrock et al.5). Many of the other antibiotics can be purified by this technique. II. Preparation of Resin Columns Ion-exchange resin columns can be made in any desired size, from a few milliliters up to many liters. Height of exchanger beds for a good separation is usually 10-20 times longer than a column diameter. In a case of difficult separation, a much longer column can be used. In a conventional method of packing the column, ~ the resin is stirred in an excess of the first eluent to be used, and after most of the resin is settled, small particles are decanted off. The remaining resin is slurried into the column and allowed to settle by gravity to form the packed bed. It is often disadvantageous to use too large a volume of resin. Larger exchanger beds need more flow time and more rinsing, and yield inconveniently large solution volumes. An efficient system employed in antibiotic industries uses a minimum amount of the resin which is divided into more than one column. The culture liquid is continuously charged on the first column which is connected to the second column. From each column on which the antibiotic is fully adsorbed, it is eluted. By continuous repetition, the antibiotic can be extracted in excellent yield. The adsorption and elution are carried out usually in laboratories by downflow, but the upflow (backflow) technique is frequently used in large-scale operations. Before adsorption of an antibiotic, commercial ion-exchange resins are treated by repeated cycles of washing with 1-2 N hydrochloric acid and sodium hydroxide to remove imourities, and converted to a desired form by treatment with 1 M solution of an appropriate salt, acid or base. After the conversion is completed, the resin is washed with distilled water. Resins shrink and swell considerably and therefore the conversion is handled most easily in a large diameter column. Then, the resin is packed into the desired size column. J. W. Rothrock, R. T. Goegelman, and F. J. Wolf, Antibiot. Annu. 1958--1959, 796 (1959). G. Zweig and J. Sherma, eds., "Handbook of Chromatography," Vol. II, p. 61. Chem. Rubber Publ. Co., Cleveland, Ohio, 1972.
268
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
Weak cation exchange resins possessing carboxylic acid as the functional group are usually converted to H ÷ form by 1 N hydrochloric acid and to Na ÷ or NH4 + form by 1-2 N sodium hydroxide or ammonia. If a partially regenerated form is necessary, H + form and alkaline form of resins are well mixed, or the resin is washed with phosphate buffer of the proper pH. III. Extraction and Purificationby Using Carboxylic Acid Resins
Since carboxylic acid resins bind very strongly with H ÷ and weakly with N a +, K +, and N H 4 +, m a n y aminoglycoside antibiotics in aqueous solution are efficiently adsorbed by ion-exchange on a column of N a +, K +, or N H 4 + form of the resins. And these antibiotics can be eluted from the column with aqueous mineral acids such as hydrochloric acid and sulfuric acid. Alkaline-stable aminoglycoside antibiotics, for example, neomycins and kanamycins, are isolated by adsorption on N H 4 + form of this resin and by elution with aqueous ammonia. A m o n g the aminoglycoside antibiotics shown in Table I, those except weakly basic antibiotics are well adsorbed on Amberlite IRC-50 resin (Na + or N H , ÷ form) from culture filtrates.The weakly basic antibiotics, for example, nojirimycin, 4-amino-4-deoxy-a,a-trehalose, kasugamycin, and validamycins, are not adsorbed on carboxylic acid resin from culture filtrates. However, there is some retention of them on Amberlite CG-50 (NH4 + form) column, and they can be purified by elution with water from the resin. Strongly basic antibiotics, adsorbed on Amberlite IRC-50 columns, are usually eluted with less than I N hydrochloric acid or aqueous ammonia in good yields. The use of ammonia for their purification is preferred, because in this case a salt-free eluate is obtained. However, alkaline-unstable antibiotics, for example, streptomycins and streptothricins, must be eluted with a proper ion strength of salts or mineral acids. Efficient separation of m a n y alkaline-stable aminoglycoside antibiotics can be accomplished by elution with a linear gradient of ammonia from a column of Amberlite CG-50 resin,4 as shown in Figs. 1 and 2. A. Isolation o f K a n a m y c i n 7
Kanamycin in a culture filtrate (more than 500 ~g/ml) of Streptomyces kanamyceticus s can be purified by a single resin process followed 7K. Maeda, "Streptomyces Products Inhibiting Mycobacteria," p. 60. Wiley, New York, 1965. s H. Umezawa, M. Ueda, K. Maeda, K. Yagishita, S. Kondo, Y. 0kami, R. Utahara, Y. Osato, K. Nitta, and T. Takeuchi, J. Antibiot. Set. A 10, 181 (1957).
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CHROMATOGRAPHY
OF
AMINOGLYCOSIDES
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60 70 8b 90 160 Fraction number FIG. 1. Separation of neomycin B (sulfate, 6.8 mg), paromomycin I (sulfate, 5.8 mg), lividomycin A (5.4 mg), and lividomycin B (5.2 mg) by a column (0.8 X 20 cm) of Amberlite CG-50 (type II, NH4÷ form, 10 ml). The column which adsorbed the antibiotics mixture in water (0.5 ml) was washed with 0.1 N NH40H (40 ml) and then eluted with a linear gradient of 0.1 N to 0.8 N NH4OH (gradient rate: 0.5%/min, flow rate: 21 ml/hr). These antibiotics in fractions (1 ml each) were detected by Rr values of thin-layer chromatography (TLC) of silica gel (Merck, Art 5721) using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as developing solvent, followed by coloration with ninhydrin reagent. Each antibiotic in fractions was assayed by agar plate method using Bacillus subtilis PCI 219 as a test organism.
by crystallization. A column of Amberlite I R C - 5 0 resin (70% Na + or NH4 ÷ form) on which kanamycin is adsorbed is eluted with 1 N aqueous ammonia. The eluate is concentrated by evaporation to remove ammonia, acidified with sulfuric acid to p H 3, and decolorized with active charcoal. The pH of the solution is then adjusted to 8.0-8.2, that is, the p H of kanamycin monosulfate solution. To this concentrate containing about 100 mg of kanamycin base per milliliter, the same volume of methanol is added with stirring and cooling. After crystallization starts, more methanol is added up to 60% methanol concentration. With continued stirring, crystals of kanamycin monosulfate are obtained in 80% yield from the culture filtrate.
B. Isolation of 4-Amino-4-deoxy-(~,~-trehalose A streptomyces antibiotic, 4-amino-4-deoxy-~,a-trehalose9 has been purified by Amberlite CG-50 column chromatography by the authors. "H. Naganawa, N. Usui, T. Takita, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 145 (1974).
270
METHODS FOR THE STUDY OF ANTIBIOTICS Paromam i ne Destomycin A Lividomycin A Kanamycin A Paromomycin l Kanamycin ]3 Ribostamycin Lividomyci n B Tobrarnycin Neamine DKI3a Gentamicin Cb Neomycin B Butirosins c
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[11]
0.2
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Fie. 2. The concentrations of ammonia for elution of aminoglycoside antibiotics from a column (0.8 × 20 cm) of Amberlite CG-50 (type II, NH~÷ form, 10 ml). The column which adsorbed antibiotics (approximately 5 mg each) was washed with 0.1 N NH,OH (40 ml) and then eluted with a linear gradient of 0.1 N to 0.8 N NH40H (gradient rate : 0.5%/min, flow rate : 21 ml/hr). These antibiotics in fractions (1 ml each) were detected by thin-layer chromatography of silica gel (Merck, Art 5721) using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as a developing solvent, high-voltage paper electrophoresis under 3300 V for 15 min using formic acid-acetic acid-water (25:75:900 in volume) as an electrolyte solution, and agar plate method using Bacillus subtilis PCI 219 as a test organism. (a) 3',4'dideoxykanamycin B; (b) gentamicin C complex; (c) butirosins A and B.
An aqueous solution (3 ml) of the crude powder of the antibiotic extracted from a culture filtrate by a carbon adsorption process is charged to a column (1.25 X 31 cm) of Amberlite CG-50 resin (type I, NH~ + form, 40 ml) and the c h r o m a t o g r a m is developed with water. The eluate in fractions 22-56 (2 ml each) showing bioaetivity against Escherichia coli N I H J or Bacillus subtilis P C I 219 are combined and lyophilized to yield a white powder (391 mg) of the pure antibiotic.
C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a 4 An aqueous solution (0.5 ml) containing neomycin B sulfate (6.8 mg), p a r o m o m y c i n I sulfate (5.8 mg), lividomycin A (5.4 rag), and lividomycin B (5 2 rag) is charged to a column (0.8 X 20 era) of Amberlite CG-50 resin (type I I , NH4 ÷ form, 10 ml). After the column is washed with 0.1 N aqueous ammonia (40 ml), the antibiotics are eluted with a linear gradient of aqueous ammonia from 0.1 to 0.8 N for 200 min (a gradient rate of 0.5% per minute) at a flow rate of 21 ml per hour using an Aerograph LC-4200 gradient system (Varian Instruments, California). With
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
271
the aid of a fraction collector approximately 1-ml fractions of the efituent are collected. Each antibiotic in the fractions is determined by thin-layer chromatography on silica gel (Merck, Art 5721), for instance, using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as a developing solvent, followed by treatment with ninhydrin reagent. Each antibiotic in the fractions can be also assayed by an agar plate method using Bacillus subtilis PCI 219 as a test organism. These four antibiotics are completely separated by this technique as shown in Fig. 1. This technique has been applied to the other strongly basic antibiotics and the concentrations of ammonia in eluent for each antibiotic are summarized in Fig. 2.
D. Separation of Nebramycins on a Large Scale I° A solution of nebramycin complex has been obtained from culture filtrates of Streptomyces tenebrarius by adsorption on Amberlite IRC-50 resin, elution with 1 N aqueous ammonia and concentration to remove excess ammonia. The following example was reported. The solution (20 liters, 82 X 106 units of the activity against Klebsiella pneumoniae FDA KL4) was applied to a 10 cm diameter column which contained 20 liters of Bio-Rex 70 resin (NH4 * form). The column was washed with 12 liters of water and then eluted with a gradient prepared by adding 0.2 N aqueous ammonia to a 50-liter constant-volume reservoir which was charged with 0.05 N aqueous ammonia. The flow rate was 20 ml per minute and fractions of 500 ml were collected. The following fractions were combined, concentrated, and freeze-dried: fractions 39-100, 102 g (35 X 10~ units) of factor 2 (apramycin); fractions 115-154, 14 g (8.1 X 10~ units) of factor 4 (6"-O-carbamoylkanamycin B) ; fractions 188-240, 24 g (30 X 10~ units) of factor 5' (6"-O-carbamoyltobramycin). Eighty-nine percent of the activity was recovered.
E. Separation of Derivatives of 3',4'-Dideoxykanamycin B In the course of chemical derivation of kanamycins based on mechanism of resistance to aminoglycoside antibiotics, 11 3',4'-dideoxykanamycin B (DKB), which inhibits sensitive and resistant organisms including ~*K. F. Koch, F. A. Davis, and J. A. Rhoades, d. Antibiot. 26, 745 (1973). 11H. Umezawa, "Progress in Antimicrobial and Anticancer Chemotherapy," Vol. II, p. 567. Univ. of Tokyo Press, Tokyo, 1970. Published also in Advan. Carbohyd. Chem. 30, 183-225 (1975).
272
METHODS FOR THE STUDY OF ANTIBIOTICS
[lll
Pseudomonas aeruginosa, was synthesized, 12 and thereafter, N-(S)-4amino-2-hydroxybutyryl (AHB) derivatives were prepared. 13,~4 These products were purified by the following resin processes. 6'-N-t-Butyloxycarbonyl DKB (1109 rag) was acylated with (S)-4-amino-2-hydroxybutyric acid and a mixture of positional isomers of AHB-DKB was obtained after removal of protecting groups. The mixture (trifluoroacetate, 3067 mg in 5 ml water) was charged to a column (14 mm diameter) of Ambertire CG-50 resin (type I, NH4 ÷ form, 50 ml). After washing with water (250 ml), the column was eluted by stepwise elution with 500 ml each of 0.5, 0.75, and 1.0 N ammonia. The unreacted DKB (297 mg, 32% yield) and 2'-AHB-DKB (238 mg, 21%) were eluted with 0.5 N ammonia, 3-AHB-DKB (72 mg, 6%), 1-AHB-DKB (131 mg, 12%) and 3"-AHB-DKB (53 mg, 4%) with 0.75 N ammonia, and 3,2'-diAHB-DKB (46 rag, 3%), 1,2'-diAHB-DKB (47 rag, 3%) and the other diacyl derivatives with 1.0 N ammonia. By this chromatography, these positional isomers 14 of AHB-DKB, were completely separated. 1-AHB-DKB and 1,2'-diAHB-DKB show strong activity against sensitive and resistant organisms. The latter organisms produced kanamycin phosphotransferases and kanamycin nucleotidyltransferase. IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins Weakly basic aminoglycoside antibiotics, for example, nojirimycin, 3-amino-3-deoxy-D-glucose, trehalosamine, mannosyl glucosaminide, 4-amino-4-deoxy-a,a-trehalose, kasugamycin, and validamycins can be extracted from culture filtrates by adsorption on strong cation exchange resins possessing sulfonic acid groups, such as Amberlite IR-120, Dowex 50, and Lewatit SP-120, and elution with dilute aqueous ammonia. Since sulfonie acid resins hold H + more weakly than Na ÷ and NH4 +, these resins are generally used as H + form. From a column of Amberlite IR-120 resin kanamycin is efficiently eluted with 1 N aqueous ammonia, but not with t N hydrochloric acid. Therefore washing the column with the acid and elution of kanamycin with ammonia gives a highly purified kanamycin. 1 Phosphonic acid resins have not been used widely. However, extraction of kanamycin from culture filtrates by adsorption on Duolite C-62, 12H. Umezawa, S. Umezawa, T. Tsuchiya, and Y. Okazaki, J. Antibiot. 24, 485 (1971). 1aS. Kondo, K. Iinuma, It. Yamamoto, K. Maeda, and H. Umezawa, J. Antibiot. 26, ¢12 (1973). ~4S. Kondo, K. Iinuma, H. Yamamoto, Y. Ikeda, K. Maeda, and H. Umezawa, J. Antibiot. 26, 705 (1973).
[11]
CHROMATOGR/kPHY OF AMINOGLYCOSIDES
273
a phosphonic acid resin formerly supplied by Diamond Shamrock Chemical Co., Resinous Products Division, Ohio, and elution with 5% aqueous ammonia has been reported? Most of aminoglycoside antibiotics can be adsorbed on phosphonic acid resins.
A. Isolation of Kasugamycin by Amberlite IR-120 Kasugamycin15 in culture filtrate of Streptomyces kasugaensis has been successfully extracted by Amberlite IR-120 resin as follows. The filtrate (1570 liters, 530 ~g/ml) was charged on Amberlite IR-120 column (H ÷ form, 300 liters). The column was washed with water and then eluted with 0.5 N aqueous ammonia. The first (53 liters), the second (200 liters), and the third (200 liters) eluates contained 28.5 g, 738 g, and 44.2 g of kasugamycin, respectively. The second eluate was adjusted to pH 6.6 with hydrochloric acid and concentrated to 6.32 liters under reduced pressure. After addition of ethanol (60 liters) to this concentrate, the crude crystals of kasugamycin hydrochloride (850 g, 90% purity) were obtained.
B. Separation of Validamycins by Dowex 5016 Validamycin E-rich fractions and F-rich fractions obtained by Dowex l-X2 resin chromatography (see Section VI) were further chromatographed on a column of Dowex 50-W X2 resin by eluting with a pyridineacetic acid buffer (pH 6.0). In this chromatography, validamycin F was eluted first and thereafter validamycin E.
V. Extraction and Purification Using Cellulose and Sephadex Exchangers Various kinds of cation exchangers having hydrophilic supporting matrix are commercially available. Cellulose and Sephadex ion-exchangers are used generally for separation of large molecules. Among these exchangers, carboxymethylcellulose, cellulose phosphate, CM-Sephadex C-25, and SE-Sephadex C-25 can be used for separation of basic antibiotics. Separation of streptothricin components by column chromatography 15H. Umezawa, Y. Okami, T. Hashimoto, Y. Suhara, M. Hamada, and T. Takeuchi, J. Antibiot. Ser. A 18, 101 (1965). S. Horii, Y. Kameda, and K. Kawahara,J. Antibiot. 25, 48 (1972).
274
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
of carboxymethyl cellulose has been reported27 Many phleomycinTM and bleomycinTM components are efficiently separated by column chromatography of CM-Sephadex C-25 with a gradient of ammonium formate. Most of the aminoglycoside antibiotics can be purified by column chromatography on carboxymethyl cellulose, cellulose phosphate, CMSephadex C-25, and SE-Sephadex C-25 using various ion strengths of salt solutions, such as sodium chloride and ammonium formate. Streptomycin is adsorbed on a column of CM-Sephadex C-25 equilibrated with 0.1 M sodium chloride or 1.0 M ammonium formate and eluted with 0.55 M sodium chloride or 2.0 M ammonium formate, s° The antibiotic, adsorbed on CM-cellulose (Brown Co., Fifth Ave., New York, 0.63 mEq/g, equilibrated with 0.1 M ammonium formate), is eluted with 0.6 M ammonium formate, s° A. Separation of Gentamicin C Complex by CM-Sephadex Maehr and Schaffneff1 have described the separation of gentamicin components by Dowex 1-X2 resin chromatography (see Section VI). The authors 2s found another powerful method using CM-Sephadex column as follows. Gentamicin C complex (800 rag) in 0.1 M ammonium formate (400 ml) was adsorbed on a column (35 mm diameter) of CM-Sephadex C-25 (800 ml, equilibrated with 0.1 M ammonium formate). The column was washed successively with 450 ml of 0.1 M, 1275 ml of 0.5 M, 4590 ml of 1.0 M and 1860 ml of 1.5 M ammonium formate and then eluted with 1.8 M ammonium formate. The effluent was cut into each approximately 15-ml fractions, and the fractions were assayed by agar plate method using Bacillus subtilis PCI 219 as a test organism and by silica gel (Merck, Art. 5721) thin-layer chromatography using butanol-ethanol-chloroform-28% ammonia (4:5:2:5 in volume) as a developing solvent followed by coloration with ninhydrin reagent. The three main peaks of gentamicins C1, C~, and C1~ appeared in fractions 569, 594, and 613, respectively. The eluates in fractions 557-575 were combined and diluted with water (6 liters). Gentamicin C~ in the diluted solution was adsorbed on a column of Amberlite CG-50 resin (type I, NH4 ÷ form, 50 ml). Elution with 0.5% aqueous ammonia gave 172 mg of pure gentamicin C1. 1~A. S. Khokhlov and P. D. Reshetov, J. Chromatogr. 14, 495 (1964). T. Ikekawa, F. Iwami, H. Hiranaka, and H. Umezawa, Y. Antibiot. Set. A 17, 194 (1964). 1~H. Umezawa, Y. Suhara, T. Takita, and K. Maeda, J. Antibiot. Ser. A 19, 210 (1966). 2oK. Yokose, Y. Suhara, M. Miyamoto, S. Kondo, and H. Umezawa, unpublished data, 1973. ~ It. Maehr and C. P. Schaffner,J. Chromatogr. 30, 572 (1967). 22M. Yagisawa,S. Kondo, and H. Umezawa,unpublished data, 1973.
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
275
B. Separation of Lividomycins by CM-Sephadex 2~ The separation of each lividomycin has been accomplished by the following processes. A crude powder of lividomycins (24 mg) which was obtained by adsorption on Amberlite IRC-84 resin (NH4 + form) from a culture filtrate of S t r e p t o m y c e s lividus and elution with 1 N aqueous ammonia was dissolved in water (5 ml) and charged to a column (1 }( 40 cm) of CM-Sephadex C-25 (NH4 ÷ form). After washing with water, the column was eluted by a gradient between 0.12 N (200 ml) and 0.35 N aqueous ammonia (25 ml) at a flow rate of 25 ml per hour at 27% All fractions (3 ml each) were assayed by the paper disk method using Bacillus subtilis ATCC 6633 as a test organism. Four peaks of active fractions appeared at fractions 30, 44, 64, and 72, which were designated as No. 2230-C (mannosyl paromomycin), lividomycin A, No. 2230-D (paromomycin), and lividomycin B, respectively.
VI. Nonionic Adsorption Chromatography by Anion Exchange Resins In using anion exchange resins for the removal of colored impurities from crude kanamyein aqueous solutions, Rothrock et al. ~ observed some retention of the antibiotic by strong anion exchange resins. Further investigations revealed that porous, strongly basic anion exchange resins with quaternary amine functional groups could be used at low loadings to separate kanamyein A from kanamycin B. Of these, Dowex 1-X2 resin (Dow Chemical Co., Midland, Michigan, 50-100 mesh) in OH- form is satisfactory for the chromatographic analysis of kanamycin mixtures. This technique by ion exclusion chromatography can be most efficiently used for separation of analogous aminoglycoside antibiotics. Complete separations of neomycins,24 paromomycins,~4 destomycins,"-5 gentamicins,21 lividomycins,2~ butirosins, 33 and validamycins16 have been reported by many researchers, and this technique can be applied £o largescale preparative operations. Recently, high-pressure liquid chromatography using a column of Aminex A-2726 or A-28 =7 resin (a quaternary amine resin, Bio-Rad Laboratories, California) was introduced into analysis or purification of aminoglycoside antibiotics. =3T. Mori, T. Ichiyanagi, H. Kondo, K. Tokunaga, T. Oda, and K. Munakata, J. Antibiot. 24, 339 (1971). ~4H. Maehr and C. P. Schaffner,Anal. Chem. 36, 104 (1964). 25S. Kondo, M. Sezaki, M. Koike, M. Shimura, E. Akita, K. Satoh, and T. Hats, I. Antibiot. Ser. A 18, 38 (1965). 2~T. Ohtake and M. Yaguchi, "Liquid Chromatography at Work," No. 5. Nippon Electric Varian Ltd., 1973. ~7H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, and T. Takeuchi, J. Antibiot. 26, 407 (1973).
276
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
A. Preparation of Nonionic Columns T M The anion exchange resin, D0wex I-X2 (CI- form, 50-100 mesh) is backwashed free of fines before being converted to the O H - form by columnwise washing with 2 resin volumes of I % sodium sulfate solution and 2 resin volumes of 10% sodium hydroxide solution. The resin shrinks and swells considerably and therefore these operations can be done most easily in a large-diameter column. The resin is washed thoroughly with C02-free distilled water to remove excess alkali. The chromatographic tube is filled up to two-thirds capacity with C02-free distilled water, and the resin in the O H - form is slurried directly into the column with C02free distilled water, so that the resin does not entrap air bubbles. The water is drained from the bottom of the column to the resin bed level. A column prepared in this manner conveniently can hold 400 ml of resin in a bed depth of I00 cm which gives efficient chromatography to handle I0 g of crude kanamycin. For smaller samples a proportionately smaller column can be employed. B. Chromatographic Procedure TM
An approximately 25% aqueous solution of aminoglycoside antibiotics is introduced to the top of the resin bed. Although hydrochloride or sulfate salts of antibiotics are usually used for the chromatography, free bases of antibiotics give more satisfactory separation. The column is developed with C02-free distilled water at the effluent flow rate of one resin volume per 2-4 hr. With the aid of an automatic fraction collector, the effluent is fractionated (usually one-tenth volume of a resin volume for each fraction). Antibiotics in the effluent are determined quantitatively by bioactivity, conductivity, refractive index, and colorations with reagents, such as ninhydrin. Antibiotics in the fractions can be also detected by paper and thin-layer chromatography, and high-voltage paper electrophoresis. C. Application to Separation of Kanamycins
Rothrock et al2 achieved a complete separation of kanamycins A and B by the technique described above. By application of this technique, crystalline kanamycin C was isolated from a mixture of kanamycins. 2s An example of quantitative separation of kanamycins is as follows. 29 A ~sM. Murase, T. Wakazawa, M. Abe, and S. Kawaji, J. Antibiot. Set. A 14, 156 (1961). 29S. Inouye and H. Ogawa, J. Chromatogr. 13, 536 (1964).
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CHROMATOGRAPHY OF AMINOGLYCOSIDES
277
mixture of kanamycin A (2.08 mg), B (0.207 rag), and C (0.143 rag) is chromatographed on a column (0.9 X 39 cm) of Dowex l-X2 resin (OH- form, 200-400 mesh) with an effluent flow rate of 30 ml per hour. The antibiotics in the effluent are automatically determined by color development of ninhydrin reaction using an amino acid analy~,er (Hitachi Type KLA-2, Hitachi Co., Tokyo). The elution peaks of these antibiotics have retention times as follows, kanamycin B: 85 min, C: 108 min, and A: 187 min.
D. Application to Separation of Neomycins Neomycins were completely separated into three components by Maehr and Schaffner.2. An approximately 25% aqueous solution of a commercial neomycin sulfate sample (2 g) containing about 30% neomycin C, 70% neomycin B, and a trace of neomycin A (neamine) is charged to a column (2.5 X 100 cm) of Dowex l-X2 resin (OH- form, 50-100 mesh) and the column is developed with distilled water at a linear flow rate of 0.4-0.6 cm per minute. The effluent is cut into fractions, approximately 40 ml each, and the antibiotics in these fractions are analyzed by a quantitative ninhydrin procedure. Three elution peaks of neomycins A, C, and B appear in fractions 11, 22, and 41, respectively.
E. Application to Separation of Destomycins 25 By application of this technique, a crude powder of destomycins which was obtained by adsorption on Amberlite IRC-50 resin (Na ÷ form) from a culture filtrate of Streptomyces rimofac~ens and elution with 2% aqueous ammonia has been purified. A crude powder (175 g) of destomycins is dissolved in 200 ml of water and charged to a column of Dowex I-X2 resin (OH- form, 50-100 mesh, 1600 ml). The column is developed with water at a flow rate of 4 ml/min. The eluate is cut into 20-ml fractions each, and the bioactivities of all fractions are determined using Bacillus subtilis ATCC 6633 and Mycobacterium smegmatis ATCC 607 as the test organisms. Destomycin B is eluted in fractions 64--72, and destomycin A starts to appear in fraction 73. Fractions 76-200 are combined and lyophilized to yield a white powder (69.5 g) of pure destomycin A. The eluate in fractions 64-75 gives a white powder (13.7 g of a mixture of destomycins) on lyophilization. The mixture is dissolved in 50 ml of water and rechromatographed on a column of the resin (OH- form, 400 ml). Destomycin B is eluted in fractions 15-26 (20 ml each), which contains 4.0 g of pure destomycin B.
278
METHODS
FOR
THE
STUDY
OF ANTIBIOTICS
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F. High-Pressure Liquid Chromatography High-pressure liquid chromatography using a column of Aminex A-2726 or A-2827 resin (a quaternary amine resin, Bio-Rad Laboratories,
California) is a powerful technique for analysis and purification of aminoglycoside antibiotics. The differential quantitative determination of kanamycin, lividomycin A, and 3 ' , 4 ' - d i d e o x y k a n a ~ c i n B TM has been accomplished by the following method? ~ A commercial ~pparatus, Aerograph LC-4200 system of Varian Instruments, Californi~ was used. A mixture of 40 ~g each of kanamycin A, 3',4'-dideoxykanafa~rcin B, and lividomycin A in water (12 ~l) is injected in the top of ~ e column (0.2 X 100 cm) of Aminex A-28 resin (8-12 ~m, OH- form)~and the column is developed with distilled water, operating at 50 °, 15 ml/hr, and 3400 psi. The amounts of these antibiotics in the effluent are automatically recorded by a refractive index detector. As shown in Fig. 3, these antibiotics can be completely separated in a short time and can be quantitatively determined by measuring the height of each peak. Such a differential assay is useful in studies on kinetics of an enzyme which phosphorylates or acetylates these antibiotics. High-pressure liquid chromatography is described in detail elsewhere. 3°
J
"N
.r I
0
:5 I
i
5 10 Minutes
I
15
FIG. 3. Separation of kanamycin, lividomycin A, and 3',4'-dideoxykanamycin B by high-pressure liquid chromatography on a column (0.2 X 100 cm) of Aminex A-28 (8-12 /zin, OH- form). A mixture of each 40 ~g of antibiotics in water (12 /~l) was injected to the top of the column connected with Varian Aerograph LC-4200 system and the column was developed with distilled water, operating at 50°, 15 ml/hour, 3400 psi. The amounts of these antibiotics in effluent were automatically recorded by the RI detector (4 × 10-5 refractive index units in full scale).
~°K. TsuJi, this volume [15].
CHROMATOGRAPHY OF AMINOGLYCOSIDES
263
Condition B. Condition B is the same as condition A except that the column is 0.6 cm by 75 cm of Chromobeads type C2, and the column effluent is monitored by an automated ninhydrin-hydrazine procedure. 2:~ The prepared reagent for this modified detection system is more stable than that used in condition A. Column flow rate is 0.9 ml/min. The retention times and color yields for various hydrolysis products and related compounds are given in Table I, and a comparison of quantitative results for different streptothricin-type antibiotics is given in Table II. 18 :~Technicon Corporation, Res. Bull. No. 20, Technicon Chromatography Corp., Chauncey, New York, 1968.
[11] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Aminoglycoside Antibiotics
By HAMAO UMEZAWA and SHINICHI KO•DO I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . II. P r e p a r a t i o n of Resin Columns . . . . . . . . . . . . . III. Extraction and Purification b y Using Carboxylic Acid Resins . . . . A. Isolation of K a n a m y c i n . . . . . . . . . . . . . . B. Isolation of 4-Amino-4-deoxy-a,a-trehalose . . . . . . . . C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a . D. Separation of N e b r a m y c i n s on a Large Scale . . . . . . . . E. Separation of Derivatives of 3 ' , ¥ - D i d e o x y k a n a m y c i n B . . . . . IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins . . . . . . . . . . . . . . . . . . A. Isolation of K a s u g a m y c i n by Amberlite IR-120 . . . . . . . B. Separation of Validamycins by Dowex 50 . . . . . . . . . V. Extraction a n d Purification Using Cellulose and Sephadex Exchangers A. Separation of Gentamicin C Complex b y CM-Sephadex . . . . . B. Separation of Lividomycins b y CM-Sephadex . . . . . . . . VI. Nonionic Adsorption C h r o m a t o g r a p h y b y Anion Exchange Resins. A. P r e p a r a t i o n of Nonionic Columns . . . . . . . . . . . B. C h r o m a t o g r a p h i c Procedure . . . . . . . . . . . . . C. Application to Separation of K a n a m y c i n s . . . . . . . . . 1). Application to Separation of Neomycins . . . . . . . . . E. Application to Separation of Destomyeins . . . . . . . . . F. High-Pressure Liquid C h r o m a t o g r a p h y . . . . . . . . . .
263 267 268 268 269 270 271 271 272 273 273 273 274 275 275 276 276 276 277 277 278
I. I n t r o d u c t i o n A m i n o g l y c o s i d e a n t i b i o t i c s ( T a b l e I ) p r o d u c e d b y Streptomyces, Micromonospora, a n d Bacillus h a v e b e e n e x t r a c t e d a n d p u r i f i e d b y a p p l i c a -
264
METHODS FOR THE STUDY OF ANTIBIOTICS
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TABLE I AMINOGLYCOSIDE ANTIBIOTICSa
1. Monosaccharide antibiotics: nojirimycin, 3-amino-3-deoxy-D-glucose, b streptozotocin, prumycinc 2. Simple disaccharide antibiotics: trehalosamine, mannosyl glucosaminide,d 4-amino4-deoxy-a,a-trehalose" 3. Inositol-containing antibiotics: kasugamycin, myomycin/ 4. Inosamine-inosadiamine-containingantibiotics: (1) Inosamine group: minosaminomycing (2) Streptomycin group: streptomycin, mannosidostreptomycin (streptomycin B), hydroxystreptomycin (reticulin), dihydrostreptomycin, glebomycin (bluensomycin) (3) Actinospectacin (spectinomycin) (4) Hybrimycin group: hybrimycin A1, A2, Aa, BI, B~, Bah (5) Neamine group: neamine (neomycin A), paromamine (6) Kanamycin group; kanamycin (A), B, C, NK-1001, NK-1012-1, NK-1012-2, NK-1013-1, NK-1013-2, NK-10O3,i tobramycinJ (nebramycin factor 6), apramycink (factor 2), 6"-0-carbamoylkanamycin Bk (factor 4), 6"-0carbamoyltobramycin~ (factor 5'), gentamicin A, C~, C~, C2, sisomicin,t verdamicin,~ G-418~ (7) Neomycin group: neomycin B, C (streptothricin BII, BI), neomycin Lps, Lpc, paromomycin I, II, lividomycin A,° B,p mannosyl paromomycin,q ribostamycinr (SF-733), butirosin A, B,* Bu-17O9 E~, E.~t (8) Destomycin group: hygromycin B, destomycin A, B, A-396-I,~ SS-56C ~ 5. Other cyclitol-containing antibiotics: v'alidamycin A,~ B, • C, D, E, F~ Most antibiotics were described in H. Umezawa, "Index of Antibiotics from Actinomycetes." University of Tokyo Press, Tokyo, 1967. b S. Umezawa, K. Umino, S. Shibahara, M. Hamada, and S. Omoto, J. Antibiot. Ser. A 20, 355 (1967). c S. Omura, M. Tishler, M. Katagiri, and T. Hata, Chem. Commun., p. 633 (1972). d M. Uramoto, N. Otake, and H. Yonehar~, J. Antibiot. Ser. A 20, 236 (1967). • It. Naganawa, N, Usui, T. Takita, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 145 (1974). I j. C. French, Q. R. Bartz, and H. W. Dion, J. Antibiot. 26, 272 (1973). a M. Hamada, S. Kondo, T. Yokoyama, K. Miura, K. Iinuma, H. Yamamoto, K. Maeda, T. Takeuchi, and H. Umezawa, J . Antibiot. 27, 81 (1974). h W. T. Shier, K. L. Rinehart, Jr., and D. Gottlieb, J. Antibiot. 23, 51 (1970). i M. Murase, T. Ito, S. Fukatsu, and H. Umezawa, "Progress in Antimicrobial and Anticancer Chemotherapy," Vol. II, p. 1098. Univ. of Tokyo Press, Tokyo, 1970. K. F. Koch and J. A. Rhoades, Antimicrob. Ag. Chemother. 1970 309 (1971). k K. F. Koch, F. A. Davis, and J. A. Rhoades, J. Antibiot. 26, 745 (1973). z D. J. Cooper, R. S. Jaret, and H. Reimann, Chem. Commun., p. 285 (1971). " P. J. L. Daniels and A. S. Yehaskel, 13th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., September, 1973. n p. j. L. Daniels, A. S. Yehaskel, and J. Morton, 13th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., September, 1973. o T. Oda, T. Mori, Y. Kyotani, and M. Nakayama, J. Antibiot. 24, 511 (1971).
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
265
P T. Mori, Y. Kyotani, I. Wutanabe, and T. Oda, J. Antibiot. 25, 149 (1972). q T. Mori, Y. Kyotani, I. Watanabe, and T. Oda, J. Antibiot. 25, 317 (1972). E. Akita, T. Tsuruoka, N. Ezaki, and T. Niida, J. Antibiot. 28, 175 (1970). P. W. K. Woo, H. W. Dion, and Q. R. Bartz, Tetrahedron Left., p. 2125 (1971). t H. Tsukiura, K. Saito, S. Kobaru, M. Konishi, and H. Kawaguchi, J. Antibiot. 26, 386 (1973). J. Shoji ~nd Y. Nakaguwa, J. Antibiot. 23, 569 (1970). " S. Inouye, T. Shomura, H. Watanabe, K. Totsugawa, and T. Niida, J. Antibiot. 26, 374 (1973). S. Horii and Y. Kameda, Chem. Commun., p. 747 (1972). T. Iwasa, Y. K~meda, M. Asai, S. Horii, and K. Mizuno, J. Antibiot. 24, 119 (1971). YS. Horii, Y. Kameda, and K. Kawahara, J. Antibiot. 25, 48 (1972). tion of an ion-exchange chromatography. Among these antibiotics, streptomycin, dihydrostreptomycin, k a n a m y c i n (which can be also called k a n a m y c i n A), k a n a m y c i n B, a mixture of gentamicins C1, C2 and Cla, a mixture of neomycins B and C, a mixture of p a r o m o m y c i n s I and I I , and ribostamycin are commercially available as chemotherapeutic agents useful in treating infections. H y g r o m y c i n B and destomycin A are used as animal anthelminties. K a s u g a m y c i n and validamycin are used for prevention of plant diseases. More than one aminoglycoside antibiotic is generally produced by the same strain as in cases of k a n a m y c i n s A, B, and C by S t r e p t o m y c e s k a n a m y c e t i c u s , 1 gentamicins C1, C,_,, and C ~ by M i c r o m o n o s p o r a purpurea"- and butirosins A and B by Bacillus circulans, a Therefore, complete separation of the analogous antibiotics which are produced in a same culture filtrate has been studied. These antibiotics are adsorbed by cation exchangers such as resin, cellulose, and Sephadex ion-exchangers, and separated. Commercially available cation exchangers used for their separation are shown in T a b l e II. A weak cation exchange resin possessing carboxylic acid as the functional group is most useful not only in extraction of these antibiotics from culture liquids, but also in separation of a mixture of these antibiotics into each components. For the first time in order to extract streptomycin ill about 1949, tile resin process was introduced into industrial extraction of natural products. This resin was a polyacrylic acid resin, Amberlite IRC-50, manufactured by R o h m and H a a s Co., Philadelphia. 1K. Maeda, M. Ueda, K. Yagishita, S. Kawaji, S. Kondo, M. Murase, T. Takeuchi, Y. Okami, and H. Umezawa, J. Antibiot. Set. A 10, 228 (1957). 2j. p. Rosselet, J. Marquez, E. Meseck, A. Murawski, A. Hamdan, C. Joyner, R. Schmidt, D. Migliore, and H. L. Herzog, Anlimicrob. Ag. Chemother. 1963, 14 (1964). H. W. Dion, P. W. K. Woo, N. E. Willmer, D. L. Kern, J. Onaga, and S. A. Fusari, Antimicrob. Ag. Chemother. 2, 84 (1972).
266
[11]
METHODS FOR T H E STUDY OF ANTIBIOTICS
TABLE II COMMERCIAL CATION EXCHANGERS FOR SEPARATION OF AMINOGLYCOSIDE ANTIBIOTICS
Functional group Matrix Resin
Sephadex Cellulose
Carboxylic acid
Phosphonic acid
Sulfonic acid
Amberlite IRC-50" Amberlite CG-50~ Amberlite IRC-84" Duolite CC-3b Lewatit CNP ~ Bio-Rex 70d CM-Sephadex C-25I CM-celluloseg
Duolite ES-63b Bio-Rex 63d
Amberlite IR-120~ Dowex 50~ Duolite C-20b Lewatit SP-120° SE-Sephadex C-25s
P-cellulosea
Manufactured by Rohm and Haas Co., Philadelphia, Pennsylvania. b Diamond Shamrock Chemical Co., Resinous Products Div., Cleveland, Ohio. c Naftone, Inc., Park Ave., New York. d Bio-Rad Laboratories, Richmond, California. DoT Chemical Co., Midland, Michigan. I Pharmacia Fine Chemicals, Division of AB Pharmacia, Uppsala, Sweden, CM-: carboxymethyl group, SE-: sulfoethyl group. g Carboxymethyl celluloses and cellulose phosphates are supplied by many different manufacturers. Even now it is widely used in antibiotic industries. Streptomycin adsorbed on a column of this resin in the Na* form was eluted with aqueous mineral acid in a good yield. Extraction of neomycins and kanamycins which are stable in alkaline solution is more efficiently accomplished by adsorption of NH4 * form of this resin and by elution with aqueous ammonia. A mixture of m a n y kinds of aminoglycoside antibiotics are efficiently separated into each components by a linear gradient elution of ammonia from a column of Amberlite CG-50 (chromatographic grade, a polyacrylic acid resin). 4 Processes using strong cation exchange resins possessing sulfonic acid as the functional group were introduced into extraction of weakly basic aminoglycoside antibiotics. In this case also, Amberlite CG-50 resin was utilized for further purification, eluting the weakly adsorbed antibiotics with water. Adsorption of aminoglycoside antibiotics on a resin is often interfered with by various kinds of cations in culture filtrates. Multivalent inorganic cations such as Ca 2÷ and Fe 3÷ are tightly bound to cation exchange resins. Therefore, these cations are first removed from culture liquids before application of the resin processes. 4H. Yamamoto, Y. Ikeda, S. Kondo, and H. Umezawa, unpublished data, 1973.
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C H R O M A T O G R A P HOF Y AMINOGLYCOSIDES
267
For separation and purification of aminoglycoside antibiotics, cellulose and Sephadex ion-exchangers are also useful. The most efficient chromatography for separation of analogous aminoglycoside antibiotics can be accomplished by the use of anion exchange resin, Dowex l-X2 (Dow Chemical Co., Midland, Michigan). This technique by nonionic adsorption chromatography was first employed for separation of kanamycins (Rothrock et al.5). Many of the other antibiotics can be purified by this technique. II. Preparation of Resin Columns Ion-exchange resin columns can be made in any desired size, from a few milliliters up to many liters. Height of exchanger beds for a good separation is usually 10-20 times longer than a column diameter. In a case of difficult separation, a much longer column can be used. In a conventional method of packing the column, ~ the resin is stirred in an excess of the first eluent to be used, and after most of the resin is settled, small particles are decanted off. The remaining resin is slurried into the column and allowed to settle by gravity to form the packed bed. It is often disadvantageous to use too large a volume of resin. Larger exchanger beds need more flow time and more rinsing, and yield inconveniently large solution volumes. An efficient system employed in antibiotic industries uses a minimum amount of the resin which is divided into more than one column. The culture liquid is continuously charged on the first column which is connected to the second column. From each column on which the antibiotic is fully adsorbed, it is eluted. By continuous repetition, the antibiotic can be extracted in excellent yield. The adsorption and elution are carried out usually in laboratories by downflow, but the upflow (backflow) technique is frequently used in large-scale operations. Before adsorption of an antibiotic, commercial ion-exchange resins are treated by repeated cycles of washing with 1-2 N hydrochloric acid and sodium hydroxide to remove imourities, and converted to a desired form by treatment with 1 M solution of an appropriate salt, acid or base. After the conversion is completed, the resin is washed with distilled water. Resins shrink and swell considerably and therefore the conversion is handled most easily in a large diameter column. Then, the resin is packed into the desired size column. J. W. Rothrock, R. T. Goegelman, and F. J. Wolf, Antibiot. Annu. 1958--1959, 796 (1959). G. Zweig and J. Sherma, eds., "Handbook of Chromatography," Vol. II, p. 61. Chem. Rubber Publ. Co., Cleveland, Ohio, 1972.
268
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
Weak cation exchange resins possessing carboxylic acid as the functional group are usually converted to H ÷ form by 1 N hydrochloric acid and to Na ÷ or NH4 + form by 1-2 N sodium hydroxide or ammonia. If a partially regenerated form is necessary, H + form and alkaline form of resins are well mixed, or the resin is washed with phosphate buffer of the proper pH. III. Extraction and Purificationby Using Carboxylic Acid Resins
Since carboxylic acid resins bind very strongly with H ÷ and weakly with N a +, K +, and N H 4 +, m a n y aminoglycoside antibiotics in aqueous solution are efficiently adsorbed by ion-exchange on a column of N a +, K +, or N H 4 + form of the resins. And these antibiotics can be eluted from the column with aqueous mineral acids such as hydrochloric acid and sulfuric acid. Alkaline-stable aminoglycoside antibiotics, for example, neomycins and kanamycins, are isolated by adsorption on N H 4 + form of this resin and by elution with aqueous ammonia. A m o n g the aminoglycoside antibiotics shown in Table I, those except weakly basic antibiotics are well adsorbed on Amberlite IRC-50 resin (Na + or N H , ÷ form) from culture filtrates.The weakly basic antibiotics, for example, nojirimycin, 4-amino-4-deoxy-a,a-trehalose, kasugamycin, and validamycins, are not adsorbed on carboxylic acid resin from culture filtrates. However, there is some retention of them on Amberlite CG-50 (NH4 + form) column, and they can be purified by elution with water from the resin. Strongly basic antibiotics, adsorbed on Amberlite IRC-50 columns, are usually eluted with less than I N hydrochloric acid or aqueous ammonia in good yields. The use of ammonia for their purification is preferred, because in this case a salt-free eluate is obtained. However, alkaline-unstable antibiotics, for example, streptomycins and streptothricins, must be eluted with a proper ion strength of salts or mineral acids. Efficient separation of m a n y alkaline-stable aminoglycoside antibiotics can be accomplished by elution with a linear gradient of ammonia from a column of Amberlite CG-50 resin,4 as shown in Figs. 1 and 2. A. Isolation o f K a n a m y c i n 7
Kanamycin in a culture filtrate (more than 500 ~g/ml) of Streptomyces kanamyceticus s can be purified by a single resin process followed 7K. Maeda, "Streptomyces Products Inhibiting Mycobacteria," p. 60. Wiley, New York, 1965. s H. Umezawa, M. Ueda, K. Maeda, K. Yagishita, S. Kondo, Y. 0kami, R. Utahara, Y. Osato, K. Nitta, and T. Takeuchi, J. Antibiot. Set. A 10, 181 (1957).
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AMINOGLYCOSIDES
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60 70 8b 90 160 Fraction number FIG. 1. Separation of neomycin B (sulfate, 6.8 mg), paromomycin I (sulfate, 5.8 mg), lividomycin A (5.4 mg), and lividomycin B (5.2 mg) by a column (0.8 X 20 cm) of Amberlite CG-50 (type II, NH4÷ form, 10 ml). The column which adsorbed the antibiotics mixture in water (0.5 ml) was washed with 0.1 N NH40H (40 ml) and then eluted with a linear gradient of 0.1 N to 0.8 N NH4OH (gradient rate: 0.5%/min, flow rate: 21 ml/hr). These antibiotics in fractions (1 ml each) were detected by Rr values of thin-layer chromatography (TLC) of silica gel (Merck, Art 5721) using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as developing solvent, followed by coloration with ninhydrin reagent. Each antibiotic in fractions was assayed by agar plate method using Bacillus subtilis PCI 219 as a test organism.
by crystallization. A column of Amberlite I R C - 5 0 resin (70% Na + or NH4 ÷ form) on which kanamycin is adsorbed is eluted with 1 N aqueous ammonia. The eluate is concentrated by evaporation to remove ammonia, acidified with sulfuric acid to p H 3, and decolorized with active charcoal. The pH of the solution is then adjusted to 8.0-8.2, that is, the p H of kanamycin monosulfate solution. To this concentrate containing about 100 mg of kanamycin base per milliliter, the same volume of methanol is added with stirring and cooling. After crystallization starts, more methanol is added up to 60% methanol concentration. With continued stirring, crystals of kanamycin monosulfate are obtained in 80% yield from the culture filtrate.
B. Isolation of 4-Amino-4-deoxy-(~,~-trehalose A streptomyces antibiotic, 4-amino-4-deoxy-~,a-trehalose9 has been purified by Amberlite CG-50 column chromatography by the authors. "H. Naganawa, N. Usui, T. Takita, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 145 (1974).
270
METHODS FOR THE STUDY OF ANTIBIOTICS Paromam i ne Destomycin A Lividomycin A Kanamycin A Paromomycin l Kanamycin ]3 Ribostamycin Lividomyci n B Tobrarnycin Neamine DKI3a Gentamicin Cb Neomycin B Butirosins c
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Fie. 2. The concentrations of ammonia for elution of aminoglycoside antibiotics from a column (0.8 × 20 cm) of Amberlite CG-50 (type II, NH~÷ form, 10 ml). The column which adsorbed antibiotics (approximately 5 mg each) was washed with 0.1 N NH,OH (40 ml) and then eluted with a linear gradient of 0.1 N to 0.8 N NH40H (gradient rate : 0.5%/min, flow rate : 21 ml/hr). These antibiotics in fractions (1 ml each) were detected by thin-layer chromatography of silica gel (Merck, Art 5721) using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as a developing solvent, high-voltage paper electrophoresis under 3300 V for 15 min using formic acid-acetic acid-water (25:75:900 in volume) as an electrolyte solution, and agar plate method using Bacillus subtilis PCI 219 as a test organism. (a) 3',4'dideoxykanamycin B; (b) gentamicin C complex; (c) butirosins A and B.
An aqueous solution (3 ml) of the crude powder of the antibiotic extracted from a culture filtrate by a carbon adsorption process is charged to a column (1.25 X 31 cm) of Amberlite CG-50 resin (type I, NH~ + form, 40 ml) and the c h r o m a t o g r a m is developed with water. The eluate in fractions 22-56 (2 ml each) showing bioaetivity against Escherichia coli N I H J or Bacillus subtilis P C I 219 are combined and lyophilized to yield a white powder (391 mg) of the pure antibiotic.
C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a 4 An aqueous solution (0.5 ml) containing neomycin B sulfate (6.8 mg), p a r o m o m y c i n I sulfate (5.8 mg), lividomycin A (5.4 rag), and lividomycin B (5 2 rag) is charged to a column (0.8 X 20 era) of Amberlite CG-50 resin (type I I , NH4 ÷ form, 10 ml). After the column is washed with 0.1 N aqueous ammonia (40 ml), the antibiotics are eluted with a linear gradient of aqueous ammonia from 0.1 to 0.8 N for 200 min (a gradient rate of 0.5% per minute) at a flow rate of 21 ml per hour using an Aerograph LC-4200 gradient system (Varian Instruments, California). With
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
271
the aid of a fraction collector approximately 1-ml fractions of the efituent are collected. Each antibiotic in the fractions is determined by thin-layer chromatography on silica gel (Merck, Art 5721), for instance, using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as a developing solvent, followed by treatment with ninhydrin reagent. Each antibiotic in the fractions can be also assayed by an agar plate method using Bacillus subtilis PCI 219 as a test organism. These four antibiotics are completely separated by this technique as shown in Fig. 1. This technique has been applied to the other strongly basic antibiotics and the concentrations of ammonia in eluent for each antibiotic are summarized in Fig. 2.
D. Separation of Nebramycins on a Large Scale I° A solution of nebramycin complex has been obtained from culture filtrates of Streptomyces tenebrarius by adsorption on Amberlite IRC-50 resin, elution with 1 N aqueous ammonia and concentration to remove excess ammonia. The following example was reported. The solution (20 liters, 82 X 106 units of the activity against Klebsiella pneumoniae FDA KL4) was applied to a 10 cm diameter column which contained 20 liters of Bio-Rex 70 resin (NH4 * form). The column was washed with 12 liters of water and then eluted with a gradient prepared by adding 0.2 N aqueous ammonia to a 50-liter constant-volume reservoir which was charged with 0.05 N aqueous ammonia. The flow rate was 20 ml per minute and fractions of 500 ml were collected. The following fractions were combined, concentrated, and freeze-dried: fractions 39-100, 102 g (35 X 10~ units) of factor 2 (apramycin); fractions 115-154, 14 g (8.1 X 10~ units) of factor 4 (6"-O-carbamoylkanamycin B) ; fractions 188-240, 24 g (30 X 10~ units) of factor 5' (6"-O-carbamoyltobramycin). Eighty-nine percent of the activity was recovered.
E. Separation of Derivatives of 3',4'-Dideoxykanamycin B In the course of chemical derivation of kanamycins based on mechanism of resistance to aminoglycoside antibiotics, 11 3',4'-dideoxykanamycin B (DKB), which inhibits sensitive and resistant organisms including ~*K. F. Koch, F. A. Davis, and J. A. Rhoades, d. Antibiot. 26, 745 (1973). 11H. Umezawa, "Progress in Antimicrobial and Anticancer Chemotherapy," Vol. II, p. 567. Univ. of Tokyo Press, Tokyo, 1970. Published also in Advan. Carbohyd. Chem. 30, 183-225 (1975).
272
METHODS FOR THE STUDY OF ANTIBIOTICS
[lll
Pseudomonas aeruginosa, was synthesized, 12 and thereafter, N-(S)-4amino-2-hydroxybutyryl (AHB) derivatives were prepared. 13,~4 These products were purified by the following resin processes. 6'-N-t-Butyloxycarbonyl DKB (1109 rag) was acylated with (S)-4-amino-2-hydroxybutyric acid and a mixture of positional isomers of AHB-DKB was obtained after removal of protecting groups. The mixture (trifluoroacetate, 3067 mg in 5 ml water) was charged to a column (14 mm diameter) of Ambertire CG-50 resin (type I, NH4 ÷ form, 50 ml). After washing with water (250 ml), the column was eluted by stepwise elution with 500 ml each of 0.5, 0.75, and 1.0 N ammonia. The unreacted DKB (297 mg, 32% yield) and 2'-AHB-DKB (238 mg, 21%) were eluted with 0.5 N ammonia, 3-AHB-DKB (72 mg, 6%), 1-AHB-DKB (131 mg, 12%) and 3"-AHB-DKB (53 mg, 4%) with 0.75 N ammonia, and 3,2'-diAHB-DKB (46 rag, 3%), 1,2'-diAHB-DKB (47 rag, 3%) and the other diacyl derivatives with 1.0 N ammonia. By this chromatography, these positional isomers 14 of AHB-DKB, were completely separated. 1-AHB-DKB and 1,2'-diAHB-DKB show strong activity against sensitive and resistant organisms. The latter organisms produced kanamycin phosphotransferases and kanamycin nucleotidyltransferase. IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins Weakly basic aminoglycoside antibiotics, for example, nojirimycin, 3-amino-3-deoxy-D-glucose, trehalosamine, mannosyl glucosaminide, 4-amino-4-deoxy-a,a-trehalose, kasugamycin, and validamycins can be extracted from culture filtrates by adsorption on strong cation exchange resins possessing sulfonic acid groups, such as Amberlite IR-120, Dowex 50, and Lewatit SP-120, and elution with dilute aqueous ammonia. Since sulfonie acid resins hold H + more weakly than Na ÷ and NH4 +, these resins are generally used as H + form. From a column of Amberlite IR-120 resin kanamycin is efficiently eluted with 1 N aqueous ammonia, but not with t N hydrochloric acid. Therefore washing the column with the acid and elution of kanamycin with ammonia gives a highly purified kanamycin. 1 Phosphonic acid resins have not been used widely. However, extraction of kanamycin from culture filtrates by adsorption on Duolite C-62, 12H. Umezawa, S. Umezawa, T. Tsuchiya, and Y. Okazaki, J. Antibiot. 24, 485 (1971). 1aS. Kondo, K. Iinuma, It. Yamamoto, K. Maeda, and H. Umezawa, J. Antibiot. 26, ¢12 (1973). ~4S. Kondo, K. Iinuma, H. Yamamoto, Y. Ikeda, K. Maeda, and H. Umezawa, J. Antibiot. 26, 705 (1973).
[11]
CHROMATOGR/kPHY OF AMINOGLYCOSIDES
273
a phosphonic acid resin formerly supplied by Diamond Shamrock Chemical Co., Resinous Products Division, Ohio, and elution with 5% aqueous ammonia has been reported? Most of aminoglycoside antibiotics can be adsorbed on phosphonic acid resins.
A. Isolation of Kasugamycin by Amberlite IR-120 Kasugamycin15 in culture filtrate of Streptomyces kasugaensis has been successfully extracted by Amberlite IR-120 resin as follows. The filtrate (1570 liters, 530 ~g/ml) was charged on Amberlite IR-120 column (H ÷ form, 300 liters). The column was washed with water and then eluted with 0.5 N aqueous ammonia. The first (53 liters), the second (200 liters), and the third (200 liters) eluates contained 28.5 g, 738 g, and 44.2 g of kasugamycin, respectively. The second eluate was adjusted to pH 6.6 with hydrochloric acid and concentrated to 6.32 liters under reduced pressure. After addition of ethanol (60 liters) to this concentrate, the crude crystals of kasugamycin hydrochloride (850 g, 90% purity) were obtained.
B. Separation of Validamycins by Dowex 5016 Validamycin E-rich fractions and F-rich fractions obtained by Dowex l-X2 resin chromatography (see Section VI) were further chromatographed on a column of Dowex 50-W X2 resin by eluting with a pyridineacetic acid buffer (pH 6.0). In this chromatography, validamycin F was eluted first and thereafter validamycin E.
V. Extraction and Purification Using Cellulose and Sephadex Exchangers Various kinds of cation exchangers having hydrophilic supporting matrix are commercially available. Cellulose and Sephadex ion-exchangers are used generally for separation of large molecules. Among these exchangers, carboxymethylcellulose, cellulose phosphate, CM-Sephadex C-25, and SE-Sephadex C-25 can be used for separation of basic antibiotics. Separation of streptothricin components by column chromatography 15H. Umezawa, Y. Okami, T. Hashimoto, Y. Suhara, M. Hamada, and T. Takeuchi, J. Antibiot. Ser. A 18, 101 (1965). S. Horii, Y. Kameda, and K. Kawahara,J. Antibiot. 25, 48 (1972).
274
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
of carboxymethyl cellulose has been reported27 Many phleomycinTM and bleomycinTM components are efficiently separated by column chromatography of CM-Sephadex C-25 with a gradient of ammonium formate. Most of the aminoglycoside antibiotics can be purified by column chromatography on carboxymethyl cellulose, cellulose phosphate, CMSephadex C-25, and SE-Sephadex C-25 using various ion strengths of salt solutions, such as sodium chloride and ammonium formate. Streptomycin is adsorbed on a column of CM-Sephadex C-25 equilibrated with 0.1 M sodium chloride or 1.0 M ammonium formate and eluted with 0.55 M sodium chloride or 2.0 M ammonium formate, s° The antibiotic, adsorbed on CM-cellulose (Brown Co., Fifth Ave., New York, 0.63 mEq/g, equilibrated with 0.1 M ammonium formate), is eluted with 0.6 M ammonium formate, s° A. Separation of Gentamicin C Complex by CM-Sephadex Maehr and Schaffneff1 have described the separation of gentamicin components by Dowex 1-X2 resin chromatography (see Section VI). The authors 2s found another powerful method using CM-Sephadex column as follows. Gentamicin C complex (800 rag) in 0.1 M ammonium formate (400 ml) was adsorbed on a column (35 mm diameter) of CM-Sephadex C-25 (800 ml, equilibrated with 0.1 M ammonium formate). The column was washed successively with 450 ml of 0.1 M, 1275 ml of 0.5 M, 4590 ml of 1.0 M and 1860 ml of 1.5 M ammonium formate and then eluted with 1.8 M ammonium formate. The effluent was cut into each approximately 15-ml fractions, and the fractions were assayed by agar plate method using Bacillus subtilis PCI 219 as a test organism and by silica gel (Merck, Art. 5721) thin-layer chromatography using butanol-ethanol-chloroform-28% ammonia (4:5:2:5 in volume) as a developing solvent followed by coloration with ninhydrin reagent. The three main peaks of gentamicins C1, C~, and C1~ appeared in fractions 569, 594, and 613, respectively. The eluates in fractions 557-575 were combined and diluted with water (6 liters). Gentamicin C~ in the diluted solution was adsorbed on a column of Amberlite CG-50 resin (type I, NH4 ÷ form, 50 ml). Elution with 0.5% aqueous ammonia gave 172 mg of pure gentamicin C1. 1~A. S. Khokhlov and P. D. Reshetov, J. Chromatogr. 14, 495 (1964). T. Ikekawa, F. Iwami, H. Hiranaka, and H. Umezawa, Y. Antibiot. Set. A 17, 194 (1964). 1~H. Umezawa, Y. Suhara, T. Takita, and K. Maeda, J. Antibiot. Ser. A 19, 210 (1966). 2oK. Yokose, Y. Suhara, M. Miyamoto, S. Kondo, and H. Umezawa, unpublished data, 1973. ~ It. Maehr and C. P. Schaffner,J. Chromatogr. 30, 572 (1967). 22M. Yagisawa,S. Kondo, and H. Umezawa,unpublished data, 1973.
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
275
B. Separation of Lividomycins by CM-Sephadex 2~ The separation of each lividomycin has been accomplished by the following processes. A crude powder of lividomycins (24 mg) which was obtained by adsorption on Amberlite IRC-84 resin (NH4 + form) from a culture filtrate of S t r e p t o m y c e s lividus and elution with 1 N aqueous ammonia was dissolved in water (5 ml) and charged to a column (1 }( 40 cm) of CM-Sephadex C-25 (NH4 ÷ form). After washing with water, the column was eluted by a gradient between 0.12 N (200 ml) and 0.35 N aqueous ammonia (25 ml) at a flow rate of 25 ml per hour at 27% All fractions (3 ml each) were assayed by the paper disk method using Bacillus subtilis ATCC 6633 as a test organism. Four peaks of active fractions appeared at fractions 30, 44, 64, and 72, which were designated as No. 2230-C (mannosyl paromomycin), lividomycin A, No. 2230-D (paromomycin), and lividomycin B, respectively.
VI. Nonionic Adsorption Chromatography by Anion Exchange Resins In using anion exchange resins for the removal of colored impurities from crude kanamyein aqueous solutions, Rothrock et al. ~ observed some retention of the antibiotic by strong anion exchange resins. Further investigations revealed that porous, strongly basic anion exchange resins with quaternary amine functional groups could be used at low loadings to separate kanamyein A from kanamycin B. Of these, Dowex 1-X2 resin (Dow Chemical Co., Midland, Michigan, 50-100 mesh) in OH- form is satisfactory for the chromatographic analysis of kanamycin mixtures. This technique by ion exclusion chromatography can be most efficiently used for separation of analogous aminoglycoside antibiotics. Complete separations of neomycins,24 paromomycins,~4 destomycins,"-5 gentamicins,21 lividomycins,2~ butirosins, 33 and validamycins16 have been reported by many researchers, and this technique can be applied £o largescale preparative operations. Recently, high-pressure liquid chromatography using a column of Aminex A-2726 or A-28 =7 resin (a quaternary amine resin, Bio-Rad Laboratories, California) was introduced into analysis or purification of aminoglycoside antibiotics. =3T. Mori, T. Ichiyanagi, H. Kondo, K. Tokunaga, T. Oda, and K. Munakata, J. Antibiot. 24, 339 (1971). ~4H. Maehr and C. P. Schaffner,Anal. Chem. 36, 104 (1964). 25S. Kondo, M. Sezaki, M. Koike, M. Shimura, E. Akita, K. Satoh, and T. Hats, I. Antibiot. Ser. A 18, 38 (1965). 2~T. Ohtake and M. Yaguchi, "Liquid Chromatography at Work," No. 5. Nippon Electric Varian Ltd., 1973. ~7H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, and T. Takeuchi, J. Antibiot. 26, 407 (1973).
276
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
A. Preparation of Nonionic Columns T M The anion exchange resin, D0wex I-X2 (CI- form, 50-100 mesh) is backwashed free of fines before being converted to the O H - form by columnwise washing with 2 resin volumes of I % sodium sulfate solution and 2 resin volumes of 10% sodium hydroxide solution. The resin shrinks and swells considerably and therefore these operations can be done most easily in a large-diameter column. The resin is washed thoroughly with C02-free distilled water to remove excess alkali. The chromatographic tube is filled up to two-thirds capacity with C02-free distilled water, and the resin in the O H - form is slurried directly into the column with C02free distilled water, so that the resin does not entrap air bubbles. The water is drained from the bottom of the column to the resin bed level. A column prepared in this manner conveniently can hold 400 ml of resin in a bed depth of I00 cm which gives efficient chromatography to handle I0 g of crude kanamycin. For smaller samples a proportionately smaller column can be employed. B. Chromatographic Procedure TM
An approximately 25% aqueous solution of aminoglycoside antibiotics is introduced to the top of the resin bed. Although hydrochloride or sulfate salts of antibiotics are usually used for the chromatography, free bases of antibiotics give more satisfactory separation. The column is developed with C02-free distilled water at the effluent flow rate of one resin volume per 2-4 hr. With the aid of an automatic fraction collector, the effluent is fractionated (usually one-tenth volume of a resin volume for each fraction). Antibiotics in the effluent are determined quantitatively by bioactivity, conductivity, refractive index, and colorations with reagents, such as ninhydrin. Antibiotics in the fractions can be also detected by paper and thin-layer chromatography, and high-voltage paper electrophoresis. C. Application to Separation of Kanamycins
Rothrock et al2 achieved a complete separation of kanamycins A and B by the technique described above. By application of this technique, crystalline kanamycin C was isolated from a mixture of kanamycins. 2s An example of quantitative separation of kanamycins is as follows. 29 A ~sM. Murase, T. Wakazawa, M. Abe, and S. Kawaji, J. Antibiot. Set. A 14, 156 (1961). 29S. Inouye and H. Ogawa, J. Chromatogr. 13, 536 (1964).
[lll
CHROMATOGRAPHY OF AMINOGLYCOSIDES
277
mixture of kanamycin A (2.08 mg), B (0.207 rag), and C (0.143 rag) is chromatographed on a column (0.9 X 39 cm) of Dowex l-X2 resin (OH- form, 200-400 mesh) with an effluent flow rate of 30 ml per hour. The antibiotics in the effluent are automatically determined by color development of ninhydrin reaction using an amino acid analy~,er (Hitachi Type KLA-2, Hitachi Co., Tokyo). The elution peaks of these antibiotics have retention times as follows, kanamycin B: 85 min, C: 108 min, and A: 187 min.
D. Application to Separation of Neomycins Neomycins were completely separated into three components by Maehr and Schaffner.2. An approximately 25% aqueous solution of a commercial neomycin sulfate sample (2 g) containing about 30% neomycin C, 70% neomycin B, and a trace of neomycin A (neamine) is charged to a column (2.5 X 100 cm) of Dowex l-X2 resin (OH- form, 50-100 mesh) and the column is developed with distilled water at a linear flow rate of 0.4-0.6 cm per minute. The effluent is cut into fractions, approximately 40 ml each, and the antibiotics in these fractions are analyzed by a quantitative ninhydrin procedure. Three elution peaks of neomycins A, C, and B appear in fractions 11, 22, and 41, respectively.
E. Application to Separation of Destomycins 25 By application of this technique, a crude powder of destomycins which was obtained by adsorption on Amberlite IRC-50 resin (Na ÷ form) from a culture filtrate of Streptomyces rimofac~ens and elution with 2% aqueous ammonia has been purified. A crude powder (175 g) of destomycins is dissolved in 200 ml of water and charged to a column of Dowex I-X2 resin (OH- form, 50-100 mesh, 1600 ml). The column is developed with water at a flow rate of 4 ml/min. The eluate is cut into 20-ml fractions each, and the bioactivities of all fractions are determined using Bacillus subtilis ATCC 6633 and Mycobacterium smegmatis ATCC 607 as the test organisms. Destomycin B is eluted in fractions 64--72, and destomycin A starts to appear in fraction 73. Fractions 76-200 are combined and lyophilized to yield a white powder (69.5 g) of pure destomycin A. The eluate in fractions 64-75 gives a white powder (13.7 g of a mixture of destomycins) on lyophilization. The mixture is dissolved in 50 ml of water and rechromatographed on a column of the resin (OH- form, 400 ml). Destomycin B is eluted in fractions 15-26 (20 ml each), which contains 4.0 g of pure destomycin B.
278
METHODS
FOR
THE
STUDY
OF ANTIBIOTICS
[11]
F. High-Pressure Liquid Chromatography High-pressure liquid chromatography using a column of Aminex A-2726 or A-2827 resin (a quaternary amine resin, Bio-Rad Laboratories,
California) is a powerful technique for analysis and purification of aminoglycoside antibiotics. The differential quantitative determination of kanamycin, lividomycin A, and 3 ' , 4 ' - d i d e o x y k a n a ~ c i n B TM has been accomplished by the following method? ~ A commercial ~pparatus, Aerograph LC-4200 system of Varian Instruments, Californi~ was used. A mixture of 40 ~g each of kanamycin A, 3',4'-dideoxykanafa~rcin B, and lividomycin A in water (12 ~l) is injected in the top of ~ e column (0.2 X 100 cm) of Aminex A-28 resin (8-12 ~m, OH- form)~and the column is developed with distilled water, operating at 50 °, 15 ml/hr, and 3400 psi. The amounts of these antibiotics in the effluent are automatically recorded by a refractive index detector. As shown in Fig. 3, these antibiotics can be completely separated in a short time and can be quantitatively determined by measuring the height of each peak. Such a differential assay is useful in studies on kinetics of an enzyme which phosphorylates or acetylates these antibiotics. High-pressure liquid chromatography is described in detail elsewhere. 3°
J
"N
.r I
0
:5 I
i
5 10 Minutes
I
15
FIG. 3. Separation of kanamycin, lividomycin A, and 3',4'-dideoxykanamycin B by high-pressure liquid chromatography on a column (0.2 X 100 cm) of Aminex A-28 (8-12 /zin, OH- form). A mixture of each 40 ~g of antibiotics in water (12 /~l) was injected to the top of the column connected with Varian Aerograph LC-4200 system and the column was developed with distilled water, operating at 50°, 15 ml/hour, 3400 psi. The amounts of these antibiotics in effluent were automatically recorded by the RI detector (4 × 10-5 refractive index units in full scale).
~°K. TsuJi, this volume [15].
