256
METHODS FOR THE STUDY OF ANTIBIOTICS
[10]
Hitachi 063GC equipped with flame ionization detector was used with the following chromatographic conditions: 1. For TMS validamycins A to F: Glass column, 3 mm i.d. X 2000 mm packed with 1% OV-1 on Chromosorb W AW DMCS. Temperature: column, 280°; injection, 300 °. Carrier gas (helium), 60 ml/min. 2. For separation of TMS-validoxylamines A and B: Glass column, 3 mm i.d. X 2000 mm packed with 3% OV-17 on Chromosorb W AW DMCS. Temperature: oven, 250c; injection, 300 °. Carrier gas (helium), 30 ml/min. 3. For separation of TMS-derivatives of degradation products: Glass column, 3 mm i.d. X 2000 mm packed with 5% OV-17 on chromosorb W AW DMCS. Column temperature: initial, 150°; final, 280 ° (10°/rain). Injection temperature, 300 °. Carrier gas (helium) : 45 ml/min.
Acknowledgment The authors are indebted to their many colleagueswho provided suggestionsand data for this chapter.
[10] 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 Streptothricin-like Antibiotics
By
DONALD B . BORDERS
I. Introduction . . . . . . . . . . . . . . . . . . II. Ion-Exchange Chromatography of Intact Antibiotics. . . . . . . III. Ion-Exchange Chromatography of Hydrolysis Fragments . . . . .
256 258 260
I. Introduction Members of the streptothricin family of antibiotics are very watersoluble, strongly basic compounds with broad antimicrobial spectra. One of the most effective means of resolving these antibiotics and their hydrolysis products is by ion-exchange chromatography. It is assumed that the ion-exchange chromatographic techniques which apply to streptothricin-type antibiotics would be of general interest since so'me of the techniques might also apply to a number of other structurally different antibiotics having similar chromatographic and ionic properties.
[10]
CHROMATOGRAPHYOF STREPTOTHRICIN ANTIBIOTICS
257
The structural relationship of some streptothricin-type antibiotics is indicated in Fig. 1. In general, the antibiotics with known structures vary by the type of amino acid side chain (R~) and methyl or hydrogen substitution (R~ and R~) on the nitrogens of the streptolidine or the aminosugar. Usually gulosamine is the aminosugar moiety. However, fucosRI~ OH ~ H , , ~ O H
streptolidine
N~NH NH
/
Rs
gufosamine
R20"~..,,,,"~OR
3
Substituents Compound
RI
R2, R3
Streptothricin
H
H, CNH2
H
CCH2 CH CH2 CH2 CH2 NH2
Streptolin
H
O H, ~NH2
H
(CCH2 C CH2 CH2 CH2 NH)2 or 3 H
LL-AC541
H
O ii H, CNH2
CH3
C CH2 NH CH
H
O H, CNH2
CH3
O C CH2 NH2
LL-AB664
CH3
O H, CNH2
CH3
O NH C CH2NH CH
Oeformimino LL-AB664
CH3
0 H>CNH2
CH3
0 C CH2NH2
H
O H,(~NH2
CH3
O ~ CH2NH CH3
R4
?
o
Deformimino LL-AC541
LL-BL136
R5
?
NH2 ,NHz
9
N,.
FIC. 1. Structures of streptothricin and some related antibiotics.
258
METHODS FOR THE STUDY OF ANTIBIOTICS
[10l
amine has been proposed as a hydrolysis product from fucothricin I and glucosamine from racemomycin 0. 2 Streptolidine is an amino acid that appears to be unique to this family of antibiotics. The relative stereochemistry of the three asymmetric centers of this moiety was deduced from chemical studies 3 and N M R spectral; subsequent X - r a y analysis of streptolidine dihydrochloride confirmed these results and established the absolute stereochemistry2 As a result of this stereochemistry, the amino and carboxyl groups of the streptolidine moiety are linked together into a strained lactam ring which probably approximates a boat conformation. This strained ring system explains one of the most labile structural features of this family of antibiotics, and ring opening with biological inactivation occurs if the antibiotics are allowed to stand several hours at room temperature in dilute acid or alkali2 ,7 When streptothricin is allowed to stand in water at room temperature for several days inactivation occurs s presumably through this same mechanism. Some of these antibiotics, LL-AC541 and LL-AB664, can also degrade by standing several days in methanol at room temperature due to a very labile formimino group. 4 These stability features should be considered when any chromatographic separations of streptothricin-type antibiotics are undertaken.
II. Ion-Exchange Chromatography of Intact Antibiotics Probably one of the best methods of separating streptothricin-type antibiotics which differby the number of fl-lysinegroups in the side chain has been by salt gradient elution on carboxymethyl cellulose2,I° In our laboratories, this same system has given excellent separation of a mixture of deformimino LL-AC541, LL-AC541, and streptothricin (listedin order 1M. J. Thirumalachar, P. V. Deshmukh, R. S. Sukapure, and P. W. Rahalker, Hindustan Antibiot. Bull. 14, 4, 1971. S. Takemura, Chem. Pharm. Bull. 8, 578 (1960). 3H. E. Carter, C. C. Sweeley, E. E. Daniels, J. E. McNary, C. P. Schaffner, C. A. 83, 4296 (1961). ' D. B. Borders, K. J. Sax, J. E. Lancaster, W. K. Hausmann, L. A. Mitscher, E. R. West, E. E. Van Tamelen, J. R. Dyer, and It. A. Whaley, J. Amer. Chem. Soc. Wetzel, and E. L. Patterson, Tetrahedron 26, 3123 (1970). B. W. Bycroft and T. J. King, Chem. Commun. 1972, 652 (1972). E. E. Van Tamelen, J. R. Dyer, H. A. Whaley, H. E. Carter, and G. B. Whitfield, Jr., J. Amer. Chem. Soc. 83, 4295 (1961). 7It. Taniyama, Y. Sawada, and T. Kitagawa, J. Antibiot. 24, 662 (1971). s A. W. Johnson and J. W. Westley, J. Chem. Soc. (London), p. 1642 (1962). 9A. S. Khokhlov and P. D. Reshetov, J. Chromatogr., 14, 495 (1964). 10p. D Reshetov and A. S. Khokhlov, Khim. Prir. Soedin. 1, 42 (1965).
[101
CHROMATOGRAPHY OF S T R E P T O T H R I C I N ANTIBIOTICS
259
of increasing retention time). This type of chromatography also provides a medium with an optimum pH for antibiotic stability. A very useful variation of this procedure has been elution with a volatile buffer of pyridine-acetic acid in the isolation of the racemomycins. H The procedure described below in the experimental section has been quite successful in our laboratories and involves the use of a KC1 gradient on a weak cationic exchanger such as Amberlite CG-5022 Recovery of the antibiotics from the salt solutions of the column affluents can be accomplished by derivatives or ion-exchange techniques. ~°,1~ Procedure Separation o] LL-AC541 and De]ormimino LL-AC541 by Salt Gradient Elution on a Weak Cationic Exchange Resin. 12 A column is prepared by suspending cycled resin (Amberlite CG-50), K ÷ form, 200-400 mesh, in water and adding with stirring 1 N HC1 until the solution over the resin remains approximately p H 7. A slurry of the resin is then poured into a column, 1.2 X 100 cm, which is rinsed with several bed volumes of water prior to addition of the antibiotic charge. A mixture, 300 mg, of LL-AC541 and deformimino LL-AC541 is dissolved in 1.5 ml of water and adsorbed onto the column. The adsorbed antibiotic is eluted from the column with a gradient formed from water (500 ml) and 2.0 M potassium chloride (500 ml) at a flow of approximately 0.17 m l / m i n while the effluent is collected in approximately 5-ml fractions with the aid of a fraction collector. The gradient is approximately linear and is formed by slow addition of the KC1 solution to water with an apparatus consisting of a reservoir and mixing chamber as described by Parr. 13,14 The deformimino LL-AC541 emerges from the column in 550-580 ml and the LL-AC541 in 610-680 ml of gradient effluent (Fig. 2). The antibiotics in these fractions are readily detected by a disc agar diffusion method against a sensitive organism such as Klebsiella pneumoniae. This is accomplished by dipping paper disks, 13.5 mm diameter, into each tube of eluate and placing the resulting discs on agar seeded with the test organism. Inhibition zones are read as zone diameters after incubation of the test organism at 37 ° overnight. 1~H. Taniyama, Y. Sawada and T. Kitagawa, Chem. Pharm. Bull. 19, 1627 (1971). ,5 V. Zbinovsky, W. K. Hausmann, E. R. Wetzel, D. B. Borders, and E. L. Patterson, Appl. Microbiol. 16, 614 (1968) 1, C. W. Parr, Biochem. J. 56, XXVII (1954); R. M. Block and N.-S. Ling, Anal. Chem. 26, 1543 (1954). 1~j. j. Wren, .l. Chromatogr. 12, 32 (1963).
260
METHODS
FOR
THE
STUDY
[10]
OF ANTIBIOTICS
SO
-g E o U
g
25 20
1.0
L
G Y
I
"5
15.-
>, o
O5 '6
I0 5 029O
0.1 420
550 610 680 ml of Solt Grodient
810
FIG. 2. Resolution of LL-AC541 and deformimino LL-AC541 by chromatography with a KCI gradient on a weak cation exchanger. The two antibiotic components emerging from the column can also be detected by monitoring the column effluent at 215 nm since these compounds have significant end absorption. III. Ion-Exchange Chromatography of Hydrolysis Fragments Quantitative and qualitative studies of hydrolysis fragments have proved to be very valuable in the identification and structure determination of streptothricin-type antibiotics. The most precise procedures for conducting these studies have utilized amino acid autoanalyzers with columns of strong cationic exchangers eluted with a buffer near pH 5. The first reported application of an amino acid autoanalyzer to the study of streptothricin hydrolysates was described by C. A. Egorov et al. 15 They found that the components of various streptothricin-type complexes had the gross structure of streptothricin but differed only by the number of #-lysine groups per molecular ranging from I to 7.16,17 In the course of these studies evidence was presented to suggest that streptolin (Fig. I) might have three #-lysine residues per molecule rather than two as originally proposed2 Subsequent studies extended the analyzer procedures to streptothricintype antibiotics differing by the type of amino acid side chain and to is C. A. Egorov, P. D. Reshetov, and A. S. Khokhlov, J. Chromatogr. 19, 214 (1965). 18A. S. Khokhlov and K. I. Shutova, J Antibiot. 2S, 501 (1972). 17p. D. Reshetov, T. A. Egorov, and A. S. Khokhlov, Khim. Prir. Soedin. I, 117 (1965).
[10]
CHROMATOGRAPHY OF STREPTOTHRICIN ANTIBIOTICS
261
resolution of the different streptolidine-sugar compounds, ls-2° An additional advantage of this modification is that some streptolidine-sugar compounds can be detected by ninhydrin whereas the corresponding sugars do not react because of substitution on the nitrogen of the sugar.4,19 Resolution of the different streptolidine-sugar compounds by paper chromatography or eleetrophoresis has been only partially successful. Although the amino acid autoanalyzer provides excellent reproducibility and separation of the hydrolysis fragments, care should be given to proper controls for each individual analyzer. Variations in resin apparently can cause a reversal of peaks. 21 Procedure Preparation o] Hydrolysates. Hydrolysates are prepared under conditions that favor formation of the streptolidine-sugar compounds. Approximately 1% solutions of the antibiotics in 3 N HC1 are heated in sealed vials at 100-110 ° for 5 hr. The resulting solutions are evaporated under reduced pressure to residues which are dissolved in water to give ,-~ 1% solutions used for autoanalyzer studies. Autoanalyzer Determinations. The compositions of the hydrolysates are determined with a Technicon amino acid autoanalyzer under the conditions described below. The columns are eluted continuously with a pH 5.0 buffer composed of 14.71 g {0.05 mole) of sodium citrate dihydrate, 900 ml of water, 25 ml (0.05 mmole) of 0.002 N sodium hydroxide, 35.07 g (0.60 mole) of sodium chloride, and 10 ml of Brij detergent [polyoxyethylene (23) lauryl ether (10 g) dissolved in 200 ml of water] adjusted to pH 5.0 with 6 N HC1 and diluted to 1 liter with water. Condition A. A column (0.6 cm by 130 cm) of Chromobeads type A (sulfonated polystyrene resin from Technicon Chromatography Corp., Chauncey, New York) is maintained at 60 ° by a water jacket and eluted with pH 5 0 buffer. The column effluent is monitored by a standard automated n!nhydrin-hydrindantin procedure. 2~ The column flow rate is 0.5 ml/min. ~8D. B. Borders, J. P. Kirby, E. R. Wetzel, M. C. Davies, and W. K. Hausmann, Antimicrob. Ag. Chemother. I, 403 (1972). 1, D. B. Borders, W. K. Hausmann, E. R. Wetzel, and E. L. Patterson, Tetrahedron Lett. p. 4187 (1967). :o K. J. Sax, P. Monnikendam, D. B. Borders, P. Shu, L. A. Mitscher, W. K. Hausmann, and E. L. Patterson, Antimicrob. Ag. Chemother. 1967, 442 (1968). ~*J. Shoji, S. Kozuki, M. Ebata, and H. Otsuka, J. Antibiot. 21, 509 (,1968). 2.-Technicon Corporation, Technicon Autoanalyzer Methodology. Section I, pp. 7-8. Technicon Chromatography Corp., Chauncey, New York, 1961.
262
[10]
METHODS FOR THE STUDY OF ANTIBIOTICS TABLE I AUTOANALYZER RETENTION TIMES FOR ANTIBIOTIC HYDROLYSIS FRAGMENTS AND RELATED COMPOUNDS Condition A (14-hr r u n )
Compound Glycine Glucosamine Galactosamine Gulosamine Lysine Streptolidine ~-Lysine Ammonia Methylamine Arginine N-guan-Streptolidyl
Retention time ° (min)
Condition B (5-hr r u n )
Color yield b (area/mmole)
Retention time (Inin)
Color yield (area/mmole)
73 159 ~ 164 ~ 173 c 204 235 258 307 344 437 605
35.3 33.3 32.6 -34.4 29.6 -12.7 10.0 31.6 --
35 61 -69 -93 99 113 --236
19.1 15.7 -14.3 -8.3 15.4 6.4 --9.8
767
31.9
302
9.9
gulosaminide N-guan-Streptolidyl
N~-methylgulosaminide a R e t e n t i o n t i m e averages for a p a r t i c u l a r c o m p o u n d were u s u a l l y + 2 m i n f r o m given value. b Area in a r b i t r a r y units. c S e p a r a t i o n of s u g a r s confirmed b y a m i x e d r u n . TABLE II AUTOANALYZER COMPARISONS OF NINHYDRIN-PosITIVE FRAGMENTS FROM HYDROLYSATES OF VARIOUS STREPTOTHR1CIN-TYPE ANTIBIOTICS a O b s e r v e d mole ratios of f r a g m e n t s Antibiotic fragment
Deformimino LL-AC541 LL-AB664 b
Streptothricin
LL-AC541
Glycine Gulosamine Streptolidine B-Lysine Ammonia
-0.2 0.6 1.0 1.1
1.0 -0.2 -2.3
1.0 -0.1 -1.3
1.0 ---2.4
N-guan-Streptolidyl
O. 4
--
--
--
--
O .8
O.6
--
gulosaminide N-guan-Streptolidyt
NP-methylgulosaminide Values o b s e r v e d f r o m h y d r o l y s e s w i t h 3 N HC1 at 100-110 °. b T h e N - I n e t h y l s t r e p t o l i d i n e a n d s t r e p t o l i d i n e - s u g a r c o m p o u n d f r o m LL-AB664 were essentially n i n h y d r i n - n e g a t i v e .
[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 -
METHODS FOR THE STUDY OF ANTIBIOTICS
[10]
Hitachi 063GC equipped with flame ionization detector was used with the following chromatographic conditions: 1. For TMS validamycins A to F: Glass column, 3 mm i.d. X 2000 mm packed with 1% OV-1 on Chromosorb W AW DMCS. Temperature: column, 280°; injection, 300 °. Carrier gas (helium), 60 ml/min. 2. For separation of TMS-validoxylamines A and B: Glass column, 3 mm i.d. X 2000 mm packed with 3% OV-17 on Chromosorb W AW DMCS. Temperature: oven, 250c; injection, 300 °. Carrier gas (helium), 30 ml/min. 3. For separation of TMS-derivatives of degradation products: Glass column, 3 mm i.d. X 2000 mm packed with 5% OV-17 on chromosorb W AW DMCS. Column temperature: initial, 150°; final, 280 ° (10°/rain). Injection temperature, 300 °. Carrier gas (helium) : 45 ml/min.
Acknowledgment The authors are indebted to their many colleagueswho provided suggestionsand data for this chapter.
[10] 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 Streptothricin-like Antibiotics
By
DONALD B . BORDERS
I. Introduction . . . . . . . . . . . . . . . . . . II. Ion-Exchange Chromatography of Intact Antibiotics. . . . . . . III. Ion-Exchange Chromatography of Hydrolysis Fragments . . . . .
256 258 260
I. Introduction Members of the streptothricin family of antibiotics are very watersoluble, strongly basic compounds with broad antimicrobial spectra. One of the most effective means of resolving these antibiotics and their hydrolysis products is by ion-exchange chromatography. It is assumed that the ion-exchange chromatographic techniques which apply to streptothricin-type antibiotics would be of general interest since so'me of the techniques might also apply to a number of other structurally different antibiotics having similar chromatographic and ionic properties.
[10]
CHROMATOGRAPHYOF STREPTOTHRICIN ANTIBIOTICS
257
The structural relationship of some streptothricin-type antibiotics is indicated in Fig. 1. In general, the antibiotics with known structures vary by the type of amino acid side chain (R~) and methyl or hydrogen substitution (R~ and R~) on the nitrogens of the streptolidine or the aminosugar. Usually gulosamine is the aminosugar moiety. However, fucosRI~ OH ~ H , , ~ O H
streptolidine
N~NH NH
/
Rs
gufosamine
R20"~..,,,,"~OR
3
Substituents Compound
RI
R2, R3
Streptothricin
H
H, CNH2
H
CCH2 CH CH2 CH2 CH2 NH2
Streptolin
H
O H, ~NH2
H
(CCH2 C CH2 CH2 CH2 NH)2 or 3 H
LL-AC541
H
O ii H, CNH2
CH3
C CH2 NH CH
H
O H, CNH2
CH3
O C CH2 NH2
LL-AB664
CH3
O H, CNH2
CH3
O NH C CH2NH CH
Oeformimino LL-AB664
CH3
0 H>CNH2
CH3
0 C CH2NH2
H
O H,(~NH2
CH3
O ~ CH2NH CH3
R4
?
o
Deformimino LL-AC541
LL-BL136
R5
?
NH2 ,NHz
9
N,.
FIC. 1. Structures of streptothricin and some related antibiotics.
258
METHODS FOR THE STUDY OF ANTIBIOTICS
[10l
amine has been proposed as a hydrolysis product from fucothricin I and glucosamine from racemomycin 0. 2 Streptolidine is an amino acid that appears to be unique to this family of antibiotics. The relative stereochemistry of the three asymmetric centers of this moiety was deduced from chemical studies 3 and N M R spectral; subsequent X - r a y analysis of streptolidine dihydrochloride confirmed these results and established the absolute stereochemistry2 As a result of this stereochemistry, the amino and carboxyl groups of the streptolidine moiety are linked together into a strained lactam ring which probably approximates a boat conformation. This strained ring system explains one of the most labile structural features of this family of antibiotics, and ring opening with biological inactivation occurs if the antibiotics are allowed to stand several hours at room temperature in dilute acid or alkali2 ,7 When streptothricin is allowed to stand in water at room temperature for several days inactivation occurs s presumably through this same mechanism. Some of these antibiotics, LL-AC541 and LL-AB664, can also degrade by standing several days in methanol at room temperature due to a very labile formimino group. 4 These stability features should be considered when any chromatographic separations of streptothricin-type antibiotics are undertaken.
II. Ion-Exchange Chromatography of Intact Antibiotics Probably one of the best methods of separating streptothricin-type antibiotics which differby the number of fl-lysinegroups in the side chain has been by salt gradient elution on carboxymethyl cellulose2,I° In our laboratories, this same system has given excellent separation of a mixture of deformimino LL-AC541, LL-AC541, and streptothricin (listedin order 1M. J. Thirumalachar, P. V. Deshmukh, R. S. Sukapure, and P. W. Rahalker, Hindustan Antibiot. Bull. 14, 4, 1971. S. Takemura, Chem. Pharm. Bull. 8, 578 (1960). 3H. E. Carter, C. C. Sweeley, E. E. Daniels, J. E. McNary, C. P. Schaffner, C. A. 83, 4296 (1961). ' D. B. Borders, K. J. Sax, J. E. Lancaster, W. K. Hausmann, L. A. Mitscher, E. R. West, E. E. Van Tamelen, J. R. Dyer, and It. A. Whaley, J. Amer. Chem. Soc. Wetzel, and E. L. Patterson, Tetrahedron 26, 3123 (1970). B. W. Bycroft and T. J. King, Chem. Commun. 1972, 652 (1972). E. E. Van Tamelen, J. R. Dyer, H. A. Whaley, H. E. Carter, and G. B. Whitfield, Jr., J. Amer. Chem. Soc. 83, 4295 (1961). 7It. Taniyama, Y. Sawada, and T. Kitagawa, J. Antibiot. 24, 662 (1971). s A. W. Johnson and J. W. Westley, J. Chem. Soc. (London), p. 1642 (1962). 9A. S. Khokhlov and P. D. Reshetov, J. Chromatogr., 14, 495 (1964). 10p. D Reshetov and A. S. Khokhlov, Khim. Prir. Soedin. 1, 42 (1965).
[101
CHROMATOGRAPHY OF S T R E P T O T H R I C I N ANTIBIOTICS
259
of increasing retention time). This type of chromatography also provides a medium with an optimum pH for antibiotic stability. A very useful variation of this procedure has been elution with a volatile buffer of pyridine-acetic acid in the isolation of the racemomycins. H The procedure described below in the experimental section has been quite successful in our laboratories and involves the use of a KC1 gradient on a weak cationic exchanger such as Amberlite CG-5022 Recovery of the antibiotics from the salt solutions of the column affluents can be accomplished by derivatives or ion-exchange techniques. ~°,1~ Procedure Separation o] LL-AC541 and De]ormimino LL-AC541 by Salt Gradient Elution on a Weak Cationic Exchange Resin. 12 A column is prepared by suspending cycled resin (Amberlite CG-50), K ÷ form, 200-400 mesh, in water and adding with stirring 1 N HC1 until the solution over the resin remains approximately p H 7. A slurry of the resin is then poured into a column, 1.2 X 100 cm, which is rinsed with several bed volumes of water prior to addition of the antibiotic charge. A mixture, 300 mg, of LL-AC541 and deformimino LL-AC541 is dissolved in 1.5 ml of water and adsorbed onto the column. The adsorbed antibiotic is eluted from the column with a gradient formed from water (500 ml) and 2.0 M potassium chloride (500 ml) at a flow of approximately 0.17 m l / m i n while the effluent is collected in approximately 5-ml fractions with the aid of a fraction collector. The gradient is approximately linear and is formed by slow addition of the KC1 solution to water with an apparatus consisting of a reservoir and mixing chamber as described by Parr. 13,14 The deformimino LL-AC541 emerges from the column in 550-580 ml and the LL-AC541 in 610-680 ml of gradient effluent (Fig. 2). The antibiotics in these fractions are readily detected by a disc agar diffusion method against a sensitive organism such as Klebsiella pneumoniae. This is accomplished by dipping paper disks, 13.5 mm diameter, into each tube of eluate and placing the resulting discs on agar seeded with the test organism. Inhibition zones are read as zone diameters after incubation of the test organism at 37 ° overnight. 1~H. Taniyama, Y. Sawada and T. Kitagawa, Chem. Pharm. Bull. 19, 1627 (1971). ,5 V. Zbinovsky, W. K. Hausmann, E. R. Wetzel, D. B. Borders, and E. L. Patterson, Appl. Microbiol. 16, 614 (1968) 1, C. W. Parr, Biochem. J. 56, XXVII (1954); R. M. Block and N.-S. Ling, Anal. Chem. 26, 1543 (1954). 1~j. j. Wren, .l. Chromatogr. 12, 32 (1963).
260
METHODS
FOR
THE
STUDY
[10]
OF ANTIBIOTICS
SO
-g E o U
g
25 20
1.0
L
G Y
I
"5
15.-
>, o
O5 '6
I0 5 029O
0.1 420
550 610 680 ml of Solt Grodient
810
FIG. 2. Resolution of LL-AC541 and deformimino LL-AC541 by chromatography with a KCI gradient on a weak cation exchanger. The two antibiotic components emerging from the column can also be detected by monitoring the column effluent at 215 nm since these compounds have significant end absorption. III. Ion-Exchange Chromatography of Hydrolysis Fragments Quantitative and qualitative studies of hydrolysis fragments have proved to be very valuable in the identification and structure determination of streptothricin-type antibiotics. The most precise procedures for conducting these studies have utilized amino acid autoanalyzers with columns of strong cationic exchangers eluted with a buffer near pH 5. The first reported application of an amino acid autoanalyzer to the study of streptothricin hydrolysates was described by C. A. Egorov et al. 15 They found that the components of various streptothricin-type complexes had the gross structure of streptothricin but differed only by the number of #-lysine groups per molecular ranging from I to 7.16,17 In the course of these studies evidence was presented to suggest that streptolin (Fig. I) might have three #-lysine residues per molecule rather than two as originally proposed2 Subsequent studies extended the analyzer procedures to streptothricintype antibiotics differing by the type of amino acid side chain and to is C. A. Egorov, P. D. Reshetov, and A. S. Khokhlov, J. Chromatogr. 19, 214 (1965). 18A. S. Khokhlov and K. I. Shutova, J Antibiot. 2S, 501 (1972). 17p. D. Reshetov, T. A. Egorov, and A. S. Khokhlov, Khim. Prir. Soedin. I, 117 (1965).
[10]
CHROMATOGRAPHY OF STREPTOTHRICIN ANTIBIOTICS
261
resolution of the different streptolidine-sugar compounds, ls-2° An additional advantage of this modification is that some streptolidine-sugar compounds can be detected by ninhydrin whereas the corresponding sugars do not react because of substitution on the nitrogen of the sugar.4,19 Resolution of the different streptolidine-sugar compounds by paper chromatography or eleetrophoresis has been only partially successful. Although the amino acid autoanalyzer provides excellent reproducibility and separation of the hydrolysis fragments, care should be given to proper controls for each individual analyzer. Variations in resin apparently can cause a reversal of peaks. 21 Procedure Preparation o] Hydrolysates. Hydrolysates are prepared under conditions that favor formation of the streptolidine-sugar compounds. Approximately 1% solutions of the antibiotics in 3 N HC1 are heated in sealed vials at 100-110 ° for 5 hr. The resulting solutions are evaporated under reduced pressure to residues which are dissolved in water to give ,-~ 1% solutions used for autoanalyzer studies. Autoanalyzer Determinations. The compositions of the hydrolysates are determined with a Technicon amino acid autoanalyzer under the conditions described below. The columns are eluted continuously with a pH 5.0 buffer composed of 14.71 g {0.05 mole) of sodium citrate dihydrate, 900 ml of water, 25 ml (0.05 mmole) of 0.002 N sodium hydroxide, 35.07 g (0.60 mole) of sodium chloride, and 10 ml of Brij detergent [polyoxyethylene (23) lauryl ether (10 g) dissolved in 200 ml of water] adjusted to pH 5.0 with 6 N HC1 and diluted to 1 liter with water. Condition A. A column (0.6 cm by 130 cm) of Chromobeads type A (sulfonated polystyrene resin from Technicon Chromatography Corp., Chauncey, New York) is maintained at 60 ° by a water jacket and eluted with pH 5 0 buffer. The column effluent is monitored by a standard automated n!nhydrin-hydrindantin procedure. 2~ The column flow rate is 0.5 ml/min. ~8D. B. Borders, J. P. Kirby, E. R. Wetzel, M. C. Davies, and W. K. Hausmann, Antimicrob. Ag. Chemother. I, 403 (1972). 1, D. B. Borders, W. K. Hausmann, E. R. Wetzel, and E. L. Patterson, Tetrahedron Lett. p. 4187 (1967). :o K. J. Sax, P. Monnikendam, D. B. Borders, P. Shu, L. A. Mitscher, W. K. Hausmann, and E. L. Patterson, Antimicrob. Ag. Chemother. 1967, 442 (1968). ~*J. Shoji, S. Kozuki, M. Ebata, and H. Otsuka, J. Antibiot. 21, 509 (,1968). 2.-Technicon Corporation, Technicon Autoanalyzer Methodology. Section I, pp. 7-8. Technicon Chromatography Corp., Chauncey, New York, 1961.
262
[10]
METHODS FOR THE STUDY OF ANTIBIOTICS TABLE I AUTOANALYZER RETENTION TIMES FOR ANTIBIOTIC HYDROLYSIS FRAGMENTS AND RELATED COMPOUNDS Condition A (14-hr r u n )
Compound Glycine Glucosamine Galactosamine Gulosamine Lysine Streptolidine ~-Lysine Ammonia Methylamine Arginine N-guan-Streptolidyl
Retention time ° (min)
Condition B (5-hr r u n )
Color yield b (area/mmole)
Retention time (Inin)
Color yield (area/mmole)
73 159 ~ 164 ~ 173 c 204 235 258 307 344 437 605
35.3 33.3 32.6 -34.4 29.6 -12.7 10.0 31.6 --
35 61 -69 -93 99 113 --236
19.1 15.7 -14.3 -8.3 15.4 6.4 --9.8
767
31.9
302
9.9
gulosaminide N-guan-Streptolidyl
N~-methylgulosaminide a R e t e n t i o n t i m e averages for a p a r t i c u l a r c o m p o u n d were u s u a l l y + 2 m i n f r o m given value. b Area in a r b i t r a r y units. c S e p a r a t i o n of s u g a r s confirmed b y a m i x e d r u n . TABLE II AUTOANALYZER COMPARISONS OF NINHYDRIN-PosITIVE FRAGMENTS FROM HYDROLYSATES OF VARIOUS STREPTOTHR1CIN-TYPE ANTIBIOTICS a O b s e r v e d mole ratios of f r a g m e n t s Antibiotic fragment
Deformimino LL-AC541 LL-AB664 b
Streptothricin
LL-AC541
Glycine Gulosamine Streptolidine B-Lysine Ammonia
-0.2 0.6 1.0 1.1
1.0 -0.2 -2.3
1.0 -0.1 -1.3
1.0 ---2.4
N-guan-Streptolidyl
O. 4
--
--
--
--
O .8
O.6
--
gulosaminide N-guan-Streptolidyt
NP-methylgulosaminide Values o b s e r v e d f r o m h y d r o l y s e s w i t h 3 N HC1 at 100-110 °. b T h e N - I n e t h y l s t r e p t o l i d i n e a n d s t r e p t o l i d i n e - s u g a r c o m p o u n d f r o m LL-AB664 were essentially n i n h y d r i n - n e g a t i v e .
[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 -
