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Levomycin. I. Isolation and chemical studies
Levomycin. I. Isolation and Chemical Studies’ Herbert From
E. Carter, Carl P. Schaffner2 and David Gottlieb
the Department-s
of Chemistry and Horticulture, Urbana, Illinois Received
May
University
of Illinois,
10, 1954
In the course of studies on antibiotics produced by streptomycetes, an unidentified species (Illinois isolate, 65-24) was found to produce a new antibiotic active against some bacteria, of both the gram-positive and the gram-negative groups. This substance has been isolated in the pure, crystalline state and named levomycin because of its high levorotation. The present, paper describes the isolation and chemical properties of levomycin. RESULTS
The streptomycete producing levomycin was isolated by methods similar to those previously described (1). Shake cultures were made in the Kelner and Morton medium (2) because of its relative simplicity and the ease of extraction from it. Both the culture broth and the mycelia possessed activity. Passage of the broth through a Seitz fiilter removed the active material completely, while a sintered bacteriological filter removed it only partially. The biological activity of levomycin against various bacteria is presented in Table I; among the organisms tested, growth was prevented at concentrations varying from 1 to 100 pg./ml., although some retardation of growth was always obtained at lower levels. When the crude yellow solid, which is described in the chemical section of this 1 The authors wish to acknowledge a generous grant in support of this work by the Abbott Laboratories, Eli Lilly and Company, and the Upjohn Company. * Part of the material in this paper was taken from the thesis submitted by Carl P. Schaffner to the Graduate College of the University of Illinois in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry. Present address: Department of Microbiology, New Jersey Agricultural Experiment Station, Rutgers University, Kew Brunswick, New Jersey. 282
LEVOMYCIA-. TABLE Inhibition
283
I
I
of Bacteria
by Lcvomycin Concentration
of levomycin.
-
Organism
10
Mycobac&erium tuberculosis 607 Mycobacterium ranae Mycobacterium avium Mycobacterium phlei Mycobacterium smegmatis Bacillus subtilis Corynebacterium diphtheriae Escherichia coli Listerella monocytogenes Streptococcus faecalis Spirillum serpens Salmonella gallinarum Pasteurella avicida Pseudomonas aeruginosa Micrococcus aureus Klebsiella pneumoniae Bacillus subtilis, etrepto-
thricin --
----
m --
f
p~./mL”
-
.-
-
+++ ++ + +++ +++ +++ + +++ zk +++ +++
f I
+++ ++ + +++ +++ +++ +++
+ f +++ +++ i +++
++ ++ f +++
resistant -’
____------
--I-
_
0 These tests were run by the agar plate dilution method; results are recorded LLB+ + + (full growth, no levomycin present) to f (trace of growth) snd - (complete inhibition).
paper, was !administered t.o mice intravenously at 44 mg./kg., all five mice in the test died. Papergrati analyses of t.he 65-24 culture broths showed a single antibiotic spot ivith the following R, values: n-butyl alcohol-water, 0.94; n-butyl alc?hol-10% acetic acid, 0.95; methyl isopropyl ketone, 0.55; methyl isopropyl ketone-2 % $-toluenesulfonic acid, 0.63; methyl isopropyl ketope-2 % pyridine, 0.84. LevoFycin was readily extracted from such broths at all pH values by several organic solvents (ethyl acetate, ether, n-butyl alcohol). Far preparative’purposes, ethyl *acetate proved most suitable, and a highly active amorphous solid was obtained on adding petroleum ether to the concentrated ethyl acetate extracts. All attempts to crystallize the ant)ibiotic from the crude amorphous material failed, and it was subsequently discovered that levomycin tends
284
CARTER,
SCHAFFNER
AND
GOTTLIEB
to coprecipitate with impurities making purification by recrystallization difficult. Chromatography with acid-washed Florisil and alumina produced poor results. Crystalline levomycin was finally obtained only after a series of steps including mild alkaline extraction, countercurrent distribution, and silicic acid chromatography. Considerable purification was achieved by extracting an ethyl acetate solution of crude material in the cold with 0.01 N sodium hydroxide. A yellow pigment was removed as an acidic impurity, and the antibiotic itself was not adversely affected by the process. The resultant tan amorphous solid was successfully fractionated by the Craig countercurrent distribution procedure, utilizing the solvent system methanol-benzene-water (5: 5 : 1) with 80 transfers. The antibiotic distributed with a partition coefficient of 0.95. Plots for bioactivity versus tube number correlated well with the plots of dry residue weight or ultraviolet absorption at 318 rnp versus tube number (Fig. 1). Levomycin taken from the peak tube was crystallized from hot isopropyl alcohol as white prisms, m.p. 213-215’C. However, some decomposition occurred giving a light-brown impurity which was difficult to remove. Crystallization from cold chloroform-methanol solution gave better results, the product melting at 218-220°C. .5
-
0
10
IO
30
.O
50
7,
64
FIN. 1. Countercurrent distribution of levomycin: benzene-methanol-water (5:5:1), 80 transfers in a 54-plate glass Craig machine. Plot: optical density at 318 nq versus transfer number.
LEVOMYCIN.
I
14oc ).
IZO( , .
400
.?O( C16 220
240
260 WAVE
FIG. 2. Ultraviolet
absorption
280 LENGTH,
300
320
340
MILLIb4IC.RON.S
spectrum of levomycin
in chloroform
solution.
Pure levomycin was obtained by subjecting the peak fractions from the Craig distribution to chromatography over silicic acid in chloroformmethanol. The major band3 passed rapidly through the column and gave crystalline levomycin melting at 222-224°C. Levomycin is very soluble in chloroform and pyridine, less so in carbon tetrachloride, ethyl acetate, and hot alcohols. It is slightly soluble in ether, benzene, cold alcohols, acetone, and dioxane, and is insoluble in water, petroleum ether, and 5 % aqueous hydrochloric acid and sodium hydroxide. In cold 6 N hydrochloric acid it is soluble but decomposes slowly. Levomycin is highly levorotatory [LX],” = -290“ (2.0 % solution in acetone), [al z5 = -323” (1.0 % solution in chloroform) and exhibits characteristic absorption in the ultraviolet with maxima at 318 rnp (I$‘&. = 185) and 243 rnp (E&. = 1200) (Fig. 2). The infrared absorption spectrum (Fig. 3a) suggests that of a peptide-type molecule with absorption in the regions of unconjugated esters and monosubstituted amides. Strong absorption is evident in the regions of 1450-1550, 1625J Minor levomycin
bands with antibacterial activity were eluted from the column after the band. The nature of these materials has not been investigated.
286
CARTER,
SCHAFFNER
AND
FIG. 3. Infrared spectra of (a) levomycin and solutions.
GO’FI’LIEB
(b)
levomycic acid in chloroform
1700, and 1725-1756 cm.+, regions of -NH bending, -C=Cstretching, and -C=N - stretching. Analysis of levomycin showed the presence of nitrogen, carbon, and hydrogen; no halogen or sulfur was detected. Elementary analysis agrees with the formula CzrHaeNs010 (molecular weight, 606.6). Molecular weight values of 550-650 by the Rast method and a saponification equivalent of 427-489 support this formulation. The antibiotic gave negative or inconclusive results in the following tests: ninhydrin, biuret, Millon’s, Hopkins-Cole, Pauly, Sakaguchi, Maltol, Benedict’s, Tollens’, 2,4-dinitrophenylhydrazine, bromine water, bromine (CCL), potassium permanganate, periodate, mcthanolic ferric chloride, Molisch, zinc-ammonium chloride, Zeisel alkoxyl, and hydroxamic acid, A positive pine splint test was obtained. On treatment with cold dilute methanolic sodium hydroxide, levomytin was converted to a water-insoluble, organic-solvent-soluble acid designated levomycic acid. This substance was isolated as a white amorphous solid (m.p. 155-160°C.) by chromatography of the crude material over
LEVOMYCIN.
287
I
silicic acid in a chloroform-methanol system. Attempts to crystallize the acid were unsuccessful. The purified product gave a neutralization equivalent of 606 and analytical data in reasonable agreement with ,the formula C%HseNsOs . Since levomycin on saponification gives an’ almost equal weight of levomycic acid, it seems likely that the hydrolysis involves the opening of a lactone ring. The infrared curves of levomycin and levomycic acid (Fig. 3b) are very similar. Vigorous alkaline degradation of levomycin produced extensive degradation of the antibiotic, yielding a volatile base, a yellow fluorescent pigment, and a volatile, ether-extractable acid. Two-dimensional paper chromatographic analysis of the hydrolyzate indicated the presence of one yellow fluorescent and four ninhydrin-positive components. Vigorous alkaline degradation of levomycic acid produced identical results. Acid hydrolysis (4 hr. under reflux in 6 N hydrochloric acid) of levomycin and levomycic acid also resulted in complete breakdown of the molecule. The hydrolyzate was faintly colored, but only a trace of the yellow fluorescent pigment observed in the alkaline hydrolyzate was present in the filtered acid hydrolyzate. Two-dimensional paper chromatographic analysis (Fig. 4) indicated the presence of the same four
n-BUTYi
FIG.
4. Two-dimensional
hydrolyzate fers; solvent velopment.
of levomycin system two,
ALCOHOL-ACETIC
paper chromatogram : solvent system one, n-butyl alcohol-acetic
ACID-WATER
of the phenol-citrate acid-water
6 N hydrochloric and phosphate 4:1:5; ninhydrin
acid buf de-
288
CARTER,
SCHAFFNER
AND
GOTTLIEB
ninhydrin-positive components as in the alkaline hydrolyzate. Traces of another component, giving yellow ninhydrin development, were also observed in some papergrams. A summary of the ninhydrin papergram analysis of the acid hydrolyzate of levomycin is given in Table II. Microbiological amino acid analysis of the acid hydrolyzate of levomycin failed to identify any of the components as one of the usual amino acids of proteins. In summary, vigorous alkaline and acidic hydrolyses of levomycin and levomycic acid have revealed the presence of a minimum of four ninhydrin-positive components, a volatile acid, and a fluorescent yellow pigment. The presence of other, as yet undetected, components in the TABLE Paper
Analysis
Chromatographic
component Ninhydrin color Fluorescence R, , phenol-citrate phosphate buffer R, , n-butyl alcoholacetic acid-water (4:1:5) R, , n-butyl alcoholethyl alcoholwater (4:l:l)
1
2
II of Acid Hydrolyzate 3
of Levomycin
4
5
6
Purple None 0.21
Purple None 0.28-0.46
Pink None 0.82
Purple None 0.87
Blue Yellow 0.93
Yellow None 0.25
0.16
0.21
0.13
0.33
0.36
-
0.10
0.20
0.00
0.29
-
0.18
hydrolyzates is possible. Mild alkaline treatment results in slight modification of the structure giving rise to an inactive acid, the structure of which appears to be very similar to that of the intact molecule. The neutral character of levomycin suggeststhe presence of a peptide structure without free amino or carboxyl groups. The fluorescent, colored degradation product suggeststhe presence in levomycin of some “chromophoric” group associated with a peptide. A cyclic peptide structure like that found in other antibiotics is possible. Levomycin appears to be another antibiotic belonging to the group characterized structurally by the presence of an aromatic chromophoric moiety in association with amino acids or peptides. Antimycin A (3) and the actinomycins (4) both belong to this group, and it should be noted that the ultraviolet absorption of levomycin is very similar to that of antimycin.
LEVOMYCIN.
I
289
EXPERIMENTAL
Fermentation of Levomycin The streptomycete which produces levomycin (65-24) was usually kept in soil culture tubes and prior to use was transferred to Emerson’s agar slants. In this medium an incubation of 5 days at 24°C. was sufficient to produce a good crop of aerial spores which were used as primary inoculum for shake flasks. The suspension of spores was then transferred to 500-ml. Erlenmeyer flasks containing 100 ml. of Kelner and Morton broth and grown for 2 days on a reciprocal shaker with a stroke of 5 cm. and a speed of 94 strokes/mm. Five milliliters of this mycelium suspension was used to inoculate each of the flasks containing the same medium. The maximum production of the antibiotic occurred within 5 days at a temperature of 26°C. After this time, the cultures from many flasks were pooled and used for extraction. Assays were made by the agar plate disk method, and in the early phases of the investigation only the size of the inhibition zone was used as a criterion of antibiotic concentration. During purification the agar dilution method was also used to assay the potency of the various materials obtained during the process.
Isolation and PuriJication of Levomycin In a typical experiment 14 1. of culture broth (pH 8) was filtered at 10” through glass wool to remove the mycelia, and the filtrate was extracted with one 0.5-vol. and two successive 0.25-vol. portions of ethyl acetate. The extracts were filtered and concentrated at 20-25” in vacua to about 1000 ml. This solution was extracted four times with lOO-ml. portions of 0.01 N aqueous sodium hydroxide, thus effectively removing most of the yellow pigment present in the crude broth extract. The concentrate was then washed three times with lOO-ml. aliquots of distilled water and concentrated to a volume of 250 ml. The crude antibiotic was precipitated from solution by the addition of excess n-hexane. In all, 460 mg. of a highly active tan amorphous solid (m.p. 195210°C.) was obtained. With the 14 1. of filtered broth, 1006 ml. of mycelia was also obtained. The myCelia were lysed and extracted with 206 ml. of a 1:l ether-acetone solution. Three additional 500-ml. extractions with acetone effectively removed all activity. After filtration through glass wool, the combined extracts were vacuum-concentrated to dryness, and the residue was dissolved in 206 ml. of hot ethyl acetate. After cooling, the yellow acidic pigment was removed by alkaline extraction (0.01 N aqueous sodium hydroxide). After washing, hexane precipitation of the concentrate produced 250 mg. of tan amorphous solid. Craig Countercurrent Distribution. The general methods outlined by Craig (5) were employed for purification. A preliminary partition study was made in a 50-ml. separatory funnel with 10 mg. of the crude solid; 20-ml. volumes of upper and lower phases of the solvent system benzene-methanol-water (5:5:1) were used. Equilibration was made with 29 complete inversions at 1 inversion/5 sec. Biological assay indicated a partition coefficient of 1.0, and weight distribution indicated values of 0.85-0.90. For the countercurrent distribution purification, a 54-tube all-glass Craig apparatus (6) was employed. All solvents were distilled before use. In a 20-l. bottle Eztraction.
290
CARTER,
SCHAFFNER
AND
GO’ITLIEB
were equilibrated 8 1. each of benzene and methanol plus 1.6 1. of distilled water. In preparing the machine for the fractionation, 30 ml. of lower phase was added to each of the tubes, with the exception of tube 0 which was left blank. Eighty milliliters of upper phase was added to each of tubes 2-5, inclusive, and an additional 50 ml. of lower phase was added to each of tubes 7-10, inclusive. The levomycin sample in one experiment (0.225 g.) was dissolved in 75 ml. of the lower-phase solvent and equilibrated in a separatory funnel with 85 ml, of the upper phase. After equilibration, the two layers were placed in tube 0. About six transfers were made per hour with the addition to tube 0 of 80 ml. of fresh upper phase plus 2% of the lower phase as cocurrent. The cocurrent was added because shrinkage of lower phase was evident. After the run was completed, the tube contents were analyzed both by biological assay with the paper disk technique (7) and by spectrophotometric analysis at 318 mr. For these assays all traces of solvents were removed. In completion, all tube fractions were concentrated to dryness by vacuum distillation and lyophilization. Levomycin, moving with a partition coefficient K of 0.95, was at peak concentration in tube 39 after 80 transfers. Of the total 225 mg. of crude antibiotic utilized, 180 mg. of material was recovered from the active regions (tubes 33-46) and 43 mg. from regions of trace activity. Two fluorescent, pigmented impurities were observed, one moving with the solvent front and the other with a partition coefficient of 0.26. Crystallization of Levomycin. Saturated hot isopropyl alcohol solutions of material from the peak tube of the countercurrent distribution (tube 39) deposited white prisms of levomycin, m.p. 213-215°C. Since some decomposition of the antibiotic occurred in this process, a new procedure was used as follows: The antibiotic was dissolved in a minimum amount of chloroform, and absolute methanol was added until a slight turbidity resulted. The turbid solution was then slowly evaporated with a jet of dry air. The more volatile chloroform evaporated, increasing the methanol concentration and promoting the crystallization of levomy& as colorless prisms, m.p. 2%2WC. The whole procedure was carried out rapidly in the cold to prevent decomposition. Silicic Acid Adsorption Chromatography. Levomycin was readily fractionated on silicic acid. In preparation, Mallinckrodt’s silicic acid No. 2847, a grade designated for partition chromatography, was activated by washing twice with 1 vol. of acetone and drying under a heat lamp. A more active silicic acid was made by washing with acetone-ether (3:l). The column was prepared by slurrying the activated silicic acid with pure anhydrous chloroform (alcohol free) into a glass column of a size designed to give a silicic acid column twice as high as its diameter. The adsorbent was thoroughly agitated to insure removal of suspended air bubbles and to produce a fine dispersion. The excess solvent was drained from the column, and the sample was applied. The levomycin preparation was completely adsorbed from pure chloroform solution, and the major active component was eluted with 2% methanol-chloroform development. Other minor active bands were eluted more slowly with the same development and more rapidly with higher concentration of methanol. For 106 mg. of antibiotic, a column prepared with 3-5 g. of silicic acid was found to be satisfactory. The major component was crystallized as colorless prisms, m.p. 222224”C., from cold chloroform-methanol solution.
LEVOMYCIN.
Anal,: CtrHtaNeO~o (606.6). C&d.: H 6.35, N 13.90.
I
C 53.46, H 6.31, N 13.86. Found:
291 C 54.05,
The Chemistry of Lmmycim Physical Properties. Bast molecular-weight determinations of levomycin were run with approximately a 10 % (by weight) solution of antibiotic in resublimed camphor. Ultraviolet absorption determinations were made in chloroform solution with both the Cary recording spectrophotometer and the Beckman spectrophotometer model D. The infrared absorption spectrum of levomycin was determined in chloroform solution with the Perkin-Delmer model 21 double beam spectrophotometer. Chemical Tests. Numerous chemical color tests and reactions were run according to directions given by Feigl (8) and by Shriner and Fuson (9). Alhdine Degradation of Levomycin. Preparation of Levomycic Acid. In one typical experiment, 71.0 mg. of levomycin was treated at room temperature with 10 ml. of 0.1 N methanolic sodium hydroxide (10% water). After 2 hr. the reaction mixture was extracted twice with 2-ml. vol. of chloroform, and the extracts were combined, washed with 1 ml. of distilled water, and evaporated to dryness. A total of 7.9 mg. of unsaponified active residue remained. The basic reaction mixture wss then acidified to pH 1.5 with 1 N hydrochloric acid. Two extractions with 2-ml. vol. of chloroform were made to remove the precipitated levomycic acid. The extracts were washed and evaporated to dryness. A residue weighing 62.8 mg. was obtained. The 62.8 mg. of crude levomycic acid in 1 ml. of pure chloroform was applied to a silicic acid column prepared with a chloroform slurry of 3 g. of silicic acid in a glass column, 23 X 1.5 cm..The acid was strongly adsorbed from the pure chloroform solution. Elution of the acid as a colorless band was effected by development with 2 % methanol-chloroform. The acid was precipitated as a white amorphous solid, m.p. l5516O”C., by addition of excessn-hexane to a concentrated solution. The hydrolysis of levomycin was followed quantitatively by allowing 0.05-g. samples to stand in 9.0 ml. of 0.1 N methanolic sodium hydroxide at room temperature for 8 hr. Back titration with 0.1 N hydrochloric acid gave saponification equivalent values of 427 and 489 for levomycin. Evidently, conversion to levomycic acid under these conditions is accompanied or followed by other degradation reactions. It was noted in the potentiometric titration of the saponification mixtures that levomycic acid first precipitated from solution at pH 4.8.
292
CARTER, SCHAFFNER AND GOTTLIEB
CompleteAlkaline Hydrolysis of Levomycin. Levomycin was degraded with excess 1.0 N sodium hydroxide by refluxing for varying periods of time. In a typical experiment, 1 mg. of levomycin was added to 3 drops of the base and the mixture was heated on a steam cone for l-2 hr. Evaporated solvent was replaced with distilled water. The volatile bases were readily detected with moist red litmus paper. Careful acidification of the hydrolyzate with 0.1 N hydrochloric acid to pH 5 resulted in liberation of a volatile acid of pungent odor. Papergram analyses were made according to methods described by Berry et al. (10). Since one-dimensional papergrams failed to resolve the components of the hydrolyzate, two-dimensional papergrams were employed. The hydrolyzates were neutralized before application to the papergram. The first solvent system generally used was phenol saturated with phosphate and citrate buffer. The second system was n-butyl alcohol-acetic acid-water (4: 1: 5), which was freshly prepared for each chromatogram. The papergram was developed by spraying with ninhydrin reagent (0.25 % in 1: 1 pyridinewater) and heating in an oven at 100°C. for 5 min. Acid Hydrolysis of Levomycin. In one experiment 20 mg. of levomycin was refluxed for 4 hr. at 100°C. with 1 ml. of 6 N hydrochloric acid. The hydrolyzate was evaporated to dryness on the water pump. The residue was dissolved in 1 ml. of distilled water, and the solution was neutralized before application to papergrams. The results are shown in Fig. 4. Acidic and basic hydrolyses of levomycic acid were carried out similarly. ACKNOWLEDGMENTS We wish to acknowledge with gratitude the assistance of Mrs. Betty Bevan and Mrs. Virginia Gallicchio in carrying out many of the biological phases of this investigation and to thank Dr. L. M. Henderson for the microbiological amino acid assays. We allso wish to thank the Upjohn Company of Kalamazoo, Michigan, for the toxicity data reported in this paper. SUMMARY
A new antibiotic, levomycin, has been isolated in pure crystalline form from the culture broth of an unidentified streptomycete. Levomycin has been assigned tentatively the empirical formula CgHasNsO, . On mild alkaline hydrolysis, an acid (levomycic acid) of approximately the same molecular weight is produced. On vigorous alkaline and acid hydrolysis, Ievomycin gives a volatile base, a volatile acid, a fluorescent pigment, and at least four ninhydrin-positive components. The physical and
LEVOMYCIN.
I
293
chemical properties of levomycin indicate that it belongs to the group of antibiotics containing an aromatic group linked to a peptide moiety. REFERENCES 1. GOTTLIEB, D., BHATTACHARYYA, P. K., ANDERSON, H. W., AND CARTER, H. E., J. Bacleriol. 66, 409 (1948). 2. KELNER, A., AND MORTON, H. E., J. Bacterial. 63, 695 (1947). 3. TENER, G. M., VAN TAMELEN, E. E., AND STRONQ, F. M., J. Am. Chem. Sot. 76, 3623 (1953). 4. BROCKMANN, H., AND GRUBHOFER, N., Naturwissenschuften 36, 376 (1949). 5. CRAIG, L. C., AND CRAIG, D., “Techniques of Organic Chemistry,” Vol. III,
p. 171. Interscience
Publishers,
New York, 1950.
6. CRAIG, L. C., Anal. Chem. 22, 1346 (1950). 7. Loo, Y. H., SKELL, P. S., THORNBERRY, H. H., EHRLICH, J., MCGUIRE, SAVAGE, G. M., AND SYLVESTER, J. C., J. Bacterial. 60, 701 (1945). 8. FEIGL, F., “Qualitative Analysis by Spot Tests.” Nordeman Publishing
J. M.,
Company, New York, 1939. of Organic 9. SHRINER, R. L., AND FUSON, R. C., “The Systematic Identification Compounds.” John Wiley and. Sons, New York, 1948. 10. BERRY, H. K., SUTTON, H. E., CAIN, L., AND BERRY, J. S., Univ. Tpas Publ. No.
6109,
22 (1951).
E. Carter, Carl P. Schaffner2 and David Gottlieb
the Department-s
of Chemistry and Horticulture, Urbana, Illinois Received
May
University
of Illinois,
10, 1954
In the course of studies on antibiotics produced by streptomycetes, an unidentified species (Illinois isolate, 65-24) was found to produce a new antibiotic active against some bacteria, of both the gram-positive and the gram-negative groups. This substance has been isolated in the pure, crystalline state and named levomycin because of its high levorotation. The present, paper describes the isolation and chemical properties of levomycin. RESULTS
The streptomycete producing levomycin was isolated by methods similar to those previously described (1). Shake cultures were made in the Kelner and Morton medium (2) because of its relative simplicity and the ease of extraction from it. Both the culture broth and the mycelia possessed activity. Passage of the broth through a Seitz fiilter removed the active material completely, while a sintered bacteriological filter removed it only partially. The biological activity of levomycin against various bacteria is presented in Table I; among the organisms tested, growth was prevented at concentrations varying from 1 to 100 pg./ml., although some retardation of growth was always obtained at lower levels. When the crude yellow solid, which is described in the chemical section of this 1 The authors wish to acknowledge a generous grant in support of this work by the Abbott Laboratories, Eli Lilly and Company, and the Upjohn Company. * Part of the material in this paper was taken from the thesis submitted by Carl P. Schaffner to the Graduate College of the University of Illinois in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry. Present address: Department of Microbiology, New Jersey Agricultural Experiment Station, Rutgers University, Kew Brunswick, New Jersey. 282
LEVOMYCIA-. TABLE Inhibition
283
I
I
of Bacteria
by Lcvomycin Concentration
of levomycin.
-
Organism
10
Mycobac&erium tuberculosis 607 Mycobacterium ranae Mycobacterium avium Mycobacterium phlei Mycobacterium smegmatis Bacillus subtilis Corynebacterium diphtheriae Escherichia coli Listerella monocytogenes Streptococcus faecalis Spirillum serpens Salmonella gallinarum Pasteurella avicida Pseudomonas aeruginosa Micrococcus aureus Klebsiella pneumoniae Bacillus subtilis, etrepto-
thricin --
----
m --
f
p~./mL”
-
.-
-
+++ ++ + +++ +++ +++ + +++ zk +++ +++
f I
+++ ++ + +++ +++ +++ +++
+ f +++ +++ i +++
++ ++ f +++
resistant -’
____------
--I-
_
0 These tests were run by the agar plate dilution method; results are recorded LLB+ + + (full growth, no levomycin present) to f (trace of growth) snd - (complete inhibition).
paper, was !administered t.o mice intravenously at 44 mg./kg., all five mice in the test died. Papergrati analyses of t.he 65-24 culture broths showed a single antibiotic spot ivith the following R, values: n-butyl alcohol-water, 0.94; n-butyl alc?hol-10% acetic acid, 0.95; methyl isopropyl ketone, 0.55; methyl isopropyl ketone-2 % $-toluenesulfonic acid, 0.63; methyl isopropyl ketope-2 % pyridine, 0.84. LevoFycin was readily extracted from such broths at all pH values by several organic solvents (ethyl acetate, ether, n-butyl alcohol). Far preparative’purposes, ethyl *acetate proved most suitable, and a highly active amorphous solid was obtained on adding petroleum ether to the concentrated ethyl acetate extracts. All attempts to crystallize the ant)ibiotic from the crude amorphous material failed, and it was subsequently discovered that levomycin tends
284
CARTER,
SCHAFFNER
AND
GOTTLIEB
to coprecipitate with impurities making purification by recrystallization difficult. Chromatography with acid-washed Florisil and alumina produced poor results. Crystalline levomycin was finally obtained only after a series of steps including mild alkaline extraction, countercurrent distribution, and silicic acid chromatography. Considerable purification was achieved by extracting an ethyl acetate solution of crude material in the cold with 0.01 N sodium hydroxide. A yellow pigment was removed as an acidic impurity, and the antibiotic itself was not adversely affected by the process. The resultant tan amorphous solid was successfully fractionated by the Craig countercurrent distribution procedure, utilizing the solvent system methanol-benzene-water (5: 5 : 1) with 80 transfers. The antibiotic distributed with a partition coefficient of 0.95. Plots for bioactivity versus tube number correlated well with the plots of dry residue weight or ultraviolet absorption at 318 rnp versus tube number (Fig. 1). Levomycin taken from the peak tube was crystallized from hot isopropyl alcohol as white prisms, m.p. 213-215’C. However, some decomposition occurred giving a light-brown impurity which was difficult to remove. Crystallization from cold chloroform-methanol solution gave better results, the product melting at 218-220°C. .5
-
0
10
IO
30
.O
50
7,
64
FIN. 1. Countercurrent distribution of levomycin: benzene-methanol-water (5:5:1), 80 transfers in a 54-plate glass Craig machine. Plot: optical density at 318 nq versus transfer number.
LEVOMYCIN.
I
14oc ).
IZO( , .
400
.?O( C16 220
240
260 WAVE
FIG. 2. Ultraviolet
absorption
280 LENGTH,
300
320
340
MILLIb4IC.RON.S
spectrum of levomycin
in chloroform
solution.
Pure levomycin was obtained by subjecting the peak fractions from the Craig distribution to chromatography over silicic acid in chloroformmethanol. The major band3 passed rapidly through the column and gave crystalline levomycin melting at 222-224°C. Levomycin is very soluble in chloroform and pyridine, less so in carbon tetrachloride, ethyl acetate, and hot alcohols. It is slightly soluble in ether, benzene, cold alcohols, acetone, and dioxane, and is insoluble in water, petroleum ether, and 5 % aqueous hydrochloric acid and sodium hydroxide. In cold 6 N hydrochloric acid it is soluble but decomposes slowly. Levomycin is highly levorotatory [LX],” = -290“ (2.0 % solution in acetone), [al z5 = -323” (1.0 % solution in chloroform) and exhibits characteristic absorption in the ultraviolet with maxima at 318 rnp (I$‘&. = 185) and 243 rnp (E&. = 1200) (Fig. 2). The infrared absorption spectrum (Fig. 3a) suggests that of a peptide-type molecule with absorption in the regions of unconjugated esters and monosubstituted amides. Strong absorption is evident in the regions of 1450-1550, 1625J Minor levomycin
bands with antibacterial activity were eluted from the column after the band. The nature of these materials has not been investigated.
286
CARTER,
SCHAFFNER
AND
FIG. 3. Infrared spectra of (a) levomycin and solutions.
GO’FI’LIEB
(b)
levomycic acid in chloroform
1700, and 1725-1756 cm.+, regions of -NH bending, -C=Cstretching, and -C=N - stretching. Analysis of levomycin showed the presence of nitrogen, carbon, and hydrogen; no halogen or sulfur was detected. Elementary analysis agrees with the formula CzrHaeNs010 (molecular weight, 606.6). Molecular weight values of 550-650 by the Rast method and a saponification equivalent of 427-489 support this formulation. The antibiotic gave negative or inconclusive results in the following tests: ninhydrin, biuret, Millon’s, Hopkins-Cole, Pauly, Sakaguchi, Maltol, Benedict’s, Tollens’, 2,4-dinitrophenylhydrazine, bromine water, bromine (CCL), potassium permanganate, periodate, mcthanolic ferric chloride, Molisch, zinc-ammonium chloride, Zeisel alkoxyl, and hydroxamic acid, A positive pine splint test was obtained. On treatment with cold dilute methanolic sodium hydroxide, levomytin was converted to a water-insoluble, organic-solvent-soluble acid designated levomycic acid. This substance was isolated as a white amorphous solid (m.p. 155-160°C.) by chromatography of the crude material over
LEVOMYCIN.
287
I
silicic acid in a chloroform-methanol system. Attempts to crystallize the acid were unsuccessful. The purified product gave a neutralization equivalent of 606 and analytical data in reasonable agreement with ,the formula C%HseNsOs . Since levomycin on saponification gives an’ almost equal weight of levomycic acid, it seems likely that the hydrolysis involves the opening of a lactone ring. The infrared curves of levomycin and levomycic acid (Fig. 3b) are very similar. Vigorous alkaline degradation of levomycin produced extensive degradation of the antibiotic, yielding a volatile base, a yellow fluorescent pigment, and a volatile, ether-extractable acid. Two-dimensional paper chromatographic analysis of the hydrolyzate indicated the presence of one yellow fluorescent and four ninhydrin-positive components. Vigorous alkaline degradation of levomycic acid produced identical results. Acid hydrolysis (4 hr. under reflux in 6 N hydrochloric acid) of levomycin and levomycic acid also resulted in complete breakdown of the molecule. The hydrolyzate was faintly colored, but only a trace of the yellow fluorescent pigment observed in the alkaline hydrolyzate was present in the filtered acid hydrolyzate. Two-dimensional paper chromatographic analysis (Fig. 4) indicated the presence of the same four
n-BUTYi
FIG.
4. Two-dimensional
hydrolyzate fers; solvent velopment.
of levomycin system two,
ALCOHOL-ACETIC
paper chromatogram : solvent system one, n-butyl alcohol-acetic
ACID-WATER
of the phenol-citrate acid-water
6 N hydrochloric and phosphate 4:1:5; ninhydrin
acid buf de-
288
CARTER,
SCHAFFNER
AND
GOTTLIEB
ninhydrin-positive components as in the alkaline hydrolyzate. Traces of another component, giving yellow ninhydrin development, were also observed in some papergrams. A summary of the ninhydrin papergram analysis of the acid hydrolyzate of levomycin is given in Table II. Microbiological amino acid analysis of the acid hydrolyzate of levomycin failed to identify any of the components as one of the usual amino acids of proteins. In summary, vigorous alkaline and acidic hydrolyses of levomycin and levomycic acid have revealed the presence of a minimum of four ninhydrin-positive components, a volatile acid, and a fluorescent yellow pigment. The presence of other, as yet undetected, components in the TABLE Paper
Analysis
Chromatographic
component Ninhydrin color Fluorescence R, , phenol-citrate phosphate buffer R, , n-butyl alcoholacetic acid-water (4:1:5) R, , n-butyl alcoholethyl alcoholwater (4:l:l)
1
2
II of Acid Hydrolyzate 3
of Levomycin
4
5
6
Purple None 0.21
Purple None 0.28-0.46
Pink None 0.82
Purple None 0.87
Blue Yellow 0.93
Yellow None 0.25
0.16
0.21
0.13
0.33
0.36
-
0.10
0.20
0.00
0.29
-
0.18
hydrolyzates is possible. Mild alkaline treatment results in slight modification of the structure giving rise to an inactive acid, the structure of which appears to be very similar to that of the intact molecule. The neutral character of levomycin suggeststhe presence of a peptide structure without free amino or carboxyl groups. The fluorescent, colored degradation product suggeststhe presence in levomycin of some “chromophoric” group associated with a peptide. A cyclic peptide structure like that found in other antibiotics is possible. Levomycin appears to be another antibiotic belonging to the group characterized structurally by the presence of an aromatic chromophoric moiety in association with amino acids or peptides. Antimycin A (3) and the actinomycins (4) both belong to this group, and it should be noted that the ultraviolet absorption of levomycin is very similar to that of antimycin.
LEVOMYCIN.
I
289
EXPERIMENTAL
Fermentation of Levomycin The streptomycete which produces levomycin (65-24) was usually kept in soil culture tubes and prior to use was transferred to Emerson’s agar slants. In this medium an incubation of 5 days at 24°C. was sufficient to produce a good crop of aerial spores which were used as primary inoculum for shake flasks. The suspension of spores was then transferred to 500-ml. Erlenmeyer flasks containing 100 ml. of Kelner and Morton broth and grown for 2 days on a reciprocal shaker with a stroke of 5 cm. and a speed of 94 strokes/mm. Five milliliters of this mycelium suspension was used to inoculate each of the flasks containing the same medium. The maximum production of the antibiotic occurred within 5 days at a temperature of 26°C. After this time, the cultures from many flasks were pooled and used for extraction. Assays were made by the agar plate disk method, and in the early phases of the investigation only the size of the inhibition zone was used as a criterion of antibiotic concentration. During purification the agar dilution method was also used to assay the potency of the various materials obtained during the process.
Isolation and PuriJication of Levomycin In a typical experiment 14 1. of culture broth (pH 8) was filtered at 10” through glass wool to remove the mycelia, and the filtrate was extracted with one 0.5-vol. and two successive 0.25-vol. portions of ethyl acetate. The extracts were filtered and concentrated at 20-25” in vacua to about 1000 ml. This solution was extracted four times with lOO-ml. portions of 0.01 N aqueous sodium hydroxide, thus effectively removing most of the yellow pigment present in the crude broth extract. The concentrate was then washed three times with lOO-ml. aliquots of distilled water and concentrated to a volume of 250 ml. The crude antibiotic was precipitated from solution by the addition of excess n-hexane. In all, 460 mg. of a highly active tan amorphous solid (m.p. 195210°C.) was obtained. With the 14 1. of filtered broth, 1006 ml. of mycelia was also obtained. The myCelia were lysed and extracted with 206 ml. of a 1:l ether-acetone solution. Three additional 500-ml. extractions with acetone effectively removed all activity. After filtration through glass wool, the combined extracts were vacuum-concentrated to dryness, and the residue was dissolved in 206 ml. of hot ethyl acetate. After cooling, the yellow acidic pigment was removed by alkaline extraction (0.01 N aqueous sodium hydroxide). After washing, hexane precipitation of the concentrate produced 250 mg. of tan amorphous solid. Craig Countercurrent Distribution. The general methods outlined by Craig (5) were employed for purification. A preliminary partition study was made in a 50-ml. separatory funnel with 10 mg. of the crude solid; 20-ml. volumes of upper and lower phases of the solvent system benzene-methanol-water (5:5:1) were used. Equilibration was made with 29 complete inversions at 1 inversion/5 sec. Biological assay indicated a partition coefficient of 1.0, and weight distribution indicated values of 0.85-0.90. For the countercurrent distribution purification, a 54-tube all-glass Craig apparatus (6) was employed. All solvents were distilled before use. In a 20-l. bottle Eztraction.
290
CARTER,
SCHAFFNER
AND
GO’ITLIEB
were equilibrated 8 1. each of benzene and methanol plus 1.6 1. of distilled water. In preparing the machine for the fractionation, 30 ml. of lower phase was added to each of the tubes, with the exception of tube 0 which was left blank. Eighty milliliters of upper phase was added to each of tubes 2-5, inclusive, and an additional 50 ml. of lower phase was added to each of tubes 7-10, inclusive. The levomycin sample in one experiment (0.225 g.) was dissolved in 75 ml. of the lower-phase solvent and equilibrated in a separatory funnel with 85 ml, of the upper phase. After equilibration, the two layers were placed in tube 0. About six transfers were made per hour with the addition to tube 0 of 80 ml. of fresh upper phase plus 2% of the lower phase as cocurrent. The cocurrent was added because shrinkage of lower phase was evident. After the run was completed, the tube contents were analyzed both by biological assay with the paper disk technique (7) and by spectrophotometric analysis at 318 mr. For these assays all traces of solvents were removed. In completion, all tube fractions were concentrated to dryness by vacuum distillation and lyophilization. Levomycin, moving with a partition coefficient K of 0.95, was at peak concentration in tube 39 after 80 transfers. Of the total 225 mg. of crude antibiotic utilized, 180 mg. of material was recovered from the active regions (tubes 33-46) and 43 mg. from regions of trace activity. Two fluorescent, pigmented impurities were observed, one moving with the solvent front and the other with a partition coefficient of 0.26. Crystallization of Levomycin. Saturated hot isopropyl alcohol solutions of material from the peak tube of the countercurrent distribution (tube 39) deposited white prisms of levomycin, m.p. 213-215°C. Since some decomposition of the antibiotic occurred in this process, a new procedure was used as follows: The antibiotic was dissolved in a minimum amount of chloroform, and absolute methanol was added until a slight turbidity resulted. The turbid solution was then slowly evaporated with a jet of dry air. The more volatile chloroform evaporated, increasing the methanol concentration and promoting the crystallization of levomy& as colorless prisms, m.p. 2%2WC. The whole procedure was carried out rapidly in the cold to prevent decomposition. Silicic Acid Adsorption Chromatography. Levomycin was readily fractionated on silicic acid. In preparation, Mallinckrodt’s silicic acid No. 2847, a grade designated for partition chromatography, was activated by washing twice with 1 vol. of acetone and drying under a heat lamp. A more active silicic acid was made by washing with acetone-ether (3:l). The column was prepared by slurrying the activated silicic acid with pure anhydrous chloroform (alcohol free) into a glass column of a size designed to give a silicic acid column twice as high as its diameter. The adsorbent was thoroughly agitated to insure removal of suspended air bubbles and to produce a fine dispersion. The excess solvent was drained from the column, and the sample was applied. The levomycin preparation was completely adsorbed from pure chloroform solution, and the major active component was eluted with 2% methanol-chloroform development. Other minor active bands were eluted more slowly with the same development and more rapidly with higher concentration of methanol. For 106 mg. of antibiotic, a column prepared with 3-5 g. of silicic acid was found to be satisfactory. The major component was crystallized as colorless prisms, m.p. 222224”C., from cold chloroform-methanol solution.
LEVOMYCIN.
Anal,: CtrHtaNeO~o (606.6). C&d.: H 6.35, N 13.90.
I
C 53.46, H 6.31, N 13.86. Found:
291 C 54.05,
The Chemistry of Lmmycim Physical Properties. Bast molecular-weight determinations of levomycin were run with approximately a 10 % (by weight) solution of antibiotic in resublimed camphor. Ultraviolet absorption determinations were made in chloroform solution with both the Cary recording spectrophotometer and the Beckman spectrophotometer model D. The infrared absorption spectrum of levomycin was determined in chloroform solution with the Perkin-Delmer model 21 double beam spectrophotometer. Chemical Tests. Numerous chemical color tests and reactions were run according to directions given by Feigl (8) and by Shriner and Fuson (9). Alhdine Degradation of Levomycin. Preparation of Levomycic Acid. In one typical experiment, 71.0 mg. of levomycin was treated at room temperature with 10 ml. of 0.1 N methanolic sodium hydroxide (10% water). After 2 hr. the reaction mixture was extracted twice with 2-ml. vol. of chloroform, and the extracts were combined, washed with 1 ml. of distilled water, and evaporated to dryness. A total of 7.9 mg. of unsaponified active residue remained. The basic reaction mixture wss then acidified to pH 1.5 with 1 N hydrochloric acid. Two extractions with 2-ml. vol. of chloroform were made to remove the precipitated levomycic acid. The extracts were washed and evaporated to dryness. A residue weighing 62.8 mg. was obtained. The 62.8 mg. of crude levomycic acid in 1 ml. of pure chloroform was applied to a silicic acid column prepared with a chloroform slurry of 3 g. of silicic acid in a glass column, 23 X 1.5 cm..The acid was strongly adsorbed from the pure chloroform solution. Elution of the acid as a colorless band was effected by development with 2 % methanol-chloroform. The acid was precipitated as a white amorphous solid, m.p. l5516O”C., by addition of excessn-hexane to a concentrated solution. The hydrolysis of levomycin was followed quantitatively by allowing 0.05-g. samples to stand in 9.0 ml. of 0.1 N methanolic sodium hydroxide at room temperature for 8 hr. Back titration with 0.1 N hydrochloric acid gave saponification equivalent values of 427 and 489 for levomycin. Evidently, conversion to levomycic acid under these conditions is accompanied or followed by other degradation reactions. It was noted in the potentiometric titration of the saponification mixtures that levomycic acid first precipitated from solution at pH 4.8.
292
CARTER, SCHAFFNER AND GOTTLIEB
CompleteAlkaline Hydrolysis of Levomycin. Levomycin was degraded with excess 1.0 N sodium hydroxide by refluxing for varying periods of time. In a typical experiment, 1 mg. of levomycin was added to 3 drops of the base and the mixture was heated on a steam cone for l-2 hr. Evaporated solvent was replaced with distilled water. The volatile bases were readily detected with moist red litmus paper. Careful acidification of the hydrolyzate with 0.1 N hydrochloric acid to pH 5 resulted in liberation of a volatile acid of pungent odor. Papergram analyses were made according to methods described by Berry et al. (10). Since one-dimensional papergrams failed to resolve the components of the hydrolyzate, two-dimensional papergrams were employed. The hydrolyzates were neutralized before application to the papergram. The first solvent system generally used was phenol saturated with phosphate and citrate buffer. The second system was n-butyl alcohol-acetic acid-water (4: 1: 5), which was freshly prepared for each chromatogram. The papergram was developed by spraying with ninhydrin reagent (0.25 % in 1: 1 pyridinewater) and heating in an oven at 100°C. for 5 min. Acid Hydrolysis of Levomycin. In one experiment 20 mg. of levomycin was refluxed for 4 hr. at 100°C. with 1 ml. of 6 N hydrochloric acid. The hydrolyzate was evaporated to dryness on the water pump. The residue was dissolved in 1 ml. of distilled water, and the solution was neutralized before application to papergrams. The results are shown in Fig. 4. Acidic and basic hydrolyses of levomycic acid were carried out similarly. ACKNOWLEDGMENTS We wish to acknowledge with gratitude the assistance of Mrs. Betty Bevan and Mrs. Virginia Gallicchio in carrying out many of the biological phases of this investigation and to thank Dr. L. M. Henderson for the microbiological amino acid assays. We allso wish to thank the Upjohn Company of Kalamazoo, Michigan, for the toxicity data reported in this paper. SUMMARY
A new antibiotic, levomycin, has been isolated in pure crystalline form from the culture broth of an unidentified streptomycete. Levomycin has been assigned tentatively the empirical formula CgHasNsO, . On mild alkaline hydrolysis, an acid (levomycic acid) of approximately the same molecular weight is produced. On vigorous alkaline and acid hydrolysis, Ievomycin gives a volatile base, a volatile acid, a fluorescent pigment, and at least four ninhydrin-positive components. The physical and
LEVOMYCIN.
I
293
chemical properties of levomycin indicate that it belongs to the group of antibiotics containing an aromatic group linked to a peptide moiety. REFERENCES 1. GOTTLIEB, D., BHATTACHARYYA, P. K., ANDERSON, H. W., AND CARTER, H. E., J. Bacleriol. 66, 409 (1948). 2. KELNER, A., AND MORTON, H. E., J. Bacterial. 63, 695 (1947). 3. TENER, G. M., VAN TAMELEN, E. E., AND STRONQ, F. M., J. Am. Chem. Sot. 76, 3623 (1953). 4. BROCKMANN, H., AND GRUBHOFER, N., Naturwissenschuften 36, 376 (1949). 5. CRAIG, L. C., AND CRAIG, D., “Techniques of Organic Chemistry,” Vol. III,
p. 171. Interscience
Publishers,
New York, 1950.
6. CRAIG, L. C., Anal. Chem. 22, 1346 (1950). 7. Loo, Y. H., SKELL, P. S., THORNBERRY, H. H., EHRLICH, J., MCGUIRE, SAVAGE, G. M., AND SYLVESTER, J. C., J. Bacterial. 60, 701 (1945). 8. FEIGL, F., “Qualitative Analysis by Spot Tests.” Nordeman Publishing
J. M.,
Company, New York, 1939. of Organic 9. SHRINER, R. L., AND FUSON, R. C., “The Systematic Identification Compounds.” John Wiley and. Sons, New York, 1948. 10. BERRY, H. K., SUTTON, H. E., CAIN, L., AND BERRY, J. S., Univ. Tpas Publ. No.
6109,
22 (1951).