ANTMICROBIAL AGENTS AND CHEMOTHERAPY, June 1980, p. 980-9870066-4804/80/06-0980/08$02.00/0

Vol. 17, No. 6

Some Chemical and Physical Characteristics of Pantomycin,an Antibiotic Isolated From Streptomyces hygroscopicust

S. GURUSIDDAIAH* AND S. 0. GRAHAM*

Bioanalytical Center, Washington State University, Pullman, Washington 99164

The production, isolation, and some structural studies of an antifungal, anti-bacterial, and antiviral substance from cultures of Streptomyces hygroscopicusare described. This material, designated pantomycin, appears to be a polypeptideantibiotic with inclusion of fatty acids and carbohydrate residues. Amino acidanalysis of pantomycin acid hydrolysates indicates that it contains threonine,serine, proline, glycine, alanine, valine, alloisoleucine, and an as-yet-unidentifiedamino acid which appears to be different from types encountered in proteinaceousmaterials. In addition to the aforementioned compounds, the antibiotic was shownto contain a-aminobutyric acid after hydrogenation. Analysis of ether extracts ofthe hydrolysate mixture indicated the presence of several fatty acids; myristic,isotridecanoic, lauric, and undecylic acids. The amino and fatty acid compositionof pantomycin is similar to the known antibiotic stendomycin. Pantomycinappears to also have at least one carbohydrate-like residue incorporated into itsstructure. The presence of carbohydrate was indicated by periodic acid-Schiffbase staining of electrophoretic patterns as well as positive color formation in thephenol-sulfuric and Molisch tests, but the carbohydrate did not appear to beeither a hexose or a pentose. The antibiotic, which appears to be dissociated inalcoholic solution, forms stable aggregates under aqueous conditions.

Pantomycin is an antifungal, antibacterial,and antiviral antibiotic produced by Strepto-myces hygroscopicus NRRL 2751 (9; S. 0. Gra-ham and S. Gurusiddaiah, U.S. Patent 3,992,528,November 1976). The culture characteristics ofthe pantomycin-producing organism have beendescribed (9; U.S. Patent 3,992,528). Methodsfor production, isolation and purification of theantibiotic, together with a description of its invitro and in vivo antiviral, antibacterial, andantifungal properties, have been reported (9;U.S. Patent 3,992,528). In laboratory trials, theantibiotic showed a strong inhibitory activityagainst 70 species of plant pathogenic fungi andalso exhibited antibacterial activity on severalspecies of pathogenic bacteria. In tissue culturetests, pantomycin exhibited antiviral activityagainst paramyxovirus, rhabdovirus, leukovirus,poxvirus, and herpesvirus (9). The present com-munication describes investigations of the phys-ical and chemical characteristics of pantomycin.

MATERIALS AND METHODSProduction and purification of pantomycin.

The antibiotic-producing organism was obtained fromthe U.S. Department of Agriculture Northern Re-gional Research Laboratory, Peoria, Ill. Culture con-ditions necessary for the production of pantomycin

t Scientific paper no. SP5118 project 0204, College of Ag-riculture Research Center, Washington State University, Pull-man, WA 99164.

t Deceased.

(U.S. Patent 3,992,528) were employed to grow S.hygroscopicus in shake cultures as well as in 10-literfermentation batches. The methods of Gurusiddaiahet al. (9) were employed for the isolation and purifi-cation of pantomycin. A sample of stendomycin wasobtained from Lilly Research Laboratories, Indianap-olis, Ind.Antifungal and antibacterial assays. Antibac-

terial activity of pantomycin was determined by em-ploying standard petri dish zonal inhibition and testtube dilution bioassay techniques. Antifungal activityof purified pantomycin and antibiotic activity in fer-mentation culture fluid were determined by a modifiedpetri dish zonal inhibition technique on the conida ofGlomerella cingulata (U.S. Patent 3,992,528).Thin-layer chromatography. Thin-layer chro-

matograms were run on Silica Gel G (Merck) withphosphor and binder, using n-butanol-acetic acid-wa-ter (3:1:1, vol/vol/vol) as the solvent. The antibioticseparated by thin-layer chromatography was detectedby bioautography using the conidia of G. cingulata.

Spectroscopic procedures. Ultraviolet and visi-ble spectra were obtained using a Beckman double-beam spectrophotometer. Infrared spectra of panto-mycin were recorded on a Beckman infrared spectro-photometer. Proton magnetic resonance spectra wereobserved in CDC16 and recorded on a Bruker-WH-90spectrometer operating at 90 MHz. Fluorescence ofpantomycin was determined on a Hitachi MPF2A flu-orescence spectrophotometer.Elemental analysis. Elemental analysis was done

on a Perkin Elmer model 240 elemental analyzer.Light scattering measurements. Determinations

of the molecular weight of the antibiotic were made980

PANTOMYCIN 981

by differential refractometry and light scattering on aBrice Phoenix light-scattering photometer at 546.1nm. A measured amount (0.1035 g) of antibiotic wasdissolved in 0.050 ml of ethanol and was further dilutedto a volume of 2.083 ml with glass-distilled water at230C.Disc gel electrophoresis. The disc gel electropho-

resis method of Brewer and Ashworth (5) for the pH8.9 system was used.

SDS-gel electrophoresis. The method of Fair-banks et al. (8) was employed for sodium dodecylsulfate (SDS)-gel electrophoresis. Gels contained 7.5%acrylamide, 1% SDS, 10 mM tris(hydroxymethyl)ami-nomethane (Tris), 20 mM sodium acetate, and 5 mMethylenediaminetetraacetic acid (EDTA) adjusted topH 8.1. The buffer in both upper and lower reservoirswas 1% SDS, 10mM Tris, 20 mM sodium acetate, and2 mM EDTA adjusted to pH 8.0. The pantomycinsamples were dissolved in 1% SDS, 10 mM sucrose, 50mM dithiothreitol, 10 mM Tris, 1 mM EDTA, and 10,ug of bromophenol blue per ml adjusted to pH 8.Electrophoresis was conducted at 12 mA per gel for 2h. Later the gels were stained with Schiff reagent forcarbohydrates and Coomassie brilliant blue for pep-tides.Amino acid analysis. In the initial studies, 2-mg

samples of pantomycin were hydrolyzed in 5 ml ofconstant-boiling HCI in evacuated sealed ampoules at110°C for periods of 24, 48, and 72 h. The hydrolysateswere lyophilized to remove acid and analyzed quanti-tatively on a Beckman 121 automatic amino acid ana-lyzer by the methods of Spackman et al. (14). Insubsequent studies, extended hydrolyses were con-ducted up to 550 h to obtain maximum yields of valineand alloisoleucine.

Separation and identification of fatty acids. A6-mg sample of pantomycin was hydrolyzed for 24 hin 10 ml of 6 N HCI at 110°C in an evacuated sealedampoule. The hydrolysate was diluted with 15 ml ofglass-distilled water and extracted three times withether. After combining the ether extracts, the solventwas removed by evaporation. The residue was dis-solved in small amounts of chloroform and spotted onthin-layer plates coated with Silica Gel G (Merck)with phosphor and binder. Ether-hexane-formic acid(80:20:2, vol/vol/vol) was used as the chromatographicsolvent system, and the running time was 2 h. Thechromatograms were dried and viewed under ultravi-olet light to define spots. The ultraviolet light-absorb-ing spots were scraped off the chromatograms andextracted with a chloroform-methanol solution (2:1,vol/vol) to elute fatty acids. After removal of thesolvent by evaporation, the fatty acids were esterified,using 14% BF3 in methanol, by refluxing in a steambath for 30 min. Double-distilled water was added tothe reflux mixture to stop the reaction, and the solu-tion was extracted with chloroform three times torecover the fatty acid esters. The chloroform extractwas evaporated to dryness, and the residue was ana-lyzed by standard procedures of gas chromatographyand mass spectrophotometry.

Qualitative carbohydrate analysis. A 6-mg sam-ple of pantomycin was hydrolyzed in 2 N H2SO4 for 6h at 940C in a sealed ampoule. The resulting solutionwas neutralized with barium hydroxide, and the pre-cipitate was removed by centrifugation. Both precipi-

tate and supernatant were tested for carbohydrateusing a reagent containing 1 ml of 5% phenol in waterand 5 ml of concentrated sulfuric acid (7).

Determination of site of attachment of fattyacid(s) to peptide chain. A 4-mg amount of panto-mycin was disolved in 7 ml of concentrated hydrochlo-ric acid and allowed to stand with occasional swirlingfor 96 h at room temperature. The digest was dilutedwith double-distilled water and extracted with hexaneto remove the fatty acids. After removal of the solventby evaporation, the residue was extracted with ether.The ether extract which contained fatty acids attachedto peptides of varying lengths was evaporated, and theresidue was dissolved in 6 N HCI and digested for 20h at 110°C. The hydrolysate was then analyzed foramino acids and fatty acids.Hydrogenation of pantomycin. A 20-mg amount

of pantomycin was dissolved in 25 ml of 95% ethanolcontaining 3 drops of glacial acetic acid. To this mix-ture was added 100 mg of palladium on charcoal (10%catalyst). The hydrogenation was carried out for 24 hat room temperature under hydrogen (25 to 30 lb/in2of pressure) with constant stirring. After removal ofthe catalyst and charcoal by filtration, the solvent wasremoved by evaporation. A 2-mg sample of hydrogen-ated antibiotic was hydrolyzed in 6 N HCI at 110°Cfor 24 h, and the hydrolysate was analyzed for aminoacids as before.

Determination of E- and L-amino acid ratios inpantomycin. A 5-mg amount of pantomycin was dis-solved in 6 ml of constant-boiling HCI to which hadbeen added 0.5 ml of norleucine solution (1.25,umol)as an internal standard. The sample was hydrolyzedfor 24 h at 110°C after which the hydrolysate wasevaporated to remove most of the acid. The residuewas dissolved in water and evaporated to dryness. Theresidue-was again dissolved in 2 ml of distilled waterand was used for D-amino acid oxidase analysis. Theincubation mixture contained 0.5 ml of the hydroly-sate, 250 jig of D-amino acid oxidase (obtained fromSigma Chemical Co.), 25 Lg of flavin adenine dinucleo-tide, and 5 mg of catalase (Sigma) in 5.75 ml of 0.1 Mphosphate buffer (pH 8.4). Treated and control sam-ples were incubated at 37°C for 24 h on a reciprocatingincubator-shaker. After the samples were cooled, 1.5ml of 5% trichloroacetic acid was added to each toprecipitate protein. The precipitate was pelleted bycentrifuging for 30 min at 8,000 rpm. The supernatantwas extracted three times with 15 ml of ether. Theether layer was discarded, and the remaining aqueouslayer was dried and redissolved in 1 ml of 0.2 N sodiumcitrate buffer (pH 2.0) for analysis of amino acids.Preparation of pantomycin acid. A 10-mg

amount of antibiotic was dissolved in 0.2 ml of meth-anol and further diluted with 19 ml of glass-distilledwater. A solution of 2 N NaOH was added dropwiseuntil a precipitate formed. The sample was acidifiedwith an excess of 1 N HCI while swirling. The panto-mycin acid was separated by centrifugation. The re-sulting pellet was washed in chloroform and recentri-fuged after which the chloroform extract was dis-carded. The pantomycin acid (pellet) was tested forbiological activity.

Determination of C- and N-terminal aminoacid residues in pantomycin. (i) The antibiotic wasdigested separately with carboxypeptidase A and B

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982 GURUSIDDAIAH AND GRAHAM

(Sigma) by the methods of Brosemer and Kuhn (6) todetermine the C-terminal amino acid of pantomycin.The amino acids released were identified by the au-tomatic amino acid analyzer.

(ii) A 6-mg amount of pantomycin acid was heatedin a sealed ampoule at 150°C for 3 h after dissolvingin a mixture of acetic anhydride (0.75 ml) and pyridine(0.5 ml) using the Dakin West (1) degradation proce-dure. After cooling, a few milliliters of water wasadded, and the mixture was evaporated to dryness.The material was hydrolyzed with 6 N HCl and ana-lyzed for amino acids.

(iii) Manual Edman (13) degradation techniqueswere followed to determine the N-terminal amino acidof pantomycin.

Saponification and transesterification of pan-tomycin. Saponification was carried out by dissolving10 mg of antibiotic in 1 N NaOH by stirring for 10 to15 min at room temperature. The solution was acidi-fied (pH 1.0) with 1 N HCl and concentrated to dry-ness, and the residue was dissolved in a solution con-taining 9 ml of acetone and 1 ml of absolute ethanol.A 10-mg amount of pantomycin was dissolved in 5 mlofanhydrous methanol and transesterified with trieth-ylamine (50 mol/mol of peptide) at room temperaturefor 20 min. Both saponified and transesterified sampleswere tested for antimicrobial activity.

RESULTSPantomycin as isolated is an amorphous, light-

colored powder soluble in methanol, ethanol,propanol, butanol, dichloromethane, 1,1-dichlo-roethane, chloroform, acetone, methylethyl ke-tone, methylisobutyl ketone, acetonitrile, di-ethyl ether, diethyl formamide, and dimethylsulfoxide. It is sparingly soluble in water, 0.1 NHCI, benzene, and carbon tetrachloride, but in-soluble in petroleum ether.

Pantomycin is stable in isopropanol and other

alcoholic solutions. There was no detectable lossin antibiotic activity after storing in isopropanol(10 mg of antibiotic per ml) for a period of 3years at room temperature. The antibiotic is alsostable for long periods of time when stored asdried material. Autoclaving of the antibiotic (10mg/ml in methanol) for 15 min at 15 lb/in2 ofpressure in an evacuated sealed ampoule pro-duced no significant loss in the biological activ-ity.

In thin-layer chromatography on Silica Gel Gwith phosphor and binder on glass plates withn-butanol-acetic acid-water (3:1:1, vol/vol/vol),the antibiotic migrated as a single compact spotwith an Rf of 0.52. Several other solvent systemsgave similar results. No minor spots were ob-served.Pantomycin (100 mg/ml) in spectral-grade

methanol exhibits strong end absorption in thelower regions of the ultraviolet spectrum. Therewere no maxima in the entire region of 230 to410 nm. Also, no absorption maxima were de-tected in the entire region of the visible spec-trum.Aqueous solutions of pantomycin (12.5 ,ug/ml)

were assessed on a fluorescence spectrophotom-eter. Excitation occurred at 380 nm, as back-ground peak with a sharp scatter peak at 445nm. In the emission scan at 460 nm, emissionoccurred as a broad peak with a sharp scatterpeak at 395 nm.

Infrared absorption spectrum of pantomycinwas obtained in a potassium bromide pellet (Fig.1). Referring to Fig. 1, the antibiotic showsstrong absorption bands at the following posi-tions, expressed in micrometers: 3.04, 3.29, 3.39,

6 7 8 9 10 12 14 16

2000 1800 1600 1400 1200 1000 800 600

Wavenumber, cm1iFIG. 1. Infrared spectra ofpantomycin and stendomycin.

3

Wavelength in microns4 5

C0

(I,cn

Ena

ANTimICROB. AGENTS CHEMOTHER.

PANTOMYCIN 983

3.44, 3.49, 3.56, 5.77, 5.82, 6.08, 6.17, 6.57, 6.86,6.91, 7.10, 7.25, 7.59, 7.78, 7.91, 8.29, 9.00, and9.45.The infrared spectrum of pantomycin is qual-

itatively similar to the stendomycin spectrum(Fig. 1), except a major absorption band at 9.00,um is absent in the stendomycin spectrum. Also,absorption bands at 10.83 and 11.50 ,um of sten-domycin are absent in pantomycin. In additionto these differences, the fingerprinting regions ofboth the spectra are different.The 'H nuclear magnetic resonance spectrum

of pantomycin is presented along with the spec-trum of stendomycin in Fig. 2. The spectra arevery similar. A large number ofresonances occurin the region of 3 to 68. Also a large methyleneenvelope is observed at 1.6. Assignment of in-dividual resonances to particular functionalitieswill require further knowledge of the structureof pantomycin and determination of spectra athigher magnetic fields. Although not proof of adifference in structure, the 90 MHz spectrumbetween 3 and 4,58 suggests that differences instructure do exist.

In the disc gel electrophoresis method ofBrewer and Ashworth, the antibiotic migratedas a single band with a relative mobility of 0.63.Using a Beckman model E analytical ultracen-trifuge at 56,000 rpm, pantomycin sedimented asa single boundary in 0.05 M KCl (Fig. 3). Thecalculated sedimentation -coefficient using the

Pantomycin

EI1

FIG. 2. Proton magnetic resonance spectra ofpan-tomycin and stendomycin.

_. _ -.1. . ,.

FIG. 3. Sedimentation-velocity pattern of panto-mycin. Photograph was taken 128 min after a speedof56,000 rpm was reached a 20°C. Antibiotic concen-tration was 7 mg/ml in 0.05M KCI.

Schlieren system was 1.45. When dissolved in95% ethanol or methanol (7 mg/ml), no bound-ary could be detected. The same sample, afterevaporation of the solvent, was redissolved in 1ml of 0.05 M KCl and exhibited a symmetricalSchlieren boundary. The calculated sedimenta-tion coefficient was 1.40s. When this sample wasevaporated and again dissolved in methanol orethanol, no boundary could be seen upon ultra-centrifugation. These data suggest that the anti-biotic is aggregated in the aqueous solution butremains dissociated in alcoholic solutions. Simi-lar patterns of association of molecules inaqueous solutions and dissociation in alcoholicsolutions were also observed upon ultracentrif-ugation of stendomycin samples.The following average elemental analysis of

pantomycin was obtained: C54.28%H8.ao4%N2.99%02A.69% (Diff), which indicates a tentative empir-ical formula of C97H172N20033, molecular weightof 2,146. No halogen or phosphorus was found.The molecular weight of the antibiotic in wa-

ter was determined by light-scattering measure-ments. The value of Qf/OC was 0.1617 ± 0.0003.The KC/A90 was plotted versus concentrationand a least-squares fit and yielded a straight linewith a slope and intercept of (0.62 0.13) x 10-3and (0.714 + 0.017) x 10-4, respectively. Themolecular weight was estimated by the recipro-cal of the intercept to be 14,000 ± 200.When 1.0-mg amounts of the antibiotic were

tested for various color reactions, the following

I'

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984 GURUSIDDAIAH AND GRAHAM

results were obtained: (i) Molisch test, positive;(ii) ninhydrin test, positive; (iii) phenol-sulfuricacid test, positive; (iv) carbazole test, weaklypositive; (v) Taubers test, negative; (vi) benzi-dine-glacial acetic acid test, negative; (vii)Moores test, negative; (viii) Liebermann-Bu-chard test, negative.On disc gel electrophoresis, pantomycin gave

a single band. A single spot was observed onthin-layer chromatography with several solventsystems. SDS-gel electrophoresis revealed threemajor bands (Fig. 4). Both Schiff reagent andCoomassie brilliant blue were bound at threeidentical Rf loci, namely, 95, 65, and 54 (Fig. 4).The migration rates of fractions compared withstandard reference compounds indicate molec-ular weights of 14,000 to 15,000, 29,000, and43,000, respectively. Since the antibiotic has 10to 15% of non-amino acid components, it is notknown to what extent these will affect the rateof migration in SDS-gel electrophoresis. Con-trary to its behavior in methanol, it appears thatthe SDS treatment causes aggregation of pan-tomycin. It is interesting to note that the anti-biotic gave positive tests for Molisch and phenol-sulfuric acid reagents. These tests suggest thatthe antibiotic molecule possesses a carbohy-drate-like constituent. Tests for sugar amines,hexoses, and pentoses were all negative.Amino acid analysis in 24-, 48-, and 72-h hy-

drolysates revealed the presence of threonine (2mol), serine (1 mol), glycine (1 mol), alanine (1mol), proline (1 mol), ailoisoleucine (1 to 1.5mol), and valine (2.4 to 3.2 mol) (amino acidratios were calculated by assigning alanine avalue of one). In addition, an unknown basicamino acid (1 mol) emerged after a high am-monia peak (1.2 to 1.5 mol) in the Spackman etal. (14) column for separation of basic aminoacids. The position of the unknown amino acidwas near to that at which a-amino-fl-guanidino-propionic acid would emerge. Tests for the pres-ence of guanidyl groups in the hydrolysates werepositive, and there was no peak at the positionexpected for arginine. The basic amino acid inpantomycin may be similar to stendomycidine(4, 10) since the proton magnetic resonance spec-tra (Fig. 2) ofboth stendomycin and pantomycinshow the presence of three N-CH3 groups. Twoof these groups of protons, one at 3.19 ppm andthe other at 3.49 ppm, can be assigned to the N-CH3 protons of stendomycidine (4, 10). Thissimilarity, coupled with its identical mobility inthin-layer chromatography and on amino acidanalyzer columns, indicates that the basic com-ponent of pantomycin may be similar if notidentical to stendomycidine.

In addition to these amino acids, small quan-tities of glutamic acid, aspartic acid, leucine, and

FIG. 4. SDS-polyacrylamide gel electrophoresispatterns ofpantomycin. Electrophoresed on gels po-lymerized from 7.5% acrylamide with 30 ug ofpanto-mycin. (Left) Gel stained with Coomassie brilliantblue; (Right) gel stained with Schiff reagent.

isoleucine were detected in the hydrolysates.Also, with an increase in hydrolysis time beyond24 h, a gradual increase in the yields of valineand alloisoleucine was observed. In subsequentstudies, extended hydrolyses were conducted upto 550 h (Table 1). As was expected, extendedhydrolysis resulted in gradual breakdown of ser-ine and threonine. Maximum yields of valineand alloisoleucine were observed after 350 h ofhydrolysis. The unidentified basic amino acid aswell as other amino acids remained intact after300 h of hydrolysis. Beyond 350 h, a consistentdecrease in yields of all amino acids and corre-sponding increase in yields of ammonia wereobserved.When pantomycin acid hydrolysates were an-

alyzed on Spackman et al. amino acid analyzersystems, a small peak was seen near the aspar-tate peak on the chromatogram. A similar peak,which was later identified to be N-methyl-L-threonine, was also observed on the chromato-grams of stendomycin hydrolysates (3). N-Methyl-L-threonine gives a strong proton peak

ALNTimICROB. AGENTS CHEMOTHER.

TABLE 1. Amino acid content ofpantomycin hydrolysatesaYield of amino acid (M ratios) at:

Amino acid24" 48 84 108 168 216 300 350 408 550

Threonine 1.60 1.70 1.63 1.60 1.59 1.62 1.50 1.51 1.24 1.19Serine 0.79 0.79 0.73 0.70 0.62 0.59 0.51 0.48 0.33 0.26Proline 0.82 0.94C 0.99 0.93 0.97 0.95 0.60 1.00 0.90 0.91Glycine 0.89 L.OOC 1.01 1.03 1.03 1.06 1.04 1.10 0.93 1.03Alanine 0.96 L.OOC 1.00 1.02 1.02 1.05 1.05 1.08 0.92 1.00Valine 2.44 3.05 3.26 3.33 3.41 3.52 3.35 3.64 3.05 3.34Alloisoleucine 1.06 1.44 1.58 1.59 1.67 1.75 1.69 1.76 1.48 1.60Isoleucine Trace Trace Trace Trace 0.07 0.07 0.08 0.08 0.08 0.11Leucine 0.07 0.07 0.07 0.08 0.07 0.08 0.08 0.08 0.07 0.08NH3 0.89 0.98 1.01 1.20 1.19 1.33 1.50 1.65 1.50 1.90Basic component 0.78 0.96 1.01 1.10 1.23 1.28 1.42 0.87 0.71 1.21

a Two-milligram quantities of pantomycin were hydrolyzed with constant-boiling hydrochloric acid at 110°Cin evacuated sealed ampoules.

b Duration of hydrolysis in hours.c Yields of alanine, glycine, or proline at 48 h are assumed as 1.

at 2.99 ppm in stendomycin proton magneticresonance spectra (Fig. 2). An identical peak isobserved in pantomycin proton magnetic reso-nance spectra (Fig. 2). This suggests that thesmall peak near aspartate on amino acid ana-lyzer chromatograms of pantomycin may be N-methyl threonine.The fatty acids identified were primarily n-

tetradecanoic acid (myristic acid; 60%), an iso-tridecanoic acid (branched-chain tridecylic acid;27%), and n-dodecanoic acid (lauric acid; 8%)and undecanoic acid (5%) (Fig. 5). The fatty acidcomponents indicate some microheterogeneityin the antibiotic sample. For every mole of poly-peptide moiety, approximately 1 mol of fattyacid was found (see Table 3). The amino acidand fatty acid components of pantomycin aresimilar to those ,of stendomycin (2).Acid hydrolysis of pantomycin released 1 to

1.5 mol of ammonia (Table 1) per mol of peptidehydrolyzed, but neither aspartic nor glutamicacid was present in the hydrolysate, nor wasthere evidence of other carbamyl residues in thechain. A labile amino group may be suggestedas the source of the ammonia. A possible expla-nation is that some unsaturated amino acid ofthe polypeptide part of the molecule was de-graded during hydrolysis and only the ammoniawas recovered. To detect the presence of anunsaturated amino acid, the antibiotic was hy-drogenated. The hydrogenation rendered theantibiotic insoluble in ethanol, dichloroethane,or 6 N HCI. Analysis of the acid hydrolysate ofthe hydrogenated antibiotic showed no changein concentration of any of the amino acids foundin the unhydrogenated sample, but there was anadditional 1 mol of an amino acid that emergedfrom the column where a-amino-n-butyric acid

should appear. Also, the acid hydrolysate re-vealed almost total absence of ammonia. This isconsistent with the hypothesis that the aminoacid was a-amino-n-butyric acid. A similar typeof unsaturated a-amino-n-butyric acid was iden-tified in the acid hydrolyates of hydrogenatedstendomycin (2). Once again, the comparison ofproton magnetic resonance spectra of both ofthese antibiotics (Fig. 2) shows the presence ofa doublet at 1.86 ppm and a multiplet at 6.60ppm. These protons were assigned to methyl(1.86 ppm) and vinyl (6.60 ppm) protons of de-hydrobutyrene (12). Therefore, the unsaturateda-amino-n-butyric acid is probably identical todehydrobutyrene of stendomycin.Hydrogenated pantomycin exhibited 40% of

the activity of native pantomycin. Saponifica-tion and transesterification of pantomycin re-sulted in total loss of biological activity, whereaspantomycin acid and pantomycin hydrochlorideretained most of its biological activity.The results of enzymatic digestion of the pan-

tomycin hydrolysate with D-amino acid oxidaseare presented in Table 2. The results suggestthat threonine, alanine, and alloisoleucine areprimarily ofD configuration, whereas valine maybe a mixture of the D and L forms. The unknownbasic amino acid component, serine, and prolinemay be in the L configuration.No amino acid residue was released even after

repeated and prolonged digestion of pantomycinwith carboxypeptidase A and B. The manualEdman degradation of pantomycin failed torelease any detectable amino acids. These re-sults suggest that both N- and C-terminals ofpantomycin are blocked. Further, the DakinWest (1) degradation technique for identifyingthe carboxyterminal amino acid was tried.

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986 GURUSIDDAIAH AND GRAHAM

Amino acid analysis of the acid hydrolysatesrevealed, as expected, complete destruction ofhydroxy amino acids threonine and serine. Pro-line and valine remained intact. Alloisoleucinewas greatly reduced, and an equal amount ofisoleucine appeared. Glycine was also reduced.The basic component was also reduced and anequal amount of arginine appeared. This sug-gests that the basic component might be a cyclicform of arginine. In addition to these aminoacids, small amounts of a-amino-n-butyric acid,aspartic acid and glutamic acid were present inthe hydrolysates. Hence, the Dakin West deg-

IOOr-

90F-

80k

70k

601-

50k

40k

30k

20

l0o

FIG.Undecacid; (

4

3

TABLE 2. Configuration of amino acids inpantomycin

Proba-Amino acid Controla Treatedb ble

(nmol) (nmol) compo-sition

Threonine 148.63 5.74 AU DSerine 85.19 85.19 AUl LProline 93.20 98.10 AlU LGlycine 75.30 74.70Alanine 100.20 8.20 All DValine 180.70 90.25 DLAiUoisoleucine 89.00 6.00 Al DBasic component 106.00 118.20

a Hydrolysate without catalase and D-amino acidoxidase.

b Quantities of amino acids (nanomoles) remainingafter treatment of the hydrolysate with D-amino acidoxidase.

radation technique did not reveal the presenceofa carboxyl terminal amino acid ofpantomycin.Pantomycin is insoluble in petroleum ether,

whereas the higher fatty acids are soluble in thissolvent. No fatty acid was detected when thehexane extract of the antibiotic was dried andtested. Also, transesterification and saponifica-tion did not release fatty acids. This implies thatthe fatty acids are covalently linked to the poly-peptide portion of the antibiotic. Also, analysisof the partial acid hydrolysate of pantomycin, todetermine the possible site of attachment offatty acid(s) to the polypeptide chain (see Ma-terials and Methods), revealed only proline andfatty acids. This suggests the possibility thatfatty acids may be attached to proline by anamide linkage and proline may be the N-termi-nal amino acid. Ifwe assume that each moleculeof proline is linked to a molecule of fatty acid,we can expect 1 mol of proline per mol of fattyacid in the acid hydrolysate of pantomycin. Ouranalytical data support this assumption (Table3).

flTQI.T T.QQT(nT

Pantomycin has been subjected to vigorouspurification techniques. The purified antibioticshows a single spot on thin-layer chromatogra-phy in several solvent systems. Other criteria,such as constant amino acid composition, singleSchlieren boundary in analytical ultracentrifu-

I I I I I I gation, and a single band in gel electrophoresis,0 1 2 3 4 5 suggest a satisfactory level of purity. The same

sample of pantomycin after acid hydrolysis re-Minutes vealed more than one fatty acid. Amino acid

5. Fatty acid components ofpantomycin. (1) analysis of pantomycin revealed 1 mol each of,ylic acid; (2) lauric acid; (3) isotridecanoic alanine, glycine, and proline in stoichiometric(4) myristic acid. ratios, whereas alloisoleucine (1.7 mol) and va-

I

ANTimICROB. AGENTS CHEMOTHER.

PANTOMYCIN 987

TABLE 3. Amino acid and fatty acid components ofpantomycin

Amino or fatty acid No. of residues permoleculea

Amino acidsD-Threonine ................ 2.00D-Alanine .................. 1.00D-Alloisoleucne ............. 1.90D-Valine .................... 1.70L-Valine .................... 1.70L-Serine .................... 100L-Proline ............. ...... 1.00L-Glycine ................... 1.00Basic amino acid ............ 1.00Unsaturated a-amino-

n-butyric acid ............. 1.00Aspartic and glutamic acids 0.01Leucine and isoleucines ...... 0.10

Fatty acidsbMyristic acid ............... 0.60Isotridecanoic acid .......... 0.27Lauric acid ................. 0.08Undecylic acid .............. 0.05a Normalized to alanine as 1.b Total yield of fatty acids was approximately 1 mol

per mol of peptide.

line (3.60 mol) yields were in nonstoichiometricratios (Table 1). This suggests the presence ofmicroheterogeneity in pantomycin. Four differ-ent fatty acids were present in the acid hydro-lysates (Table 3), but analyses indicate only onefatty acid could be present per peptide moietyof pantomycin. It is possible that there may beat least four different pantomycins each havinga different fatty acid component. The total pos-sible number of different pantomycins increasesif one considers pantomycins in which valine isreplaced by alloisoleucine or leucine, which is acommon phenomenon observed in several poly-peptide antibiotics (11). It will be of interest toattempt the fractionation of the pantomycinpreparation to determine the antiviral and an-timicrobial activity of different components.

Although the amino acid and fatty acid com-positions ofpantomycins as well as the elementalanalysis data (15) are similar to the stendomycinfamily of antibiotics (2), only pantomycinsshowed antiviral activity. Other characteristicssuch as the principal infrared and nuclear mag-netic resonance spectral bands (Fig. 1 and 2),the presence of microheterogeneity, valine-allo-isoleucine ratios, the time course of gradualrelease of these constituents during hydrolysis,the presence of the same amino acids in the Dconfiguration, and the point of attachment of

fatty acids are common characteristics of sten-domycins (2) and pantomycins.The structural and biological properties of

pantomycins and stendomycins are similarenough to suggest that pantomycins may bemembers of the stendomycin family of antibi-otics. However, further studies such as aminoacid sequence determinations will be required tocompletely elucidate the structures of panto-mycins.

ACKNOWLEDGMENTSWe thank C. M. Stevens, R. W. Brosemer, and J. A.

Magnuson of the Department of Chemistry, Washington StateUniversity, Pullman, Wash., for critical review of this manu-script. We also thank R. L. Hamill of the Lilly ResearchLaboratories, Indianapolis, Ind., for a sample of stendomycin.

This work was funded in part by Public Health Servicegrants A113296-01 and 11H, 2940, project 8889 from the Na-tional Institutes of Health to the Graduate School of Wash-ington State University.

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