biosynthesis ofdaunorubicin glycosides: role...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1980, p. 454-464 Vol. 18, No. 30066-4804/80/09-0454/1 1$02.00/0

Biosynthesis of Daunorubicin Glycosides: Role ofE-Rhodomycinone

JEFFREY C. McGUIRE,t* MONICA C. THOMAS, RONALD M. STROSHANE,BRUCE K. HAMILTON,t AND RICHARD J. WHITE

Chemotherapy Fermentation Program, Frederick Cancer Research Center, Frederick, Maryland 21701

Daunorubicin (daunomycin; NSC 82151) is a fermentation-derived anthracyc-line antibiotic that is clinically useful in the treatment of human leukemias.Daunorubicin itself is found rarely in microbial fermentations, but is presentnormally in the form of glycoside derivatives that yield the free drug on simpleacid hydrolysis. A major by-product of daunorubicin fermentations is usually thestructurally related anthracycinone e-rhodomycinone. We have used mutants ofa daunorubicin-producing Streptomyces species to study the biosynthetic rela-tionship between E-rhodomycinone and daunorubicin. We found that exogenouslyadded E-rhodomycinone can be converted to daunorubicin glycosides by a non-producing mutant and by a mutant that produces daunorubicin glycosides butnot E-rhodomycinone. Molar conversion efficiencies were in the 15 to 30% range.The latter mutant was also shown to convert exogenous "C-labeled E-rhodomy-cinone to "C-labeled daunorubicin glycosides, again at conversion effitiencies ofabout 25%. The same biotransformation was observed with daunorubicin produc-tion strain C5, which normally accumulates both E-rhodomycinone and dauno-rubicin glycosides. A significant percentage (16 to 37%) of exogenously added e-['4C]rhodomycinonewas metabolized by strain C5, and 22 to 32% of the metabo-lized radioactivity could be recovered as daunorubicin glycosides. A mathematicalmodel of E-rhodomycinone metabolism was constructed based on plausible as-sumptions concerning the kinetics of E-rhodomycinone accumulation and catab-olism. When analyzed according to this model, our data indicate that most (63 to73%), but not all, of the daunorubicin glycosides accumulated in the experimentswith production strain C5 derived from e-rhodomycinone. A pathway network forthe biosynthesis of daunorubicin glycosides is proposed that is in agreement withthese data. In this proposed pathway network, e-rhodomycinone is an interme-diate in one of at least two pathways which yield daunorubicin glycosides.

Daunorubicin (Fig. 1) is an anthracycline anti-biotic that is elaborated by several Streptomycesspecies (J. Lunel, J. Florent, D. Mancy and J.Renaut, Abstr. 174th Natl. Meet. Am. Chem.Soc., 1977, MICR 041). At present, interest indaunorubicin and related compounds is a resultof their important antitumor activity, especiallyagainst leukemias (9, 30). Details on discovery,clinical use, toxicity, mode of action, search forsuperior natural analogs, structure modification,and chemical synthesis programs related to dau-norubicin have been extensively reviewed (1-3,9, 10, 15, 19, 20, 25, 26, 30).

Several groups have studied the biosynthesisof daunorubicin. The anthracyclinone moietyderives from the sequential condensation of onepropionate and nine acetate units (22, 24). Otherstudies indicate that the 4-methoxy carbon atomderives from methionine and the carbon skele-

t Present address: Genex Corp., Rockville, MD 20852.

ton of the daunosamine moiety derives directlyfrom glucose (22, 23). Evidence exists for bio-transformation of the exogenously added puta-tive intermediates daunorubicinone and 13-di-hydrodaunorubicinone to daunorubicin by non-producing mutants of Streptomyces coeruleoru-bidus (7). However, little else is known aboutthe intermediates involved in daunorubicin bio-synthesis.

In our laboratory, we have been concernedwith increasing the production of daunorubicinby a streptomycete unidentified as to species(18). This strain accumulates very little, if any,daunorubicin directly, but instead synthesizes ahigher glycoside tentatively identified as bau-mycin A1/A2 (21). This glycoside can be cleavedto yield daunorubicin by simple acid hydrolysis.In normal fermentations baumycin A1/A2 ande-rhodomycinone (Fig. 1) are the major anthra-cyclines produced, with e-rhodomycinone pre-dominating. As a result of this, the biosynthetic

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BIOSYNTHESIS OF DAUNORUBICIN GLYCOSIDES 455

0 OH 0

A 1 12 11 10 13 4

A< HCH3

H3CO 0 OH -

H °

HO H

NH2 H

B

OH 0 OH OH

FIG. 1. Structures of daunorubicin (A) and E-rho-domycinone (B).

relationship of these two key metabolites is ofparticular importance. There are conflicting re-

ports in the literature on the ability of S. coe-

ruleorubidus mutants to convert e-rhodomyci-none to daunorubicin (7, 28). We have isolatedmutants of our daunorubicin-producing Strep-tomyces species, and in this paper we report on

the use of these to study the role of E-rhodomy-cinone in daunorubicin biosynthesis.

MATERIALS AND METHODS

Strains. All strains used in this study derive froma culture designated HD-1. Derivation of strains andtheir abilities to accumulate anthracyclines are sum-

marized in Table 1. Strains were maintained as sus-

pensions (viable counts typically 108 colony-formingunits per ml) or spores or mycelial fragments in 10%glycerol-0.5% Tween 80 in the vapor space of a liquidnitrogen freezer.

Culture conditions. Cultures were initiated bytransferring 0.05 to 0.1 ml of a spore or mycelialfragment suspension to a 250-ml baffled shake flaskcontaining 50 ml of medium S. Medium S contained(wt/vol): 1.5% corn starch (STA-Rx USP starch; A.E.Staley Mfg. Co., Decatur, Ill.), 0.5% Pharmamedia(Traders Protein, Fort Worth, Tex.), 0.5% bakers' Nu-trisoy defatted soy flour (Archer Daniels Midland Co.,Decatur, Ill.), 0.1% Red Star autolyzed yeast (Univer-sal Foods Corp., Milwaukee, Wis.), 0.25% sodium chlo-ride, and 0.5% calcium carbonate. Seed cultures were

incubated for 3 days at 28 to 30°C with rotary shakingat 250 rpm (1-inch [ca. 2.54-cm] stroke). Seed cultures

were used to inoculate (10% vol/vol) baffled shakeflasks containing one-fifth of the nominal flask volumeof medium P. Medium P contained (wt/vol): 5% glu-cose monohydrate, 1.2% herring meal (The MearlCorp., Eastport, Maine), 0.5% Red Star autolyzedyeast, 1.25% bakers' Nutrisoy defatted soy flour, 0.33%sodium chloride, 1.0% calcium carbonate, and 0.2%(vol/vol) Prochem 51 (Prochem Co., Gurnee, Ill.).Production cultures were incubated at 28 to 30°C withrotary shaking at 250 rpm (1-inch stroke).Daunorubicin assay. A 300-mg amount of oxalic

acid dihydrate was dissolved in a 10-ml sample offermentation broth and the solution was heated for 45min at 50°C to hydrolyze higher glycosides of dauno-rubicin to daunorubicin (Lunel et al., Abstr. 174thNatl. Meet. Am. Chem. Soc. 1977, MICR 041, andR.M. Stroshane, E.C. Guenther, J.L. Piontek, and A.A.Aszalos, Abstr. 178th Natl. Meet. Am. Chem. Soc.1979, ANAL 044). The heated broth was then centri-fuged, and the supernatant was filtered through a filter(Millipore Corp, type HAWP; 0.45 ,um). The filtratewas injected into a high-performance liquid chroma-tography (HPLC) system comprising a Waters Asso-ciates M600A pump, a 710A WISP automatic sampler,a ,uBondapak C18 column (3.9 mm by 30 cm; WatersAssociates, Milford, Mass.), and an ISCO UA5 absorb-ance monitor equipped with a type 6 optical unit anda 10-,ul flow cell. Detection was at 254 nm with a

sensitivity of 0.05 absorbance units full scale. Metha-nol-water-PIC-B-7 (Waters Associates) (65:35:1.8)was used as eluant at 2 ml/min. Samples of daunoru-bicin in organic solvents were filtered through a Mil-lipore filter (type FHLP; 0.5 ,um) and chromato-graphed as above. Daunorubicin was quantitated bycomparing the height of the daunorubicin peak on a

strip chart recording, with the heights of peaks givenby standard solutions of authentic daunorubicin hy-drochloride injected the same day. The sample injec-tion volume was chosen such that the sample peakheight fell within the linear portion of the standardcurve.

e-Rhodomycinone assay. E-Rhodomycinone was

quantitated by using the same HPLC system as fordaunorubicin. Aqueous samples containing both dau-norubicin and e-rhodomycinone were hydrolyzed as

described above. Under these conditions, e-rhodomy-

TABLE 1. Derivation and properties of strains usedAccumulation ofa:

Strain Parent Mutagen Dauno-rubicin E-Rhodomy-gly- cinone

cosides

V8 HD-1 Spontaneous + +R33 V8 UVb + +NZ4 R33 UV - -C5 V8 UV + +26-4 C5 NGC + +R28 V8 UV - +

a Indicated metabolites accumulate when strain isgrown in medium P.

b UV, ultraviolet light.'NG, N-methyl-N'-nitro-N-nitrosoguanidine.

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ANTIMICROB. AGENTS CHEMOTHER.

cinone was essentially insoluble and was recoveredquantitatively in the pellet after centrifugation. Thepellet was resuspended in n-butanol to the originalsample volume. After mixing for 15 min and clearingby centrifugation, the butanol extract was filtered andinjected onto the HPLC system. The peak correspond-ing to E-rhodomycinone was quantitated relative to adaunorubicin standard curve run the same day, andthe E-rhodomycinone titer was obtained by using acorrection factor relating apparent daunorubicin titerto actual E-rhodomycinone titer. This correction factorwas determined experimentally by using authentic E-rhodomycinone.Daunorubicin purification. For purification of

daunorubicin from more than 1 liter of broth, thebroth was adjusted to pH 8.6 and extracted with 23volume of n-butanol, and the insoluble cake was re-moved by filtration. The cake was re-extracted with 'Avolume of n-butanol and again filtered. The butanoland water phases of the pooled filtrates were allowedto separate, and the butanol phase was concentrated20-fold in vacuo at 40°C. Two volumes of n-hexanewere added to the butanol concentrate, and the mix-ture was extracted six times with '/A2 volume of water(pH 1.8). After separation of the phases, the pooledacid water was extracted twice with equal volumes of30% (vol/vol) acetone in toluene. The aqueous phasewas adjusted to pH 8.6 and extracted twice with equalvolumes of n-butanol. The pooled butanol extract waswashed several times with deionized water. The waterwashes were back-extracted with butanol, and thepooled butanol extract was adjusted to pH 5.2 with 1N ethanolic hydrochloride and concentrated 80-fold invacuo at 40°C. After addition of 1Ao volume of deion-ized water, the butanol concentrate was adjusted topH 1.4 with ethanolic hydrochloride and was heatedat 45°C for 40 min to hydrolyze daunorubicin glyco-sides to daunorubicin. About 10% of the solvent wasremoved by evaporation, and daunorubicin was al-lowed to crystallize as the hydrochloride salt at 4°Cfor about 24 h. Crystals were collected by filtrationand lissolved in a 1:17 mixture of methanol andethanol (80 ml of solvent per g of crystals). Afteraddition of 1.2 volumes of chloroform, daunorubicinhydrochloride was allowed to recrystallize for 3 daysat 4°C. Crystals were collected by filtration andwashed with cold chloroform before drying in a vac-uum oven.

For purification of radioactive daunorubicin, brothwas hydrolyzed by treatment with 30 mg of oxalic aciddihydrate per ml at 50°C for 45 min. Hydrolyzed brothwas cleared by centrifugation, and the supernatantwas adjusted to pH 8.6 and extracted with an equalvolume of n-butanol. The butanol extract was concen-trated in vacuo at 40°C and was then subjected topreparative thin-layer chromatography (TLC) on sil-ica gel plates with solvent A (see below). The dauno-rubicin band was scraped from the plate and elutedwith acetone. The acetone eluate was filtered, theacetone was removed by evaporation, and the dauno-rubicin was dissolved in methanol.e-Rhodomycinone purification. E-Rhodomyci-

none was purified as a side product from a large-scaledaunorubicin recovery operation. Fermentation brothwas adjusted to pH 8.6 and extracted with n-butanol.After filtration, the butanol and water phases were

allowed to separate, and the butanol phase was con-centrated 20-fold by vacuum evaporation. Two vol-umes of heptane were added to the butanol concen-trate, and the mixture was extracted several timeswith water (pH 1.8). Forty liters of the butanol-hep-tane phase was concentrated by vacuum evaporationto a final volume of 4 liters. The concentrate wasfiltered, and the filter cake was washed with dilutesulfuric acid (pH 2.0) and hexane. A portion of thesolids (6.8 g) was suspended in 100 ml of a mixture ofchloroform and methanol (80:20) and filtered througha 0.45-Mm Millipore Fluoropore filter. The filtrate waschromatographed on a Waters Associates Prep LC/System 500 with two Prep PAK/500/Silica columnsand chloroform-methanol (80:20) as eluant at a flowrate of 50 ml/min. Of the seven fractions collected,those containing primarily E-rhodomycinone werepooled and concentrated to dryness in vacuo at 40°C.The residue was dissolved in methanol (40°C) andfiltered through Whatman no. 1 filter paper. Thefiltrate was maintained at room temperature for 1 hand then was transferred to 5°C for 16 to 48 h. Theresulting crystals were recovered by filtration, washedwith cold methanol, and then recrystallized twice asdescribed above.The following physicochemical characteristics of

the final recrystallized product were determined: melt-ing point (Kofler hot stage apparatus; uncorrected),ultraviolet absorption spectrum (GCA McPhersonmodel 100 recording spectrophotometer), infrared ab-sorption spectrum (Perkin-Elmer model 180 recordingspectrophotometer), electron impact mass spectrum(Finnigan 3300 GC/MS with a 6000 MS data systemat 70 eV, solid probe), 'H nuclear magnetic resonancespectrum (Varian XL-100 spectrometer with tetra-methylsilane as internal reference standard), and 13Cnuclear magnetic resonance spectrum (JEOL/FX-60FT-NMR). All results were in accord with literaturevalues (11, 12) and with the structure shown for E-rhodomycinone in Fig. 1.

"4C-labeled E-rhodomycinone was isolated from cul-tures of strain R28 (Table 1) that had been exposed to[2-"4C]acetic acid (58 mCi/mmol; Amersham) for 3days, starting 3 or 4 days after inoculation into mediumP. Low-specific-activity E-['4C]rhodomycinone wasprepared from a culture treated with 0.5 MCi of [14C]-acetic acid per ml, and high-specific-activity E-[14C]-rhodomycinone was prepared from a culture treatedwith 12 ,uCi of ['4C]acetic acid per ml. To isolate E-['4C]rhodomycinone, the broth was adjusted to pH 2.0with HCI and extracted with an equal volume ofchloroform. The chloroform phase was recovered andconcentrated by evaporation in vacuo at 40°C. Ali-quots of the concentrate were then run on preparativeTLC in solvent A (see below). The E-rhodomycinoneband was scraped from the plate and eluted withacetone. After filtration, acetone was removed byevaporation, and E-['4C]rhodomycinone was dissolvedin ethanol.

In feeding experiments, E-['4C]rhodomycinone wasrecovered from the pellet obtained after centrifugationof hydrolyzed broth (see above). The pellet was ex-tracted with n-butanol, and after concentration E-rho-domycinone was isolated from the butanol extract bypreparative TLC as above.TLC. For preparative TLC, plates with a 1-mm-

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thick layer of silica gel were used (type Anasil H;Analabs, Inc., New Haven, Conn.). For analytical TLC,plates with a silica gel layer 0.25 mm thick and with apre-absorbent area were used (type LK; Kontes, Vine-land, N.J.). Solvent A contained heptane-chloroform-methanol-acetic acid (40:40:18:2). TLC plates werescanned for absorbance at 480 nm with a Schoeffel SD3000 spectrodensitometer, a Schoeffel SDC 300 Den-sity Computer and a conventional strip chart recorder.TLC plates were scanned for radioactivity using aPackard model 7201 Radiochromatogram scanner.

Radioactivity measurement. Radioactivity wasmeasured with a Searle ISOCAP 300 Liquid Scintil-lation System. The scintillation fluid used was PCS(Amersham). Counting efficiency was determined bythe sample channels ratio method.

RESULTSDaunorubicin fermentation. Figure 2A

shows the ultraviolet absorption profile of bu-tanol-soluble compounds, separated by HPLC,that accumulated during a typical fermentationof daunorubicin production strain C5. The pre-dominant metabolite was e-rhodomycinone,which has been observed in daunorubicin fer-mentation broths by a number of workers (8, 12,14,21). The other major component was a higherglycoside of daunorubicin, which has been ten-tatively identified as baumycin A1/A2 (21).When the butanol extract was subjected to mild

C.)

0 5

R.1

10 15 20 25 30 35

Time (min)

hydrolysis (pH 1.5, 40°C, 40 min), the higherglycoside of daunorubicin was converted to dau-norubicin (Fig. 2B). Accumulation of higher gly-cosides during daunorubicin fermentations hasalso been reported by other groups (8, 14, 21;Lunel et al., Abstr. 174th Natl. Meet. Am. Chem.Soc. 1977, MICR 041).

Biosynthetic considerations (29) implicate e-rhodomycinone or the closely related demethyl-E-rhodomycinone as likely intermediates in thesynthesis of daunorubicin. We have carried outa series of experiments to investigate the possi-ble role of e-rhodomycinone as an intermediatein daunorubicin biosynthesis.Strain NZ4. e-Rhodomycinone feeding exper-

iments were first done with strain NZ4. Sincethis strain does not accumulate any detectableanthracycline-related compounds (Table 1), thefate of exogenous e-rhodomycinone can be fol-lowed without interference from endogenouscompounds. A shake flask culture of strain NZ4that had grown for 4 days in medium P wassupplemented with solid E-rhodomycinone at aconcentration of 280 t,M. After 3 more days ofincubation, the culture was found by HPLCassay to contain 45 ,uM daunorubicin glycoside,corresponding to a conversion efficiency of 16%.A portion of this culture broth was hydrolyzed,and the hydrolysate was adjusted to pH 8.5 and

B

ca)

U)

C.)

0

A.A

R

35

t

5 10 15 20 25 30

Time (min)

FIG. 2. Ultraviolet absorption profile ofbutanol-soluble compounds, separated by HPLC, that accumulatedduring a typical fermentation of strain C5. HPLC was done as described in the text, except that the solventwas methanol-water, pH 2.0 (65:35). (A) Unhydrolyzed sample; (B) sample hydrolyzed atpH 1.5, 40°C for 45min, (G) daunorubicin glycoside; (R) E-rhodomycinone; (D) daunorubicin.

A

C

0c,)

I I

t

%I L.)

I

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extracted with butanol. Aliquots of the butanolextract were analyzed by TLC for the presenceof daunorubicin and E-rhodomycinone. The re-

sults of this experiment are shown in Fig. 3. It isapparent that strain NZ4 converted exogenous

E-rhodomycinone into a compound that co-chro-matographed with daunorubicin on TLC as wellas on HPLC. A control flask showed that E-

rhodomycinone was stable in uninoculated me-

dium P.A larger-scale feeding experiment was con-

ducted to verify that the product of strain NZ4biotransformation of e-rhodomycinone was in-deed daunorubicin glycoside. Four 400-ml shakeflask cultures of strain NZ4 were supplementedwith a total of460 Lmol ofsolid e-rhodomycinoneafter 3 days of growth in medium P. After 4more days of incubation, the cultures werepooled and found by HPLC assay to contain atotal of 100 Amol of daunorubicin glycoside, cor-

responding to a conversion efficiency of 22%.Daunorubicin was isolated from this broth asdescribed under Materials and Methods. The

0 D RF

+R,RSPIKE

+R,D SPIKE

+ R- R-____________ v__

FIG. 3. TLC analysis of daunorubicin glycosideaccumulation in strain NZ4 fermentations conductedin the absence (-R) or in the presence (+R) of exog-enous e-rhodomycinone from day 4 to 7 of fermenta-tion. Samples were hydrolyzed as described in thetext and were in some cases spiked with authentic e-rhodomycinone (R SPIKE) or daunorubicin (DSPIKE) before chromatography. (0) Origin of chro-matography; (F) solvent front; (D) position of stan-dard daunorubicin; (R) position of standard e-rho-domycinone.

crystallized product was identical to authenticdaunorubicin by the following criteria: retentiontime on HPLC (daunorubicin assay system; seeMaterials and Methods), relative mobility onTLC with solvent system A, ultraviolet spec-trum, infrared spectrum, electron impact massspectrum, and proton nuclear magnetic reso-nance spectrum.Strain 26-4. The experiments just described

established that a nonproducing mutant wascapable of transforming e-rhodomycinone todaunorubicin glycoside. We wished to testwhether this might also be true for daunorubi-cin-producing strains. For this study we chosestrain 26-4, which accumulates nearly normallevels of daunorubicin glycoside but only lowlevels of E-rhodomycinone (Table 1). We rea-soned that a hypothetical pathway from E-rho-domycinone to daunorubicin would probably besaturated in strains which accumulate highlevels ofendogenous e-rhodomycinone. Additionof exogenous e-rhodomycinone to such strainswould therefore have no effect on accumulationof daunorubicin glycloside. Strain 26-4, however,does not accumulate significant levels of E-rho-domycinone and might therefore show a re-sponse to exogenous e-rhodomycinone.A culture of strain 26-4 that had grown for 4

days in medium P was divided into several 10-ml aliquots. Three were assayed for daunorubi-cin glycosides, three were incubated for anotherday and then assayed, and three were incubatedfor 1 day in the presence of 210 ,uM e-rhodomy-cinone and then assayed. The results of thisexperiment are shown in Table 2. The culturessupplemented with E-rhodomycinone accumu-lated substantially more daunorubicin glyco-sides during the 1-day test period than did theunsupplemented cultures. Assuming that the ex-cess of daunorubicin glycosides in supplementedcultures was synthesized from the added E-rho-domycinone, we calculate that the average con-version efficiency in this experiment was 16%.Other experiments have confirmed that addi-

TABLE 2. Effect of exogenous e-rhodomycinone onaccumulation of daunorubicin glycosides by

strain 26-4

Time of addi-tion of 210 uM Time of Titer at harvest of dauno-e-rhodomyci- harvest (day) rubicin glycosidesb (uM)none (day)'_ 4 10.3 ± 0.8- 5 29.3±2.74 5 63.7±8.0

a E-Rhodomycinone was added from a finely dividedstock suspension of 10.5 mM E-rhodomycinone inethanol. Control cultures received only ethanol.

b Mean ± standard error of the mean of three flasks.

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BIOSYNTHESIS OF DAUNORUBICIN GLYCOSIDES

tion of E-rhodomycinone to cultures of strain 26-4 results in increased titers of daunorubicin gly-cosides. Conversion efficiencies of up to 30%have been observed in cultures exposed for 3days (instead of 1 day) to exogenous E-rhodo-mycinone (data not shown).

Feeding experiments were also conductedwith radioactively labeled E-rhodomycinone toconfirm that the anthracyclinone ring system ofdaunorubicin glycosides derives from the ringsystem of E-rhodomycinone. "4C-labeled E-rho-domycinone was prepared as described in Ma-terials and Methods and had a specific activityof 34 mCi/mol. A shake flask culture of strain26-4 that had grown in medium P for 4 days wassupplemented with e-[4C]rhodomycinone (2.33,umol, 79 nCi in a 48-ml culture). After 3 moredays of incubation, an aliquot of the culture wasremoved for assay of daunorubicin glycoside (ob-served titer was 62 ,iM), and daunorubicin waspartially purified from the rest of the culture.Partially purified daunorubicin solution wasfound, by liquid scintillation counting, to contain31,000 dpm/mnl and, by HPLC assay, to contain1.5 mM of daunorubicin. Initial E-[14C]rhodo-mycinone and partially purified daunorubicinwere also analyzed by TLC and radioscanning.Figure 4 demonstrates the radiochemical purityof the starting E-['4C]rhodomycinone and alsoshows that daunorubicin synthesized by strain26-4 in the presence of radioactive e-rhodomy-cinone was radioactive. By cutting out andweighing the peaks from a photocopy of thetrace shown in Fig. 4B, we estimated that about70% of the radioactivity in the daunorubicinpreparation was associated with daunorubicin.Thus, the specific activity of [14C]daunorubicinwas 70% of (31,000 dpm/ml)/(1.5,mol/ml), or6.6 mCi/mol. From this data and from the finaltotal amount of daunorubicin glycoside in theculture broth (62 ,uM x 48 ml = 2.98 Lmol), wecalculated that daunorubicin glycosides con-tained 19.7 nCi of radioactivity at the end of theexperiment, corresponding to 25% of the radio-activity that had been added as e-rhodomyci-none. This conversion efficiency is similar tothat observed in experiments in which strain 26-4 was allowed to convert exogenous unlabelede-rhodomycinone for 3 days.

Strain C5. To assess the significance of E-rhodomycinone in daunorubicin production fer-mentations, feeding experiments were conductedwith radioactive e-rhodomycinone and strain C5.Data from two independent experiments are pre-sented in Table 3. The experiments were donewith different preparations of e-['4C]rhodomy-cinone having different specific activities.From the data in Table 3, it is apparent that

most of the radioactivity added as e-rhodomy-

AR F

BFO D

4141

FIG. 4. Conversion of E-[f4C]rhodomycinone to['4C]daunorubicin glycoside by strain 26-4. (A) Start-ing E-['4C]rhodomycinone; (B) partiallypurified con-version product (daunorubicin). In each case, analy-sis was by TLC and radioscanning as described inthe text. (0) Origin of chromatography; (F) solventfront; (R) position of e-rhodomycinone; (D) positionof daunorubicin.

cinone remained as e-rhodomycinone over thecourse of the experiments. Of the radioactivitythat was metabolized, the fraction that appearedas daunorubicin glycosides was 22% in experi-ment 1 and 32% in experiment 2.

It would be interesting to know what fractionof the aglycone moiety of daunorubicin glyco-sides synthesized during the course of a fermen-tation arises from e-rhodomycinone. If a single,linear biosynthetic pathway leads to daunoru-bicin via E-rhodomycinone, then this fractionwill be 1.0 (Fig. 5A). If, however, E-rhodomyci-

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460 McGUIRE ET AL.

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none is not an obligatoiy intermediate, thenthere are more than one pathway leading todaunorubicin glycosides, and the fraction will beless than 1.0 (Fig. 5B). Several examples of mul-tiple pathways leading to secondary metaboliteshave been proposed by other workers (4, 13, 16,17, 27). The data presented in Table 3 can beused to explore this question.Our approach was to calculate the amount of

E-rhodomycinone which was catabolized duringan experiment, to measure the fraction of catab-olized E-rhodomycinone that appeared as dau-norubicin glycosides, and then to calculate theamount of daunorubicin glycosides derived frome-rhodomycinone. This latter calculated valuewas compared with the total amount of dauno-rubicin glycosides that accumulated during theexperiment, so that the fraction of accumulateddaunorubicin glycoside that derived from E-rho-domycinone was evident.The amount of E-rhodomycinone catabolized

during the experiment was calculated by makingassumptions concerning the kinetics with whichE-rhodomycinone was catabolized and the timecourse with which E-rhodomycinone accumu-lated. We considered the cases of zero-order andfirst-order kinetics of E-rhodomycinone catabo-lism and the cases of linear and exponential ratesof E-rhodomycinone accumulation. We also as-sumed that endogenous and exogenous E-rho-domycinone constituted a single well-mixedpool. Equations modeling these situations aregiven in Table 4. The expressions for loss ofradiolabel from the E-rhodomycinone pool withtime are derived as described below.

(i) Cases 1 and 2 (zero-order kinetics fore-rhodomycinone catabolism). In the case ofzero-order kinetics, the molar efflux from the E-rhodomycinone pool (rR, nanomoles/hour) istaken to be equal to a constant (C, nanomoles/hour) whose value is independent of the amountof E-rhodomycinone in the pool (R): rR = C(constant). The change in the amount of radio-label in the E-rhodomycinone pool with time(d*R/dt, disintegrations per minute/hour) isthen obtained by multiplying the constant molarefflux (C, nanomoles/hour) by the specific radio-activity of the E-rhodomycinone pool (*R/R,disintegrations per minute/nanomole): d(*R)/dt= -C(*R/R). In the above kinetic expressionsand in kinetic expressions which follow, amountscan be used instead of concentrations becausevolume is constant.

(ii) Cases 3 and 4 (first-order kinetics fore-rhodomycinone catabolism). In the case offirst-order kinetics, the molar efflux from the e-rhodomycinone pool is taken to be proportionalto the amount of E-rhodomycinone in the pool:rR = kR. Again, the change in the amount ofradiolabel in the E-rhodomycinone pool with


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A 1 propionate+

9 acetate

|r COOH

0 0II 11

o 0 0

11 1 l 10 0 0 0

E£rhodomycinone

Idaunorubicinglycosides

B 1 propionate

9 acetate

|r COOH

O J0>

011 11 0

r1oc

\E-rhodomycinone

daunorubicinglycosides

FIG. 5. Hypothetical pathways to daunorubicin glycosides. (A) Linearpathway in which E-rhodomycinoneis an intermediate; (B) proposed pathway network in which E-rhodomycinone is an intermediate in only oneof multiple pathways which yield daunorubicin glycosides. Arrows do not represent single enzymatic steps,but merely denote pathway connectivity.

TABLE 4. Equations modeling the catabolism and accumulation of E-rhodomycinone undervarious assumptionsa

Assumptions Equations modeling:Rate law for e-

Case Kinetics of e-rhodo- Time course of rhodomycinone Loss of radiolabel from e- Accumulation ofE-rhodo-mycinone catabo- e-rhodomycinone catabolism rhodomycinone pool with mycinone with time

lim accumulation time

1 Zero order Linear rR = C d(*R) (*R) R = R,, + mt-C-dt R

2 Zero order Exponential rR = C d(*R) (*R) R = Roek'tdt R

3 First order Linear rR = kR d(*R) R = Ro + mtdt

4 First order Exponential rR = kR d(*R) k('R) R = Roek"'dt

a Nomenclature as follows: rR, molar efflux from e-rhodomycinone pool in nanomoles per hour; C, zero-orderrate constant for e-rhodomycinone catabolism in nanomoles per hour; k, first-order rate constant for E-rhodomycinone catabolism in hours-'; *R, disintegrations per minute in E-rhodomycinone pool at time t; R,nanomoles of E-rhodomycinone at time t, Ro, nanomoles of E-rhodomycinone at zero time; m, rate of E-rhodomycinone accumulation in nanomoles per hour (linear accumulation with time); k', rate constant for E-rhodomycinone accumulation in hours-' (exponential accumulation with time); t, time in hours.

time is obtained by multiplying the molar efflux(now kR) by the specific radioactivity of the E-rhodomycinone pool (still *R/R): d(*R)/dt=-k (*R).The following steps were taken to calculate

the amount of e-rhodomycinone catabolized dur-ing each experiment. In step 1, the constants mand k' in the expressions for E-rhodomycinoneaccumulation (Table 4) were evaluated from theobserved initial and final amounts of e-rhodo-mycinone and the run times listed in Table 3. Instep 2, the differential equations modeling theloss of radiolabel from the E-rhodomycinone pool

were integrated, with R being replaced by theappropriate expressions for e-rhodomycinone ac-cumulation on a case-by-case basis (cases 1 to 4,Table 4). The integrated forms of these equa-tions are given in Table 5. In step 3, the constantsC and k (Table 4) were evaluated by substitutingdata from Table 3 for *Ro, *R, Ro, R, and t intothe integrated expressions of Table 5. Values form and k' obtained in step (1) were used whereneeded. The values obtained for k, k',-C, and min each of the two experiments are listed inTable 6. In step 4, in cases 1 and 2 the amountof e-rhodomycinone metabolized was simply the

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TABLE 5. Integrated expressions modeling loss ofradiolabel from the e-rhodomycinone pool for the

four cases listed in Table 4'Caseb Expression

1 In*R C RoRo m R

2 *R C(Ro-R)ln -

*Ro k'RRo3 *3ln*R =-kt

Ro

ln-R--ota *Ro, Disintegrations per minute in e-rhodomyci-

none pool at zero time. For other nomenclature, seeTable 4.

b See Table 4.

TABLE 6. Calculated values of constants inequations of Table 4 for two experiments in which

strain C5 was fed e-[`4CJrhodomycinone"Value of constants

Case Expt c m k k'(nmol/h) (nmol/h) (h-') (h-W)

1 1 114 1202 54 179

2 1 112 0.0112 52 0.0081

3 1 120 0.0112 179 0.0024

4 1 0.011 0.0112 0.0024 0.0081

aValues were calculated from the data in Table 3as described in the text.

b See Table 4.

product of the constant C and the run time ofthe experiment. In cases 3 and 4, the integral ofkR dt was evaluated over the time of the exper-iment, with the appropriate expression for R asa function of time (Table 4) being substitutedbefore integration.Table 7 lists the calculated amounts of e-rho-

domycinone catabolized during each of the twoexperiments described in Table 3 for each of thefour cases of assumed kinetics (Table 4) consid-ered. It can be seen that the different kineticassumptions made in each of the four cases hadlittle effect on the calculated amount of c-rho-domycinone catabolized. Therefore, we used theassumptions of case 1 for the rest of the analysis.Table 8 (column 6) shows the calculated frac-

tion of* accumulated daunorubicin glycosidesthat derived from f-rhodomycinone in each ex-periment. The fraction of metabolized c-rhodo-mycinone recovered as daunorubicin glycosides(Table 8,' column 3) was taken from Table 3.The amount of daunorubicin glycosides accu-mulated during each experiment (Table 8, col-

umn 5) was taken to be the difference betweenthe final and initial amounts of daunorubicinglycosides (Table 3).The calculations in Table 8 indicate that mrost,

but not all, of the daunorubicin glycoside thataccumulated during the fermentations of pro-duction strain C5 (Table 3, experiments 1 and 2)was synthesized via c-rhodomycinone. Thus,daunorubicin glycosides may be synthesized viamore than one pathway, one of which includesE-rhodomycinone as a internediate.A report in the literature suggests that dau-

norubicin glycosides may be transformed duringproduction fermentations into compounds fromwhich daunorubicin cannot be obtained by sim-ple acid hydrolysis (12). If this possibility isincluded in our model, it can be shown thatthere is no effect on the calculated fraction ofdaunorubicin glycosides that derives from c-rho-domycinone.

DISCUSSIONConversion of e-rhodomycinone to dau-

norubicin glycosides by mutants. Our resultsestablish that e-rhodomycinone can be con-verted to daunorubicin glycosides by a dauno-rubicin-producing Streptomyces species and bymutants of the same strain affected in anthra-cycline biosynthesis. Blumauerova et al. (7) haveattempted similar experiments with nonprod-ucing mutants of S. coeruleorubidus, anotherdaunorubicin-producing microorganism. Theyfound that e-rhodomycinone was not convertedto daunorubicin glycosides. It is possible thatstrain differences account for this discrepancy,since preliminary information indicates that theorganism we used is not S. coeruleorubidus.However, it should be noted that Umezawaclaims to have attempted similar experimentswith independently isolated mutants of S. coe-ruleorubidus and to have observed conversionof e-rhodomyciione to daunorubicin glycosides(28).Some 15 to 30% ofthe e-rhodomycinone added

to cultures of our mutants was transformed todaunorubicin glycosides. TLC analysis (data not

TABLE 7. Calculated amount ofe-rhodomycinonecatabolized during each ofthe two experiments

listed in Table 3, based upon assumptions listed inTable 4

e-Rhodomycinone catabolizedCase W(mol)

Expt 1 Expt 2

1 4.9 3.82 4.8 3.63 5.2 3.84 5.1 3.7

462 McGUIRE ET AL.


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TABLE 8. Analysis of e-['4C]rhodomycinone conversion experiments with strain C5Fraction of catabolized Daunorubicin

catabolizeda e-rhodomycinone A x Bc glycosideExpt camold recovered as dauno- (panol) accumulated C/Dd

('Amol) rubicin glycosidesa (C) (ttmol) b

(A) (B) (D)1 4.9 0.22 1.1 1.5 0.732 3.8 0.32 1.2 1.9 0.63

Case 1, Table 7.b Table 3.c Calculated amount of daunorubicin glycoside that derived from e-rhodomycinone.d Calculated fraction of accumulated daunorubicin glycoside that derived from e-rhodomycinone.

shown) indicated that little residual E-rhodo-mycinone remained at the end of such experi-ments. This raises the question of the fate of theother 70 to 85% of the catabolized e-rhodomyci-none. Two possibilities are: (i) some e-rhodo-mycinone was converted, perhaps via daunoru-bicin glycosides, to products lacking the char-acteristic anthracycline chromophore; (ii) somee-rhodomycinone was converted, perhaps viadaunorubicin glycosides, to anthracyclines oraglycones which do not yield daunorubicin uponacid hydrolysis. Such compounds might includedaunorubicinone, 13-dihydrodaunorubicinone,and 7-deoxy-13-dihydrodaunorubicinone, all ofwhich have been isolated from daunorubicinfermentation broths (8, 12, 14, 21; Lunel et al.,Abstr. 174th Natl. Meet. Am. Chem. Soc. 1977,MICR 041). These possibilities are currentlybeing evaluated.Role ofe-rhodomycinone in daunorubicin

production fermentations. Since mutants ofour daunorubicin-producing Streptomyces spe-cies converted exogenous e-rhodomycinone todaunorubicin glycosides, and since daunorubicinproduction fermentations contain substantialamounts of endogenous e-rhodomycinone, it ap-peared likely that E-rhodomycinone could serveas a source of the anthracyclinone moiety ofdaunorubicin in production fermentations. Thiswas confirmed by experiments in which fermen-tations of production strain C5 were supple-mented with radioactive E-rhodomycinone. Al-though most of the E-rhodomycinone appearedto be metabolically inert, a significant fraction(one-seventh to one-third; Table 3) was metab-olized. Of this fraction, about 20 to 30% appearedas daunorubicin glycosides (Table 8). Again, asubstantial portion of the metabolized e-rhodo-mycinone must give rise either to anthracyclinesthat are not converted to daunorubicin by acidhydrolysis or to nonanthracycline products.Our experiments with radioactive e-rhodo-

mycinone provided data with which we couldtest the hypothesis that e-rhodomycinone is anessential intermediate in daunorubicin biosyn-thesis. This hypothesis was originally suggested

by Vanek et al. (29), but was subsequently dis-avowed by that group (6) due to the inability oftheir strains of S. coeruleorubidus to convertexogenous E-rhodomycinone to daunorubicin.The mathematical analysis presented in Re-

sults suggests thAt more than half of the dau-norubicin glycoside accumulated during fermen-tations of production strain C5 was synthesizedvia e-rhodomycinone (Table 8). It should beemphasized that this conclusion is based uponseveral assumptions. Two of these assumptionsconcern the kinetics of E-rhodomycinone metab-olism and the rate at which E-rhodomycinoneaccumulated. Our conclusion, that most dauno-rubicin glycosides come from E-rhodomycinone,holds under zero-order or first-order kinetics ofe-rhodomycinone metabolism and under linearor exponential rates of e-rhodomycinone accu-mulation.The assumption that endogenous and exoge-

nous E-rhodomycinone constitute a single, ho-mogeneous pool is more critical, and, perhaps,less plausible than the kinetic assumptions justmentioned. Since e-rhodomycinone is insolublein water, it is not obvious whether or not exog-enously added e-rhodomycinone exists in thesame state in fermentation broths as endoge-nously synthesized e-rhodomycinone. Althoughthe data presented here do not allow a criticaltest of our assumption, they are consistent withit. The similar conversion efficiencies (20 to 30%)for transformation of E-rhodomycinone to dau-norubicin that were observed over a wide rangeof concentrations of exogenous e-rhodomyci-none, both radiolabeled and unlabeled, suggestthat e-rhodomycinone is metabolized in a uni-form manner over at least this concentrationrange. This range extends from 9 ,uM (experi-ment 2 in Table 3) through 220,uM (Table 2) to500,M (unpublished data with strain 26-4).Proposed biosynthetic pathway to dau-

norubicin glycosides. Our results indicate thatthe e-rhodomycinone pool is a major source ofthe anthracyclinone moiety of daunorubicin gly-cosides accumulated during daunorubicin pro-duction fermentations. We have developed a

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464 McGUIRE ET AL.

proposed pathway for daunorubicin glycosidesbiosynthesis, based upon the results reportedhere and by other workers (24). This pathway isillustrated in Fig. 5B. Our proposed pathwaydiffers from those of Blumauerova et al. (4-6),since those authors consider e-rhodomycinoneto be a branch product of the daunorubicinpathway, which cannot be converted to dauno-rubicin glycosides.Our overall goal in studying daunorubicin bio-

synthesis is to glean information that might beuseful in increasing the titer of our daunorubicinproduction fermentations. Since E-rhodomyci-none can apparently be converted to daunoru-bicin glycosides by our production strain, con-ditions that maximize the extent of this conver-sion might be usefully employed to increasedaunorjibicin titer. We are presently exploringstrategies to optimize the conversion of c-rho-domycinone to daunorubicin glycosides in pro-duction fermentations.

ACKNOWLEDGMENTS

We thank Eva Guenther for daunorubicin assay support,Margaret Toussaint and Ramesh Pandey for providing HPLCtraces, and John Douros for encouragement.

This work is supported by Public Health Service contractNO-1-CO-75380 with Litton Bionetics., Inc., from the NationalCancer Institute.

LITERATURE CITED1. Arcamone, F. 1977. New antitumor anthracyclines. Lloy-

dia 40:45-66.2. Arcamone, F. 1978. Daunomycin and related antibiotics,

p. 89-229. In P.G. Sammes (ed.), Topics in antibioticchemistry, vol. 2. Ellis Horwood, Ltd., Chichester.

3. Bernard, J., R. Paul, M. Boiron, C. Jacquillat, and R.Maral. 1969. Rubidomycin. Springer-Verlag, NewYork.

4.. Blumauerova, M., J. Jizba, K. Stajner, and Z. Vanek.1979. Effect of D,L-ethionine on the biosynthesis ofanthracyclines in Streptomyces coeruleorubidus. Bio-tech. Letters 1:471-476.

5. Blumauerova, M., E. Kraloveova, Z. Hostalek, and Z.Vanek. 1979. Intra- and interspecific cosynthetic activ-ity of mutants of Streptomyces coeruleorubidus andStreptomyces galilaeus impaired in the biosynthesis ofanthracyclines. Folia Microbiol. 24:128-135.

6. Blumauerova, M., E. Kraloveova, J. Mateju, Z. Hos-talek, and Z. Vanek. 1979. Genetic approach to thebiosynthesis of anthracyclines, p. 90-96. In 0. K. Sebekand A. I. Laskin (ed.), Genetics of industrial microor-ganisms. American Society for Microbiology, Washing-ton,. D.C.

7. Blumauerova, M., E. Kralovcova, J. Mateju, J. Jizba,and Z. Vanek. 1979. Biotransformation of anthracyc-lines in Streptomyces coeruleorubidus and Strepto-myces galilaeus. Folia Microbiol. 24:117-127.

8. Blumauerova, M., J. Mateju, K. Stajner, and Z. Va-nek. 1977. Studies on the production of daunomyci-none-derived glycosides and related metabolites inStreptomyces coeruleorubidus and Streptomyces peu-

cetius. Folia Microbiol. 22:275-285.9. Davis, H. L., and T. E. Davis. 1979. Daunorubicin and


adriamycin in cancer treatment: an analysis of theirroles and limitations. Cancer Treat. Rep. 63:809-815.

10. DiMarco, A., F. Arcamone, and F. Zunino. 1975. Dau-nomycin (daunorubicin) and adriamycin and structuralanalogues: biological activity and mechanism of action,p. 101-128. In J. W. Corcoran and F. E. Hahn (ed.),Antibiotics III: mechanism of action of antimicrobialand antitumor agents. Springer-Verlag, New York.

11. Essery, J. M., and T. W. Doyle. 1979. The synthesis ofe-rhodomycinone and carminomycinone-11-methyl-ethers. J. Antibiot. 32:247-249.

12. Kern, D. L., R. H. Bunge, J. C. French, and H. W.Dion. 1977. The identification of e-xhodomycinone and7-deoxy-daunorubicinol aglycone in daunorubicin beers.J. Antibiot. 30:432-434.

13. Kominek, L. A. 1967. Novobiocin, p. 231-239. In D.Gottlieb and P. D. Shaw (ed.), Antibiotics II: biosyn-thesis. Springer-Verlag, New York.

14. Komiyama, T., Y. Matsuzawa, T. Oki, T. Inui, Y.Takahashi, H. Naganawa, T. Takeuchi, and H.Umezawa. 1977. Baumycins, new antitumor antibioticsrelated to daunomycin. J. Antibiot. 30:619-621.

15. Kraska, A. R. 1979. Antineoplastic agents. Annu. Rep.Med. Chem. 14:132-145.

16. Lancini, G. C., and R. J. White. 1976. Rifamycin bio-synthesis, p. 139-153. In K. D. MacDonald (ed.), SecondInternational Symposium on the Genetics of IndustrialMicroorganisms. Academic Press, Inc., New York.

17. McCormick, J. R. D. 1967. Tetracyclines, p. 113-122. InD. Gottlieb and P. 1). Shaw (ed.), Antibiotics II: biosyn-thesis. Springer-Verlag, New York.

18. McGuire, J. C., B. K. Hamilton, and R. J. White. 1979.Approaches to development of the daunorubicin fer-mentation. Process Biochem. 14:2-5.

19. Neidle, S. 1978. Interactions of daunomycin and relatedantibiotics with biological receptors, p. 230-278. In P.G. Sammes (ed.), Topics in antibiotic chemistry, vol. 2.Ellis Horwood, Ltd., Chichester.

20. Oki, T. 1977. New anthracycine antibiotics. J. Antibiot.30:570-584.

21. Pandey, R. C., C. C. Kalita, R. J. White, and M. W.Toussaint. 1979. Process development in the purifica-tion of daunorubicin from fermentation broths. ProcessBiochem. 14:6-13.

22. Paranosenkova, V. I., and V. L Karpov. 1975. Thestudy ofrubomycin biosynthesis. Bioorg. Khim. 1:1755-1759.

23. Paranosenkova, V. I., and V. L Karpov. 1975. Studieson biosynthesis of carbohydrate fragment of rubomycin.Antibiotiki 21:299-301.

24. Paulick, R. C., M. L Casey, and H. W. Whitlock. 1976.A "3C nuclear magnetic resonance study of the biosyn-thesis ofdaunomycin from '3CH313CO2Na. J. Am. Chem.Soc. 98:3370-3371.

25. Remers, W. A. 1979. The chemistry of antitumor anti-biotics, vol. 1. John Wiley and Sons, New York.

26. Rozeneweig, M., C. DeSloover, D. D. Von Hoff, H. J.Tagnor, and F. M. Muggia. 1979. Anthracycline de-rivatives in new drug development programs. CancerTreat. Rep. 63:807-808.

27. Tanenbaum, S. W. 1967. Some acetate derived antibi-otics, p. 82-112. In D. Gottlieb and P. D. Shaw (ed.),Antibiotics II: biosynthesis. Springer-Verlag, New York.

28. Umezawa, H. 1977. Recent advances in bioactive micro-bial secondary metabolites. J. Antibiot. 30:S138-S163.

29. Vanek, Z., J. Tax, I. Komersova, P. Sedmera, and J.Vokuon. 1977. Anthracyclines. Folia Microbiol. 22:139-159.

30. Von Hoff, D. D., M. Rozencweig, and M. Slavik. 1978.Daunomycin: an anthracycline antibiotic effective inacute leukemia. Adv. Pharmacol. Chemother. 15:1-50.


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