CA1206901A - Biosynthesis of unnatural cephalosporins - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A process for converting a peptide precursor of the ACV type,in which the valine moiety may be replaced by any readily available amino acid,to an unnatural cephalosporin of the type

Description

~2C3 69~

BIOSYNTHESIS OF UNNATURAL CEPHALSOPORINS
Background of the In~ention F~eld o~ the invention Thi~ invention relates to a cell-free proce~
for producing cephalosporin antibiotic3 from peptide~ and derivative~ thereof. -The Prior Art The beta lactam family of natural products includes the penicillins:
RCONH

J N ~
CO;~Hcephalosporin~:
RCONH ~ S

O ~ ~ R
and cephamycins CO2H
~Me RCONH ~ R

in which the beta-lactam ring is fused to a five or six membered sulfur-containing ring; together with clavulanic .
acid OH

:' o C02H

in which the beta-lactam i5 fused to a five membered oxygen-containing ring;

3~20690~
I the carbapenems R~ ~ Rl J~N~
o C02H

in which the beta-lactam is fused to a five membered carbon containing ring;
and the RCONH P~ OH RCONH~

which are monocyclic compounds.
Althouqh there are many naturally occurring members of this famlly, only two can be used directly in medicine without structural change. These are penicillin G, the penicillin in whlch R = benzyl, and clavulanic acid. All other clinically important beta-lactam compounds have been prepared from one or other of the natural products by structural change. For many years the changes have been generaly effected by substitution around the peripheries of the various ring systems and not in the rlng systems themselves. Since 1974, however~ effoxts have been concentrated on nuclear modification of a beta-lactam natural product. Such efforts have generally re~ulted in complex chemical processes containlng upwards of 16 steps with the result that the products are obtained in generally low yield and at extremely high cost. Moxalactam~, for example, a third generation cephalosporin, is approximately 1~069~

five times more expensive than cephalothin, a first genera-tion cephalosporin; and cephalothin is, in turn, approximately fifty times more expensive than ampicillin, a semi-synthetic penicillin (Drug Topics Red Book 1981).
Attention has therefore turned to alternative methods of synthesis, and in particular to microbiological methods. Cell-free syntheses of penicillins and the related cephalosporins are known in the art and attention is directed to U.S. Patent No. 4,178,210 issued December 11, 1979 to A.L. Demain et al, which teaches conversion only of the D-form, penicillin N, to a cephalosporin compound. In U.S.
Patent No. 4,248,966 issued February 3, 1981, A.L. Demain et al teach the production of isopenicillin derivatives, in a cell-free system using an extract from Cephalosporiumacremonium, rom a tripeptide composed of unsubstituted or B sub~tituted D-valine, unsubstituted or substituted L-cysteine, and L-a-aminoadipic acid or its analogs. Freezing of the cell-free extract resulted in inactivation of certain enzymes ~o that conversion did not proceed past the isopenicillin stage.
In U.S. Patent 4,307,192 issued December 22, 1981, A.L. Demain et al teach the use of a fresh (i.e. not frozen) cell-free extract of C. acremonium so as to preserve the racemase ~epimerase), agent or agents necessary for the conversion of isopenicillin N to penicillin N, a necessary intermediate step in the proce~s for conversion of L-aminoadipyl-L-cysteinyl-D-valine (abbreviated to LLD in the reference but hereinafter ACV), via an oxidative cyclization step to isopenicillin N, epimerization to penicillin N and oxidative ring expansion to de~acetoxycephalosporin C.

12~6gOl H SH
H2N ~--D' N~ ~ CH3 (1) COOH O N~
H - COOH
5 L-aminoadipyl L-cysteinyl D-valine oxidative cyclization ~ H
H2N ~ N ~ S ~ CH3 (2) COOH J~N~
a- ~OOH
_ isopenicillin N
1 epimerize and ring expand H

H2N ~ ~ (3) D-aminoadipyl COOH
desacetoxycephalosporin C

The activity of the racema~e agent in a cell-free extract of C. acremonium was first recognized by Konomi et al, Biochem.
J. Vol. 184, p 427-430, 1979, and confirmed by Baldw~n et al, Biochem J. Vol. 194, 649-651, 1981, and Jayatilake et al, Biochem. J. VolO 194, 649-647, 1981 who also recognized the extreme lability of the racemase agent so that recovery of the racemase agent per se is believed to be impossible. The lability of the racemase agent is believed to preclude use of ~2~6~0~

cell-free extracts of C. acremonium for high yield commercial production of cephalosporins from peptide precursors.
Since about 1978, 6-aminopenicillanic acid has been produced commercially by the deacylation of benzyl penicillin using immobilized penicillin acylase (Proc. lST. European Congress of Biotechnology, Dechema Monographs, Volume 82, 162, 1~78), and numerous other reactions nave been suggested using immobilized biomaterials such as enzymes (Enzyme Engineerlng Vol. 6, 1982, Plenum).

It is, therefore, an object of the present invention to provide an integrated cell-free process for producing a cephalosporin compound from a peptide of the general ormula ~ S~ H
1~ 2N ~ ~ ~CH 2 R

J ~ ' H COOH

where Rl is hydrogen, a lower alkyl or functionalized carboxylic group, and R2 is hydrogen or a lower alkyl group, using stable cell-free extracts from prokaryotic organisms.
It is another object of the present invention to provide immobilized cell-free extracts from prokaryotic orqanisms 80 as to permit continuous production of cephalo-spoxins.
These and other objects of the invention will be apparent from the following description of the preferred embodiments.
Summary of_the Invention It has now been discovered that certain cell-free extracts of prokaryotic organisms such as Streptomyces ~:~IJ6~01 lavuligerus, StreP~omyces cattl~ya and Stre~tomyces lipmanii, can be separated into three fractions by a three stage treat-ment to provide three stable and separate enzymes:
(a) epimerase (MW approx. 60,000) which may be used, for example, to epimerize isopenicillin N to penicillin N;
~ b) cyclase (MW approx. 36,500) which may be used, for example, to cyclize ACV to isopenicillin N; and (c) ring expansion enzyme (MW approx. 29,000) which may be used, for example, to ring expand penicillin N to desaceto~y-1~ cephalosporin C. It has also been discovered that the threeenzymes may be immobilized on a suitable column material and employed for the continuous production of cephalosporins.
Thus, by one aspect of this invention there is provided a process for producing unnatural cephalosporins 15 of the formula J

D-Aad-NH ~ ~ 1 O ~ 2 where Rl = ~l, lower alkyl, or functionalized carboxylic group and R2 = H or lower alkyl and derivatives thereof, compri~ing reacting a starting material comprising L-a-aminoadipyl-~ cysteinyl-D-valine and analoys thereof in 25 which an amino acid is substituted for the valine moiety, with cyclase, epimerase and a ring expansion enzyme isolated from a cell-free extract of a prokaryotic organism for sufficient time and in the presence of sufficient co-factors to produce said cephalosporins.

1;~069(~

By another aspect of this invention there is provided a process for isolating cyclase, epimerase and a ring expansion enzyme from a cell-free extract of a prokaryotic organism comprising:
(a) precipitating contaminating proteins from said cell-free extract by addition of ammonium sulfate to 40%
saturation;
(b) separating precipitated protein from a supernatant;
(c) adding further ammonium sulfate to 70% saturation to said supernatant thereby precipitating desired said enzymes;
(d) suspending said precipitated enzymes in pH 7 buffer; and (e) chromatographically separating the desired enzymes from each other.
By yet another aspect of this invention there is provided an immobilized enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV and analogs thereof to desacetoxycephalosporin and the respective analogs thereof, comprising an epimerase having a molecular weight of about 60,000 a cyclase having an MW of about 36,500 and a ring expanRion enzyme havlng a molecular weight of about 29,000, derived from a prokaryotlc organism, immobilized on a diethylaminoethyltrisacryl chromatographic resin.
Brief Description of the Drawings Figures la - lf are HPLC chromatographs of reaction mixtures at O mins, 15 mins, 30 mins, 45 mins, 60 mins and 75 mins, respectively.

~20~ii9~31 Descr ption of the Preferred Embodiments In the following description, reference will be made particularly to the conversion of ACV (1) to desacetoxycephalo-sporin C which isuseful as an antibiotic as such or as a starting compound for the production of cephalosporin antibiotics such as Cephalexin~. It will be appreciated, however, that the 1~ biochemical techniques of the present invention are equally applicable to other starting materials and it is within the purview of the present invention to substitute the valine 10 moiety in the preferred ACV starting material with any of the readily available amino acids for conversion to the analogous cephalosporins which are useful as antibiotics, or as starting materials for antibiotics such as Ceftizoxime~- Thus, the starting material may be regarded ~.~ as having the general formula ~4) H2N ~ ~ ~ z (4) H COOH

where Rl and R2 are as hereinbefo.re described.
The amino acids which may be used thus include:

12069(~

. .
Rl R2 Compound H CH3 Valine H H a-aminobutyric acid H C2H5 allo isoleu~ine CH3 CH3 isoleucine COOH H glu~amic acid C~2NH-CNH2 H arginine NH
CONH2 H glutamine CH2CH2NH2 H lysine . . , ~ . _ The naturally-occurring beta-lactam compounds are formed as secondary metabolites of both eukaryotic and prokaryotic organisms. Simply stated, a eukaryote i5 a higher life form, and it has a moxe complicated cell structure, which restricts the types of compounds that can be synthesized or metabolized. Examples of eukaryotic beta-lactam-producing organisms are the fungi Penicillium chrysogenum and Cephalosporium acremonium. A prokaryote, on the other hand, is a lower, earlier, life form, with a more primitive cell ~tructure, whlch allows a greater variety of chemical transformations to take place. Thls suggest~, again simply, that prokaryote~ are more versatile at organic synthesis than are eukaryotes, provided that this versatility can be understood and controlled. Examples of prokaryotic beta-lactam-producing organisms are the actinomycetes Streptomyces clavuli~erus, S. cattleya and S. lipmanii.
As an illustration of the differing capabilities lZ069(31 of eukaryotic and prokaryotic beta-lactam-producing organisms, P. chrysogenum, a eukaryote, synthesizes ACV and converts this peptide to penicillin as the only stable beta-lactam-containing end product. C. acremonium, also a euk~ryote, synthesizes the 5 same tripeptide and converts this peptide sequentially to penicillin and cephalosporin. In contrast, the prokaryote S. clavuligerus synthesizes penicillin, cephalosporin and cephamycin from one amino acid-containing precursor and, at the same_time, clavulanic acid, from a different 10 precursor. The prokaryote S. cattleya synthesizes penicillin and cephalosporin from one precursor and, at the same time, the carbapenem,thienamycin, from a different precursor.
S. clavuliqerus, for example, is a well known micro-organism and several strains are available, on an unrestricted 15 basis, from the Northern Regional Research Laboratory, Peoria, Illinois, U.S.A. under the name NRRL 3585, among others. Other prokaryotic organisms, as described above, are equally freely availableO The NRRL 3585 organism must be cultured in a medium and under condition~ conducivs to 20 the production of ~-lactam compounds, as described in more detail hereinafter.
There are several methods for cell breakage prior to obtaining a cell-free extract, including French pressure cell,Omnimixer-plastic beads and the preferred sonication.
The preferred treatment comprises sonication for 30 seconds on 48 hour washed cells, followed by centrifugation. The supernatant from this treatment is designated "crude cell-free extract". The crude extract may be separated into three 12~69(~1 enzyme fractions in a three stage treatment. In the first stage, contaminating proteins are precipitated by addition of ammonium sulfate to 40~ saturation, and separated from the supernatant by centrifugation or other conventional means.
5 Addition of more ammonium sulfate to 70~ saturation precipi-tates the desired enzymè activities. The resulting pellet, suspended in pH 7 buffer is termed "salt-precipitated cell-free extract" (SPCFX). This SPCFX retains all the desired enzyme activities, and shows reduced baseline contamination 10 in HPLC assay~. In the second stage, the epimerase (isopenicillin N ~ pencillin N) (MW 60,000) is cleanly separated from the cyclase (ACV ~ isopenicillin N) (MW 36500) and ring expansion (penicillin N ~ desacetoxycephalosporin C) (MW ZO,OOO) enzymes, by gel filtration chromatography of the 15 SPCFX on, for example, Sephadex~ G-200 (Pharmacia, Sweden).
J In the third stage, the cyclase and ring expansion enzymes are separated by ion exchange chromatography on, for example, DEAE Trisacryl resin (sold by ~.K.B., Sweden). A 100-fold purification of the cyclase i5 achieved in this manner.
20 Thus, for the fir~t time three distinct enzyme reagents each having a different enzymatic activity and physical characteristics (e.g. different molecular weights) and which are stable over an extended period of time (of the order of months) under suitable storage conditions of 25 temperature and pH ~preferably about -20C and pH7) have been prepared. The enzymes may be ~tored and used quite separately or may be stored and used as a mixture or immobilzed on a column as required.
Analogous treatment using SPCFX from C. acremonium yields the cyclase and ring expansion enzymes only. As noted 12069(~
above the epimerase is entirely absent due to its extreme lability.
Following prepara~ion of the three enzymes, ACV
dimer or an analog thereof as described above, may be reacted there~ith under aerobic conditions, in the presence of the required co-factors such as ferrous ions usually in the form of ferrous sulfate, an antioxidant such as ascorbic acid, a reducing agent such as dithiothreitol (DTT) and a cosubstrate such as a-ketoglutarate, for sufficient time at about 20C and at a suitable pH of about 7 in either batch or continuous mode to produce desacetoxycephalosporin C or an analog thereof.
Example 1 Production of SPCFX
(a) Culture of S. clavuliqerus Stre~tom~ces clavuli~rus NRRL 3585 was maintained 4 on a sporulation medium composed of tomato paste, 20g; oat-meal, 20g; agar, 25g, in 1 litre of distilled water, pH 6.8.
Inoculated plates were incubated 7~10 days at 28C.
Spores were scraped off into sterile distilled water (5ml/
plate) and used to inoculate, 2% v/v, 25ml/125ml flask, seed medium of the following composition: glycerol, lOml;
sucrose, 20g; soy flour, 15g; yeast extract, lg; tryptone, 5g; K2HPO4, 0.2g in 1 litre of distilled water, pH 6.5.
Inoculated seed medium was incubated 3 days and used to inoculate, 2~ v/v, 100 ml amounts of production medium in 500 ml flasks. Production medium consisted of soluble starch, lOg; L asparagine, 2g; 3-N-morpholinopropane-sulfonic acid, 21g: MgSO4.7H2O, 0.6g; K2HPO4, 4.4g; FeSO4.7H2O, lmg;
MnC12 4H20, lmg; ZnS04.7H20, lmg; and CaC12.2H20, 1.3mg 120~9~D1 in l litre of H20, pH 6.8. Inoculated production medium was incubated 40-48h and the cells were then collected by filtration andused to prepare cell-free extracts. All incubations were at 27C on a gyrotory shaker (250rpm, l9mm eccentricity).
(b) Preparation of Cell-Free Extracts Cell-free extracts were prepared by washing 40-48h cells of S. clavuli~erus in O.OSM Tris-HCl buffer, pH 7.0+0.1mM dithiothreitol (DTT) (lOOml/lOOml culture).
lO Washed cells were resuspended to 1/10 of the original culture volume in the same buffer and disrupted by sonication in an ice water bath for 2x15 sec at maximum intensity (300 watts, Biosonik III, Bronwill Scientific). Broken cell suspensions were centrifuged lh at lOO,OOOxg. All cell-free extracts were stored frozen at -20C.
Salt-precipitated cell-free extract was prepared by gradual addition of ~treptomycin ~ulfate to cell-free e~tract with gentle stirring at 4C to a final concentration of 1%, w/v. After 15 min at 4C, precipitated nucleic acid 20 was removed by centrifugation for 15 min at 15~000xg. Solid ammonium sulfate was then gradually added to the supernatant with gentle stirring at 4C until 40% saturation was reached.
After 15 min at 4C the suspension was centrifuged as above and the pellet discarded. Additional ~mmonium 8Ul fate w~s then added to the supernatant, as above, until 70% saturation was reached. Following centrifugation, the pellet was resuspended to its original volume in 0.05M Tris-HC1 buffer pH 7.0 containing O.lmM DTT. The enzyme solution was then concentrated to l/lO of the original volume by ultrafiltration 12069(~1 with an Amicon~PM~10 filter.
Cyclization Assay System Cyclization activ;ty of enzyme preparations was measured in reaction mixtures containing: bis-~ -(L-a-amino-adipyl-L-cysteinyl-D-valine) (ACV~2 0.306mM, DDT 4mM, Na ascorbate 2.8mM, FeSO4 45~ M, tris-HCl buffer 0.05M, pH 7.0, enzyme preparation 0.03-0.3ml, final volume 0.4ml. Reaction mixtures were incubated at 20C for up to 4 hours and stopped by cooling on ice or by the addition of 0.4ml methanol.
Ring Expansion Assay System Ring expansion activity was followed using the cyclization assay system described above but supplemented with ATP 0.5mM, a-ketoglutarate lmM, KCl 7.5mM, and MgSO4 7.5mM. Total volume and incubation conditions were the same as for the cyclization assay.
Example 2 Separation of Enzyme Fractions (a) Separation of Epimerase by Gel Filtration Chromatography of SPCFX

2.5ml of SPCFX was applied to a Sephadex~ G-200 superfine column (2.5cm x 40cm) which had been equilibrated in 0.05M Tris-HCl buffer pH 7.0 containing O.lmM DTT. The column wa~ eluted with the same buffer and 2.5ml fractions were collected. Fractions were monitored for protein by mea8uring W absorption at 280nm, and were assayed for ~5 cyclase, epimera~e and ring expansion activities. Active fractions were pooled and concentrated by ultrafiltration using an Amicon~ PM-10 filter.

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(b) Separation of Cyclase and Ring Expansion Enzyme by Ion Exchange Chromatography of SPCFX
2.5ml of SPCFX was applied to a diethylaminoethyl (DEAE) -Trisacryl~ column (1.6 x 25cm) which had been equili-brated in 0.1M Tris-HCl buffer pH 7.0 containing O.lmM DTT.
The column was washed with 50ml of the above buffer and then eluted with a linear gradient of 150ml each of initial starting buffer vs 0.4M Tris-HCl buffer pH 7.0 containing O.lmM DTT.
2.5ml fractions were collected and monitored for protein content by measuring UV-absorption at 280nm. The ring expansion enzyme eluted at about llOmM Tris-chloride, the cyclase eluted at about 150mM Tris-chloride and epimerase at about 175mM
Tris-chloride. Fractions were also monitored for conductivity and were assayed for cyclization, epimerase and ring expansion activity. Actlve fractions were pooled and concentrated and desalted by ultrafiltration using an Amicon~ PM-10 filter. Use of aTris-chloride gradient is believed to better preserve enzyme activity as compared to the more usual NaCl gradient.
Both separations were performed at 4C, and the enzyme products were stored at -20C or lower as they were found to lose activity overnight at room temperature.
ExamPle 3 Preparation of Cell-Free Extract for Immobilization Cell-~ree extracts were prepared by washing ~0-48h cells of S. clavuli~erus in 0.05M Tris-HCl buffer, pH 7.0 +
O.ln~l dithiothreitol + O.OlmM ethylenediaminetetracetic acid (EDTA buffer)(lOOml/lOOml culture). Washed cells were resus-pended to 1/10 of the original culture volume in EDTAbuffer and disrupted by sonication in an ice water bath for 2x15 sec 12~69~

at maximum intensity (300 watts, Biosonik III, Bronwill Scientific). sroken cell suspensions were centrifuged lh at 100,000xg. All cell-free extracts were stored at -20C.
Salt-precipitated cell-free extract was prepared by gradual addition of streptomycin sulfate to cell-free extract with gentle stirring at 4C to a final concentration of 1%, w/v. After 15 min at 4C, precipitated nucleic acid was removed by centrifugation for 15 min at 15,000xg. Solid ammonium sulfate was then gradually added to the supernatant 1~ with gentle stirring at 4~ until 40% saturation was reached.
After 15 min at 4C the suspension was centrifuged as above and the pellet discarded. Additional ammonium sulfate was then added to the supernatant, as above, until 70% satuxation was reached. Following centrifugation, the pellet was resuspended to its original volume inEDTA buffer. The enzyme solution was then concentrated to 1/10 of the original volume by ultrafiltration with an Amicon~ PM-10 filter.
Immobilization of Salt-Precipitated Cell-Free Extract DEAE-trisacryl resin was loaded into a column 0.4 x 5.8cm ~packed bed volume, lml), washed with 3 x 2ml of the sameEDTA buffer, and allowed to drain to dryness by gravity.
One milliliter of thesalt-precipitated cell-free extract above was applied to the column. The effluent wa~ collected and reapplied to the column twice to ensure complete enzyme loadlng. The column was washed with 2 x lml of the sameEDTA
buffer, drained dry and centrifuged for 3 min. at 500xg to remove excess buffer. This immobilized enzyme reactor was stored at 4C when not in use.

120~

Example 4 Preparation of ACV and Related Compounds N-BoC-S-trityl-L-cysteine was coupled with the benzhydryl ester of D-valine to give a fully protected dipeptide~5).
STr BoCNH ~
(5) N--~

A 15 minute treatment with anhydrous formic acid at room temperature led to crystalline, partially protected peptide (6).
5Tr H2N ~ (6) N ~
H CO2CHPh2 Conversion to ully protected ACV (7) STr BoCNH ~ NH
O N ~
CO2CHPh2 C2CH 2 was achieved by coupling peptide (6) with (8) BcCNH ~ CO2H (8) C02CHPh2 Deprotection of (7) was achieved in two stages:

~Z~ti9~1 (a~ removal of the trityl group, with iodine in methanol;
(b) removal of all other protecting groups by overnight treatment with formic acid, leading to ACV disulfide ~9).
The ACV is best stored in this form and may be readily con-S verted to ACV (1), as needed, with dithiothreitol. Thissynthesis is readily adaptable to systematic modifications of the aminoadipyl moiety and compounds such as N-acetyl-ACV and its cyclic analog N-acetyl isopenicillin N, may be similarly prepared from N-acetyl-L-a-aminoadipic acid alpha-benzhydryl ~0 ester as the startlng material.
Example 5 Preparation of ~-(L-a-aminoadipyl)-L-cysteinyl-D-alloisoleucine ~ACI) RlNH ~ ~ N

This compound was prepared from L-a-aminoadipic acid, ~-cysteine and ~-alloisoleucine, a~ de~cribed for the synthesis of the natural cephalosporin precursor ~-(L-a-aminoadipyl)-L-cysteinyl-D-valine by S. Wolfe and M.G. Jokinen, Canadian Journal of Chemistry, Volume 57, page~ 1388-1396, 1979.
Thia led, auccessively, to the fully protected tripeptide (Rl = t-butoxycarbonyl, R2 = benzhydryl, R3 - trityl), m.p.
91-93 ~ethyl acetate-petroleum ether), Rf 0.54 (methylene chloride-ethyl acetate, 9:1; yellow with palladium chloride), the detritylated compound (Rl = t-butoxycarbonyl, R2 =
benzhydryl, R3 = disulfide), m.p. 114-116 (methanol), Rf 120~g~1 0.76 (methylene chloride-ethyl acetate, 4:1, yellow with palladium chloride), and the completely deprotected compound (Rl = R2 = H, R3 = disulfide), Rf = 0.22 (methyl ethyl ketone-water-acetic acid, 4:1:1, purple with ninhydrin), lHmr (D2O) 5 ~: 0.90 (3H, d, 6Hz), 0.91 ~3H, 5, 7Hæ), 1.30 l2H, m), 1.73 (2H, br t), 1.88 (2H, br t~, 2.01 (lH, m), 2.39 (2H~ br t),

3.00 (lH, q, 8, 15Hz), 3.16 (lH, q, 5, 15Hz), 3.76 (lH, t, 6Hz), 4.40 (lH, d, 4Hz), 4.73 (lH, br s). The latter compound is converted into the active form (Rl = R2 = R3 = H) upon 1~ treatment with dithiothreitol.

Example 6 PreParation of ~- (L-a-aminoadipyl)-L-cysteinyl-D-a-amino-butyrate (ACAb) ~ SR3 RlNH ~ N ~
C2R2 ~ ~CH2CH3 H

This compound was prepared, as in Example 5, via the intermediates Rl = t-butoxycarbonyl, R2 = benzhydryl, R3 = trityl: Rf 0.63 (toluene-ethyl acetate, 2:1); Rl = t-butoxycarbonyl, R2 = benzhydryl, R3 = disulfide: Rf 0.48 (toluene-ethyl acetate, 2:1); and Rl = R2 = H; R3 = disulfide~
Rf = 0.1 (methyl ethyl ketone-water-acetic acid, 4:1:1), Hmr ~D2O) ~: 0.91 (3H, t, 7.5Hz), 1.59-2.00 (~H, m), 2.41 (2H, t, 7HZ), 3.97 (lH, q, 8.5, 14Hz), 3.21 (lH, q, 5, 14Hz), 3.75 (lH, t, 7Hz), 4.18 (lH, q, 5, 8.5Hz), 4.73 (lH, m).

This last compound is converted into the active form (Rl =

R2 = R3 = H) upon treatment with dithiothreitol.

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Exam~e 7 Cyclization of ACV
To 0.4ml of reaction mixture were added O.9mM of ACV
dimer as produced in Example 4, 50.0mM Tris-HCl pH 7.0 buffer and a mixture of the three enzymes as produced in Example 1 from a cell-free extract of S. clavuligerus~ together with 45.0 ~ M ferrous sulfate and 2.8mM ascorbic acid as optimized amounts of essential co-factors. DTT was added in excess of the amount required to reduce ACV dimer to ACV monomer.
The reaction was continued for approximately 2 hours at 20C
and then terminated by addition of 0.4ml methanol to precipitate protein. It was found, by bioassay and HPLC
procedures (described in more detail hereinafter) that the peptide had been converted to a mixture of isopenicillin N
and penicillin N. Ring expansion to a cephalosporin did not occur. The experiment was repeated with the addition of lmM
of a standard oxygenase type enzyme co-factor, alpha-ketoglutarate, and in this case it was found that the ACVwas converted to desacetoxycephalosporin C.
Example_8 The procedures of Example 7 were repeated using L-aspartyl, L-glutamyl, D-a-aminoadipyl, adipyl, glycyl-L-a-aminoadipyl and N-acetyl-L-a-aminodipyl-containing peptides.
It was found that the L-aspartyl, L-glutamyl and D-a-amino-adipyl-containing peptides did not cyclize. Cyclization was observed with adipyl, glycyl-L-a-aminoadipyl and N-acetyl-L-a-aminoadipyl-containing peptides. The adipyl compound gave ca 20~ cyclization to the corresponding penicillin, carboxybutylpenicillin,but SPCFX converted the glycyl and 12069~1 N-acetyl compounds to penicillin N and isopenicillin N, via an initial deacylation of these peptides to ACV. Purified cyclase from S. clavuligerus did not cyclize the glycyl-L~a-aminoadipyl-containing peptide. These results suggest that the enzymatic conversion of an ACV analog to an unnatural cephalosporin nucleus requires (i) a ~-L-a~aminoadipyl side chain and (ii) an enzyme system containing the epimerase. A
prokaryotic system is, therefore, required. Modification of the valinyl moiety, as noted above, has been considered in detail. Substrates modified in the valinyl moiety such as:
Sll L-Aad-NH ~

O ~CH 2 R 1 where Rl i8 H, a lower alkyl or functionalized carboxylic

4 group; and R2 is H or a lower alkyl group may be cyclized with carbon-sulfur bond formation with retention of configuration at the beta carbon of the valine analog, leading to isopenicillin N analogs of the type:

L-Aad-NH ~ ~ CH2Rl I 1~ (10) ~ - N
O C02~

Pollowing epimerization to penicillin N analogsof the type:

D-Aad-N W S ~ ~CH2Rl L N J~ ( 1 1 , ~ L2~9V~
ring expansion leads to cephalosporin analogs of the type:
D-Aad-NH ~ ~ ~1 R (12) with transfer of the beta carbon atom attached to C2 of ~11) into C2 of the six membered ring.
Example 9 The penicillin and cephalosporin~forming ability of the immobilized en2yme reactor as prepared in Example 3 was demonstrated using reaction mixtures containing: bis- 8 -(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV)2 0.306mM, dithio-threitol 4mM, Na ascorbate 2.8mM, FeS04 45 M, a-ketoglutarate 1~2, KCl 7.SmM, MgS04 7.5mM, in TDE buffer, final volume 2.Oml.
2ml of the reaction mixture was applied to the immobilized enzyme reactor by means of a peristaltic pump operating at 40ml/h. Effluent was collected into a 13xlOOmm test tube from which the original reaction mixture was pumped, and therefore was recycled continuously through the enzyme reactor. The enzyme reactor was operated at 21C and 20 ~1 aliquots were removed at 15 minute time intervals for analysis for antibiotic formation. (Table I).
TABLE I
BIOASSAY OF REACTION MIXTURES

Sample Zone of Cephalosporin C
Time Inhibition "equivalents"
~min) (mm) (~?
O O O
.031 19.5 .086 18.5 .062 21.5 .136 21.5 .136 - 2~ -12069~1 * One microgram of cephalosporin C "equivalent" gives a zone of inhibition equal to that produced by 1 ~g of actual cephalosporin C.
Antibiotic levels increased for 60 min. before leveling off.
Since the bioassays were performed in the presence of penicillinase, the antibiotic activity detected was due to cephalosporin antibiotics only. We show hereinafter that cephalosporins can also arise from ACV via the production of the penicillin intermediates, isopenicillin N and penicillin N. The immohilized enzyme reactor similarly must form cephalosporins by the sequential cyclization, epimexiæa~ion and ring expansion of the ACV peptide substrate.
Analysis of reaction mixture time samples by HPLC
is shown in Figure l(a-f~. With increasing reaction time the ACV peak ~13.8-14.26 min) declined while a new peak at 5.2-

5.3 min. increased. The new peak is due to a mi~ture ofisopenicillin N, penicillin N and desacetoxycephalosporin C.
This peak decreases in area gradually from 60 min onwards due to the further oxidation of desacetoxycephalosporin C to desacetylcephalosporin C. Desacetylcephalosporin C has antibiotic activity, so bioassay results remain constant, but this compound elutes with a retention time of 2.2-2.5 min.
under the HPLC conditions used in this study.
Based on these studies we conclude that the immobilized enzyme reactor is converting ACV via a multi-step reaction lnvolving penicillin intermediates into cephalosporin products. Since previous studies have demonstrated that ~-ketoglutarate is absolutely required for the ring expansion of penicillinstocephalosporins, omission of ~-ketoglutarate from reaction mixtures should stop the reaction at the level of penicillin N.

1~)69~

_ ample 10 Bio~ y of Beta-lactam Compounds Antibiotic in reaction mixtures was estimated by the agar diffusion method. Cyclization reaction mixtures were bio-assayed using Micrococcus luteus ATCC 9341 and Escherichia coli Es~ as indicator organisms. Ring expansion reaction mixtures were bioassayed using E. coli Ess as indicator organism in agar __ plates supplemented with penicillinase at 2x105 units/ml.
Hi~h Performance Li~uid Chromatography (HPLC) Methanol inactivated reaction mixtures (from Examples 7 and 8) were centrifuged at 12,000xg for 5 min to remove precipitated protein before analysis. Reaction mixtures from Example 9 were examined directly. The chromatographic equipment used was: M-6000A pump, UK-6 injector, M-480 variable wave-length director, M-420 data module and Bondapak-C18 column (Rad Pak A ln a Z module) as stationary phase. All equipment was from Waters Scientific Co., Mississauga, Ontario. The mobile phase consisted of methanol/0.05M potassium phosphate buffer, pH 4.0 (5/95). The methanol content of the mobile phase depended upon the particular separation and the source of the material e.g. Examples 7 and 8 or Example 9. A short precolumn (packed with BondapakC18/Corasil) was used to guard the main column.
UV-absorbing material was detected at 220nm at a sensitivity of 0.02 AUFS.
ExamPle 11 CYcllzation and Rin~ E ~ Unnatural Peptide Substrates The procedure of Example 7 was repeated with ACV
analogs in which valine was replaced by alpha-aminobutyric acid tRl = R2 = H) and allo-isoleucine (Rl = H~ R2 = C2H5)~ as follows lZC~901 (AC-aminobutyrate)2 (ACAB)2 and (AC-alloisoleucine)2tACIJ2 were dissolved in water, neutralized~ and lyophilized in 0.1 and l.Omg amounts. These peptides were then used as substrates in cyclization and ring expansion assays as follows: One hundred micrograms of (ACV)2 from Example 6 was used as substrate in a cyclization and a ring expansion assay system using O.lml of salt-precipitated cell-free extract as enzyme source in each case. (Final concentration of (ACV)z is 0.306mM). Identical cyclization and ring expansion assays were set up in which 100 ~g (ACAB~2 or l.Omg (ACI)2 replaced the (ACV)2 as substrate and 0.3ml of salt precipitated cell-free extract was used as enzyme source. No substrate controls were also prepared. The reaction mixtures were incubated for 2h at 20C. At the end of incubation 20 ~1 amounts of the cyclization reaction mixtures were bioassayed versus M. luteus and E. coli Ess; 20 ~1 amounts of the ring expansion reaction mixtures were bioassayed versus E. coIi Ess plus and minus penicillinase.
The remaining reaction m~xtures were then mixed with an equal volume of methanol and centrifuged in preparation for HPLC analysis.
Cyclization and ring expansion reaction mixtures containing (ACI)2 as substrate and also the no substrate controls were analysed using a mobile phase of 20% Methanol/
25- 80% KH2P04, 0.05M adjusted to pH 4.0 with H3P04. Twenty microlitre amounts were injected and eluted at a flow rate of 2ml/min.
Cyclization and ring expansion reaction mixtures containing (ACV)2, (ACI)2 and (ACAB)2 as substrates and also lZ~169(~
the no substrate controls were then analysed using a mobile phase of 5~ Methanol/95~ KH2P04, O. 05M adjusted to pH 4.0 with H3P04. Twenty microlitre amounts were injected and eluted at a flow rate of-2ml/min for 5 min rising to 3ml/min 5 by 7 min and remaining at 3ml/min for the rest of the analysis time.
Results and Discussion Results of biological assays of the reaction mixtures from Examples 7 and 8 are seen in Table 2. Cyclization of (ACV)2 re-10 sults in formation of a bioactive product. The zone size pro-duced on E. coli Ess agar plates (2~.Omm) is equivalent to the zone size which a cephalosporin C solution at 29.3~ g/ml would produce. Cyclization of (AcAs)2 produces a bioactive product with antibiotic activity equivalent to a 0.9 ~g/ml 15 solution of cephalosporin C against E. coli Ess. Similarly q cyclization of (ACI)2 produces a bioactive product with antibiotic activity equivalent to a 4.85 ~g/ml solution of cephalosporin C against E. coli Ess. Ring expansion assays containing (ACV)2 result in formation of penicillinase-20 insensitive antibiotlc which produces a zone size on E. coli Ess ~ penicillinase plates (22mm) equivalent to a 7.6~ g/ml ~olution of cephalosporin C. Ring expansion assays containing (ACAB)2 do not form penicillinase-insensitive antibiotic nor do they form any antibiotic affecting E. coli 25 E~s. Since antibiotic activitv was seen in (ACAB)2-containing cyclization assay system3, this implies one of two things:
1. The additional components in a ring expansion reaction mixture inhibit cyclization of ACAB, or 2. Ring expansion assays containing (ACAB)2 produce a cephalosporin which does 12069(~i not affect E . coli Ess . Ring expansion assays containing (ACI)2 for~ penicillinase-insensitive antibiotic which produces a zone size on E. coli Ess + penicillinase plates (12.5mm) equivalent to a 0.9 ~g/ml solution of cephalosporin C.
HPLC analysis of cyclization reaction mixtures containing (ACI)2 as substrate was carried out with a mobile phase of 20% methanol/80% KH2PO4, 0.05M pH 4.0~ When compared with the no substrate control, (ACI)2 containing reaction mixtures showed a new peak at 2.66 min. Analysis of ring expansion reaction mixtures under the same conditions did not show any new peak because the region around 2.66 min was obscured by UV absorbing material (a-ketoglutarate), present in both the no substrate control and in the test.
When the mobile phase was changed to 5% Methanol/
95% K~2PO4, 0.05M pH 4.0, cyclization reaction mixtures containing (ACI)2 now showed the new peak to be at 11.26 min.
Ring expansion reaction mixtures cont~ining (ACI)2 showed the new peak to be somewhat (~ 50%) reduced in size with a smaller peak running ju~ in front of the main peak.
This is expected since cephalosporirls typically run close to, but just in front of, their corresponding penicillin.
Cyclization reaction mixtures containing (ACAB)2 as substrate showed a new peak in the region of 2~33 min.
The corresponding ring expansion reaction mixtures also show their new peak at 2.3 min. Since ring expansion reaction mixtures do not show bioactivity despite the presence of this new peak, we conclude that the cephalosporin is being formed but is of lower antibiotic activity against E. coli Ess than 12069C~1 the corresponding penicillin. Analysis of (ACV)2 containing reaction mixtures shows that the natural product formed in cyclization reaction mixtures, a mixture of isopenicillin N
and penicillin N [(iso)penicillin N], elutes at a retention time of 5.23 min. Ring expansion results in conversion of some of the penicillin to desacetoxy cephalosporin C which runs with a retention time of 4.76 min and does not separate from (iso)penicillin N under these conditions.
Based on these studies, it is concluded that salt precipitated cell-free extract from S. clavuli~erus, can cyclize (ACI)2 and (ACAB)2 to form penicillins, in addition to being able to cyclize the natural substrate, (ACV)2. The unnatural penicillins so formed have chromatographic characteristics distinct from (iso)penicillin N and there is no evidence for production of ~iso)penicillin N in reaction mixtures containing unnatural peptide substrates.
~ The same enzyme preparation can cause ring expansion of the penicillin formed from (ACI)2, resulting in formation of a new cephalosporin.
Table 2 Zone of Inhibition(mm) Substrate and E~ coli E. coli Ess Assay Condltion~M. luteus Ess~ penicillinase ~ACV)2 cyclization29.0 28.0 (ACV)~ ring expansion 28.5 22.0 25 (ACAB)2 cyclization 8.0 12.5 (ACAB)2 ring expansion 8.0 0 (ACI)2 cyclization13.0 20.0 ~ACI)2 ring expansion 20.0 12.5 no substrate cyclization + + +
no substrate ring expansion + + +

_ 28 _ lZ069~'1 Example 12 The procedure of Example 9 was repeated by passing two reaction mixtures each containing lmg of ACV (from Example 4) through a DEAE trisacryl column ~2ml bed vol.) containing 2ml of immobilized SPCFX (prepared as in Example 3). Each reaction mixture was cycled through the column for 1.5 hours at 40 ml per hour. This resulted in approximately 90~ conver-sion of ACV into a mixture of isopenicillin N, penicillin N, desacetoxycephalosporin C, and desacetylcephalosporin C as determined by HPLC.

Claims (3)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An immobilized enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV
and analogs thereof to desacetocycephalosporin and the respective analogs thereof, comprising an epimerase having a molecular weight of about 60,000, a cyclase having an MW
of about 36,500 and a ring expansion enzyme having a molecular weight of about 29,000, derived from a prokaryotic organism, immobilized on a diethylaminoethyltrisacryl chromatographic resin.

2. A process for preparing an immobilized enzyme reagent capable of continuously cyclizing, epimerizing and ring expanding ACV and analogs thereof to deacetocycephalosporin and the respective analogs thereof, comprising:
(a) precipitating contaminating proteins from a cell-free extract of a prokaryotic organism by addition of ammonium sulphate to 40% saturation;
(b) separating said precipitated protein from a supernatant;
(c) adding ammonium sulphate to 70% saturation to said supernatant thereby precipitating said cyclizing epimerizing and ring expanding enzymes;
(d) suspending said precipitated enzymes in a pH 7 50mM buffer; and (e) loading said enzymes onto a c h r o m a t o g r a p h i c c o l u m n c o m p r i s i n g diethylaminoethyltrisaoryl.

3. A process as claimed in claim 2 wherein said buffer is a Tris-chloride buffer.