Note: Descriptions are shown in the official language in which they were submitted.
~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
C02H
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~
CO2H SO3
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.
- 3 -
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
-- 5 --
~:~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
CO2H
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
-- 10 --
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
-- 11 --
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
- 12 -
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
- 13 -
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.
- 14 -
lZO~
(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
- 15 -
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.
- 16 -
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--~
H CO2 2
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
C2 R2
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
- 18 -
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
C2R2
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.
- 19 -
~z~o~
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
- 20 -
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
C02H
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:
20R2
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 ,
O CO2H
- 21 -
~ L2~9V~
ring expansion leads to cephalosporin analogs of the type:
D-Aad-NH ~ ~ ~1
R (12)
C02H
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.
- 23 -
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
- 24 -
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
- 25 -
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
- 26 -
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
- 27 -
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.
- 29 -
