Title of Invention

"NOVEL CEPH-3-EM COMPOUND AND PROCESS FOR PREPARING THE SAME"

Abstract A ceph-3-em compound characterized by formula [I]: or a salt thereof, wherein R and R' are as described in the specification.
Full Text The present invention relates to novel ceph-S-em compound and process for preparing the same.
A ceph-3-em compound according to the present invention is characterised by formula [I]:
(Formula Removed)
or a salt or ester thereof,
wherein R is selected from the group consisting of a) HOOC-X-CO-
wherein X is defined as (CH2)4
or wherein X Is defined as (CH2)p-A-(CH2)q, wherein
p and q each individually are 0,1,2, 3 or 4, and
A is CH=CH, C=C, CH8, C=0, O, S, NH. the nitrogen optionally being
substituted or the sulfur optionally being oxidized, and B is hydrogen, halogen,
C1-3 alkoxy, hydroxy!, or optionally substituted methyl,
with the proviso that p+q should be 2 or 3, when A is CH=CH or C=C, or p+q
should be 3 or 4, when A is CHB, C=0, O, S or NH
or wherein X is (CH2)m-CH=A-(CH2)n or (CH2)m-OC-(CH2)n, wherein
m and n each individually are 0,1, 2 or 3 and m+n = 2 or 3, and
A is CH or N,
or wherein X is (CH2)p-CH=CH-CH=C-{CH2)q, wherein
p and q each individually are 0 or 1 and p+q = 0 or
b) (carboxymethylthlo)propionyI
c) (carboxyethyIthIo)proplonyl
and wherein R' is selected from the group consisting of
d) OH

e) O-(alkyl 1-6C) wherein the alkyl can be straight or branched and
f) 0-C(alkyl 1-6C)-O-(alkyl 1-6C) wherein the alkyl groups can be straight or
branched.
This ceph-3-em compound [ I ] can be used as an intermediate in the
production of commercial ceph-3-em antibiotics. Alternatively, this ceph-3-em
compound [ I ] can be converted into another irftermediate, namely 7-amino-3-
carbamoyloxymethyl-3-cephem-4-carboxylic acid or a salt or ester thereof.
A particular advantage of this ceph-3-em compound [ I ] is its
enhanced stability under the conditions of purification of the compound and/or the
further synthesis of commercially attractive ceph-3-em antibiotics as compared to
cephalosporin C. By virtue thereof the preferred compound can be isolated more easily
than cephalosporin C.
Examples of commercial ceph-3-em antibiotics are cefacetril,
cefaclor, cefaloglycin, cefalonium, cefaloridin, cefalotin, cefamandole, cefapirin,
cefapyrin, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene pivoxil,
cefdinir, cefditoren pivoxil, cefepime, cefixime, cefmenoxime, cefmetazole, cefminox,
cefodizime, cefonicid, cefoperazone, ceforanid, cefotaxime, cefotiam, cefotiam hexetil,
cefpiramide, cefpirome, cefpodoxime proxetil, cefprozil, cefroxadin, cefsulodin,
ceftazidime, cefteram pivoxil, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone,
cefuroxime, cefuroxime axetil, cefuzonam.
Cefuroxime, cefoxitin and cefcapene pivoxil are examples of
cephalosporin antibiotics, which share a 3-carbamoyloxymethyl group, and which
cannot readily be produced from the currently available ceph-3-em intermediates, such
as 7-ACA. The ceph-3-em compounds according to the present invention possess a 3-
carbamoyloxymethyl group and can readily be converted into these cephalosporin
antibiotics as well as in other ceph-3-em antibiotics using well-known techniques.
Cefazolin, ceftazidine and ceftriaxone are further most preferred
cephalosporin antibiotics which can be prepared from a compound of formula [ 1 ]
according to well known techniques.
Furthermore, the compound according to formula [ I ]can be used
itself as an antibiotic.
A most preferred compound according to the present invention is the
compound of the formula [II] (adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-
carboxylic acid).
(Figure Removed)The ceph-3-em compound according to the present invention can be
produced chemically according to methods known in the art, or by fermentation or by a
combination of one or more biotransformation steps and one or more fermentation
steps and/or one or more chemical conversion steps.
The present invention also comprises a fermentative method for the
production of a ceph-3-em compound [I] as a secondary metabolite from a suitable
genetically altered microorganism.
For the fermentative production of the ceph-3-em compounds
according to the present invention preferably use can be made of microorganisms,
which inherently possess at least part of the metabolic pathway for the production of (3-
lactam. For example, microorganisms can be used which possess at least part of the
metabolic pathway for the production of penam or ceph-3-em (3-lactam compounds.
Suitable organisms for this purpose are for example fungi of the genus Penicillium,
such as P. chrysogenum or of the genus Acremonium, such as A. chrysogenum or of
the genus Aspergillus such as A. nidulans, or bacteria of the genus Streptomyces, such
as S. cJavuligeris or of the genus Nocardia, such N. lactamdurans or of the genus
Lysobacter such as L. lactamgenus.
Biosynthesis of the O-carbamoylated ceph-3-em compound
cephamycin C in microorganisms is believed to take place according to the scheme
outlined in Fig. 1. Cephalosporin C synthesis is outlined in the scheme according to
Fig.2.
Preferably, such genetically altered organisms are cultured under
conditions in which in the resulting ceph-3-em compound the a-amino-adipyl side chain
which is present at the 7-position in natural ceph-3-em compounds will be replaced by
a desired side chain according to the present invention. To this end a composition able
to deliver this side chain is supplied to the in vivo enzymatic conversion.
Suitably the in vivo enzymatic conversion can be supplied by a side
chain precursor selected from the group consisting of
i) S-carboxymethylthiopropionic acid or a salt or ester thereof,
ii) 3,3-thiodipropionic acid or a salt or ester thereof,
iii) a compound p Y- CH2-COOH or a salt or ester thereof
wherein Y is phenyl, phenoxy or tetrazolyl,
iv) a compound of the general formula HOOC-X-COOH or a salt or ester
thereof,
wherein X Is defined as (CH2)4
or wherein X is defined as (CH2)p-A-(CH2)q, wherein
p and q each individually are 0, 1, 2, 3 or 4, and
A is CH=CH, C=C, CHB, C=O, O, S, NH, the nitrogen optionally being
substituted or the sulfur optionally being oxidized, and B is hydrogen,
halogen, C alkoxy, hydroxyl, or optionally substituted methyl,
with the proviso that p+q should be 2 or 3, when A is CH=CH or C=C, or
p+q should be 3 or 4, when A is CHB, C=O, O, S or NH
or wherein X is (CH2)m-CH-A-(CH2)n or (CH2)ro-CsC-(CH2)n, wherein
m and n each individually are 0, 1, 2 or 3 and m+n = 2 or 3, and
A is CH or N,
or wherein X is (CH2)p-CH=CH-CH=C-(CH2)q, wherein
p and q each individually are 0 or 1 and p+q = 0 or 1
If A chrysogenum is used as the producing genetically engineered
organism penDE, encoding isopenicillin N acyltransferase (Alvarez, E., B.
Meesschaert, E. Montenegro, S. Gutierrez, B. Diez, J. L. Barredo, and J. F. Martin.
1993. Eur. J. Biochem. 215:323-332) and cmcH, encoding carbamoyltransferase
(Coque, J.J. R., F. J. Perez-Llarena, F. J. Enguita, J. L. Fuente, J. F. Martin, and P.
iiras. 1995. Gene 162:21-27) should be introduced. Additionally, at least the ce/G gene
encoding DAC acetyltransferase (Felix, H.R., J. Neusch, and W. Wehrli. 1980. FEMS
Microbiol. Lett. 8: 55-58; Fujisawa, Y., and T. Kanzaki. 1975. Agric. Biol. Chem.
39:2043-2048) and preferably also the cefD1 and cefD2 gene together encoding IPN
epimerase (Ullan RV, Casqueiro J, Banuelos O, Fernandez FJ, Gutierrez S, Martin JF.
2002. J Biol Chem 277(48):46216-25) should be inactivated in order to avoid undesired
side products.
If N. lactamdurans or S. clavuligerus is chosen as the producing
genetically engineered organism the introduction and expression of thepewDEis
required and preferably the ce/Dgene (Jayatilake, S., J.A. Huddleston, and E.P.
Abraham. 1981. Biochem. J. 195:645-647; Konomi, T., S. Herchen, J.E. Baldwin, M.
Yoshida, N.A. Hunt, and A.L. Demain. 1979. Biochem. J. 184:427-430) and the cmcl
gene encoding OCDAC hydroxylase (Xiao, X., G. Hintermann, A. Hausler, P. J. Barker,
F. Foor, A. L. Demain, and J. Piret. 1993. Agents Chemother. 37:84-88) and optionally
also the cmcJ gene encoding methyl transferase or cephamycin C synthetase (Coque,
JJ. R., F. J. Perez-Llarena, F. J. Enguita, J. L. Fuente, J. F. Martin, and P. Liras. 1995.
Gene 162:21-27), of which the latter two are the cephamycin biosynthesis late
enzymes (such as described in WO95/29253) may be inactivated in order to avoid
undesired side products.
If L lactamgenus is chosen as host, at least penDE and cmcH should
be introduced and ce/D preferably should be inactivated.
Insertion and inactivation of genes in organisms in order to provide
the genetic make-up for the production of a compound according to the present
invention can be carried out by methods known in the art. For the insertion use can be
made of genomic DNA sequences or if desired use can be made of cDNA.
Preferred microorganisms for the fermentative production of the
compound of the present invention are P. chrysogenum and A. chrysogenum, -which by
genetic engineering has been provided with DNA fragments encoding enzymes
suitable for the production of the instant compound and wherein in the case of A
chrysogenum the appropriate genes have been inactivated.
More preferably, the fermentative production of the compound
according to the present invention takes place in P. chrysogenum genetically
engineered in a suitable way.
For this purpose a P. chrysogenum strain capable of producing
isopenicillin N has been provided with the following set of DNA fragments:
1) DNA encoding an expandase enzyme
2) DNA encoding a hydroxylase enzyme
3) DNA encoding a O-carbamoyl transferase enzyme
Alternatively, DNA encoding the bifunctional expandase/hydroxylase
enzyme may be introduced instead of DNA's encoding the separate expandase and
hydroxylase enzymes, respectively.
In a preferred embodiment the invention relates to a process for the
fermentative production of a compound of formula [ I ]:
(Figure Removed)or a salt or ester thereof,
wherein R is selected from the group consisting of
a) HOOC-X-CO wherein
X is defined as (CH2)4
or wherein X is defined as (CH2)p-A-(CH2)q, wherein
p and q each individually are 0,1,2, 3 or 4, and
A is CH=CH, CEC, CHB, C=O, O, S, NH, the nitrogen optionally being
substituted or the sulfur optionally being oxidized, and B is hydrogen, halogen,
C_3 alkoxy, hydroxyl, or optionally substituted methyl,
with the proviso that p+q should be 2 or 3, when A is CH=CH or C=C, or p+q
should be 3 or 4, when A is CHB, C=O, O, S or NH
or wherein X is (CH2)m-CHB!A-(CH2)n or (CH2)m-C«C-(CH2)n, wherein
m and n each individually are 0,1, 2 or 3 and m+n = 2 or 3, and
A is CH or N,
or wherein X is (CH2)p-CH=CH-CH=C-(CH2)q, wherein
p and q each individually are 0 or 1 and p+q = 0 or
b) (carboxymethylthio) propbnyl
c) (carboxyethylthio) proplonyl
d) Y-CHrCOwherein
Y is phenyl, phenoxy or tetrazolyl
and wherein R' is selected from the group consisting of
e) OH
f) O-(alky) 1-6C) wherein the alkyl can be straight or branched and
g) O-C(alkyl 1-6C)-O-(alkyl 1-6C) wherein the alkyl groups can be straight or
branched.
comprising the steps of:
A) maintaining in a culture medium capable of sustaining its growth, a strain of P.
chrysogenum which produces isopenicillin N and adding to said culture medium
a feedstock comprising any one or more side chain precursors selected from
the group consisting of
i) S'-carboxymethylthiopropionic acid or a salt or ester thereof,
ii) 3,3-thiodipropionic acid or a salt or ester thereof,
iii) a compound p Y- CH2-COOH or a salt or ester thereof
wherein Y is phenyl, phenoxy or tetrazolyl,
iv) a compound of the general formula HOOC-X-COOH or a salt or ester
thereof,
wherein X is defined as (CH2)4
or wherein X is defined as (CHaJp-A-fCHJq, wherein
p and q each individually are 0,1, 2, 3 or 4, and
A is CH=CH, C=C, CHB, C=O, O, S, NH, the nitrogen optionally being
substituted or the sulfur optionally being oxidized, and B is hydrogen,
halogen, C^alkoxy, hydroxyl, or optionally substituted methyl,
with the proviso that p+q should be 2 or 3, when A is CH=CH or C=C, or
p+q should be 3 or 4, when A is CHB, C=O, O, S or NH
or wherein X is (CH2)m-CH=A-(CH2)n or (CH2)m-CeC-(CH2)n, wherein
m and n each individually are 0,1, 2 or 3 and m+n = 2 or 3, and
A is CH or N,
or wherein X is (CHdp-CHsCH-CH-fCHzJc,, wherein
p and q each individually are 0 or 1 and p+q = 0 or 1
or its salts and esters which are capable of being assimilated and utilized
by said strain of P. chrysogenum to produce a suitable acyl-6-
aminopenicillanic acid (acyl-6-APA), whereby said acyl-6-APA is produced;
B) carrying out the following enzymatic conversions by in situ expression of the
corresponding genes:
I) the acyl-6-APA is in situ ring-expanded to form the corresponding acyl-7-
amino-desacetoxycephalosporanic acid (acyl-7-ADCA) by expandase
enzyme, wherein said strain of P. chrysogenum has been transformed by
DNA encoding the expandase enzyme capable of accepting said acyl-6-
APA as a substrate, whereupon as a result of its expression, said acyl-6-
APA produced by said strain is also thereafter in situ ring-expanded to form
the corresponding acyl-7-ADCA;
ii) the 3-methyl side chain of said acyl-7-ADCA is in situ hydroxylated to yield
-8-
the corresponding acyl-7-ADAC by hydroxylase enzyme, wherein said strain
of P. chrysogenum has been transformed by DMA encoding the hydroxylase
enzyme capable of accepting said acyl-7-ADCA as a substrate, whereupon
as a result of its expression, said acyl-7-ADCA produced by said strain is
also thereafter in situ hydroxylated to form the corresponding acyl-7-ADAC;
iii) the 3-hydroxymethyl side chain of said acyl-7-ADAC is in situ Ocarbamoylated
to yield the compound according to formula [ I ] by Ocarbamoyl
transferase enzyme, wherein said strain of P. chrysogenum has
been transformed by DMA encoding the O-carbamoyl transferase enzyme
capable of accepting said acyl-7-ADAC as a substrate, whereupon as a
result of its expression, said acyl-7-ADAC produced by said strain is also
thereafter in situ carbamoylated to form a compound according to the
present invention.
The DNA encoding the expandase enzyme, hydroxylase enzyme, or
O-carbamoyl transferase enzyme for use according to the present invention can be
obtained from micro-organisms reported to contain this DNA and which are available
from culture collections or it may be obtained from micro-organisms isolated from
appropriate natural sources.
Examples of microorganisms reported to contain the expandase
enzyme are A chrysogenum (cefEF), S. clavuligerus (cefE), N. lactamdurans (cefE), L.
lactamgenus (cefE).
Examples of microorganisms reported to contain the hydroxylase
enzyme are A. chrysogenum (cefEF), S. clavuligerus (cefF), N. lactamdurans (cefF), L
lactamgenus (cefF).
Examples of microorganisms reported to contain the O-carbamoyl
transferase enzyme are species from the Order Actinomycetes, and In particular from
the Genus Streptomyces. Based on the disclosure in US Patent No. 4,075,061,
particularly suitable species which may be employed to provide the DNA encoding the
desired O-transcarbamoylase activity include S. clavuligerus for example strain NRRL
3585 as described in British Patent No. 1,315,177; S. wadayamensis, for example
strain ATCC 21948 as described in Dutch Patent Application No. 7308948; S.
albogriseolus, for example strain NRRL 5735 as described in U.S. Patent No.
3,914,158; S. lactamdurans for example strain NRRL 3802 as described in British
Patent No. 1,321,412 and S. jumonjinensis, for example strain NRRL 5741 as
described in British Patent No. 1,387,965.
According to the disclosure of Patent Publication WO 95/29253
further suitable sources of the desired O-carbamoyl transferase encoding DNA may be
N. lactamdurans, S. lipmanii, S. panayensis, S. cattleya, S. griseus, S. todorominensis,
S. filipinensls cephamldni and S. heteromorphus.
Transformation of host cells, for example of P. chrysogenum or other
fungi can, in general, be achieved by different means of tiNA delivery, like PEG-Ca
mediated protoplast uptake, electroporation or particle gun techniques, and selection of
transformants. See for example Van den Hondel and Punt, "Gene and Transfer and
Vector Development for Filamentous Fungi", in: Applied Molecular Genetics of Fungi
(Peberdy, Laten, Ogden, Bennett, eds.), Cambridge University Press (1991). The
application of dominant and non-dominant selection markers has been described (Van
den Hondel et a/., supra). Selection markers of both homologous (P. chrysogenum
derived) and heterologous (non-P. chrysogenum derived) origin have been described
(Gouka et a/., J. Biotechnol. 20 (1991) 189-200).
The application of various homologous or heterologous transformant
selection markers, in the presence or absence of vector sequences, physically linked or
not.to the non-selectable DNA, in the selection of transformants is well known.
The DNA sequence encoding the expandase activity, the hydroxylase
activity and the O-carbamoyl transferase activity are introduced into and expressed in
this way in P. chrysogenum, for instance in strain Wisconsin 54-1255 (deposited at
ATCC under accession number 28089). Other strains of P. chrysogenum, including
mutants of strain Wisconsin 54-1255, having an improved beta-lactam yield, are also
suitable. Examples of such high-yielding strains are the strains CBS 455.95, Panlabs
P2 and ASP-78 (Barredo JL, Alvarez E, Cantoral JM, Diez B, Martin JF. 1988.
Antimlcrob Agents Chemother32(7):1061-7).
Furthermore, the cmcH gene together with the cefE and ce/F or
ce/EFgene are placed under the transcriptional and translational control of
heterologous or homologous control elements, preferably under control of fungal gene
control elements. Those elements can be obtained from cloned fungal genes like the
P.chrysogenum IPNS orpobC gene, the B-tubulin gene, the A. nidulans gpdA gene, or
the A. nigerglaA gene.
As an alternative to a complete fermentative production, the
compound according to the present invention can be prepared by a combination of one
or more fermentative steps and one or more biotransformation steps and/or one or
more chemical conversion steps.
For example, in a first step a suitable penicillin derivative, such as
penicillin G or penicillin V, or cephalosporin derivative such as adipoyl-7-ADCA or
adipoyl-7-ADAC can be produced fermentatively.
A suitable penicillin derivative optionally can be subjected to
exchange of the 6-acyl group before conversion into the corresponding 7-acyl-3-
* methyl-ceph-3-em compound. The latter can in turn be converted into the
corresponding 7-acyl-3-hydroxymethyl-ceph-3-em, and finally into the compound of
formula [I].
For these conversions the necessary enzymes (acyltransferase,
expandase, hydroxylase and carbamoyltransferase, or, alternatively, acyltransferase,
exandase-hydroxylase and carbamoyltransferase, encoded by the penDE, cefE, cefF
and c/ncH genes orpenDE, ce/EFand cmcH genes, respectively) can be produced
separately in any suitable host. Preferably, this is done in Escherichia co//according to
a process known in the art. Enzymes can be purified and if needed immobilized, or
used in whole cell biotransformations or crude cell-free extracts, in single and
consecutive reaction vessels, or, in a one-pot reaction, to form the compound of
formula [I].
Single steps in this multi-step route (e.g. penicillin G - acyl-6-APA -
acyl-7-ADCA -acyl-7-ADAC -» compound [I]) can be exchanged for fermentative or
chemical conversion steps. E.g. the first step can be omitted if one uses adipoyl-7-
ADCA produced by P. chrysogenum transformed with cefE (e.g. according to the
method described in EP0523341), or alternatively, the first two steps can be omitted by
starting from acyl-7-ADAC produced by P. chrysogenum transformed with cefE and
cefF (e.g. according to the method described in EP0540210). As an alternative,
chemical conversions known in the art for each single step can be applied.
Brief description of the Figures:
Fig. 1.: Schematic representation of fermentative production of cephamycin C
in S. clavuligerus.
Fig. 2.: Schematic representation of fermentative production of cephalosporin
C in A. chrysogenum.
Fig. 3.: Plasmld used to clone the gene cefE and for transformation of
Wisconsin 54-1225.
Fig. 4.: Plasmid used to clone the gene cefF and for transformation of
Wisconsin 64-1225.
Fig. 5.: Plasmid used to clone the gene cefEF and for transformation of
Wisconsin 54-1225.
Fig. 6.: Plasmid used to clone the gene c/ncH and for transformation of
Wisconsin 54-1225.
Fig. 7.: Plasmid used to clone the gene atndS and for transformation of
Wisconsin 54-1225.
Fig. 8.: Schematic representation of the fermentative process according to
the present invention.
Fig. 9.: The NMR spectrum of adipoyl-7-amjno-3-carbamoyloxymethyl-3-
cephem-4-carboxylic acid.
Fig. 10.: The NMR spectrum of the adipoyl-7-amino-3-carbamoyloxymethyl-3-
cephem-4-carboxylic acid prepared according to Example 3
Fig. 11.: Plasmid pGK105.
Fig. 12.: HPLC/MS analysis of the byconversion product by carbamoyl
transferase yielding adipoyl-7-amino-3-carbamoyIoxymethyl-3-
cephem-4-carboxylic acid.
Fig. 13.: Intracellular (A) and extracellular (B) distribution of adipoyl-7-amino-3-
carbamoyloxymethyl-3-cephem-4-carboxylic acid and other p-lactams
(in mmol) in P. chrysogenum transformed according to Example 2
Fig. 14.: Growth inhibition by adipoyl-7-ADCA, adipoyl-ACCA and cefuroxime
on B. subtilis.
Fig. 15.: Growth inhibition by adipoyl-7-ADCA, adipoyl-ACCA and cefuroxime
on E. coll
Fig. 16.: ' Growth inhibition by adipoyl-7-ADCA, adipoyl-ACCA and cefuroxime
on M. luteus.
Example 1
Transformation of a P. chrysogenum strain with the genes encoding expandase,
hydroxylase and 3'-hydroxymethylcephem O-carbamoyltransferase activity
Common techniques used in gene cloning procedures are used in the
present application. These techniques include polymerase chain reactions (PCR),
synthetic oligonucleotide synthesis, nucleotide sequence analysis of DNA, enzymatic
ligation and restriction of DNA, E. co//vector subcloning, transformation, and
transformant selection, isolation and purification of DNA, DNA characterization by
Southemblot analysis. These techniques are all very well known in the art and
adequately described in many references.
Transformations were carried out with the P. chrysogenum strain
Wisconsin 54-1255 (ATTC 28089). All constructs introduced in P. chrysogenum are
under the control of the P. chrysogenum IPNS promoter and AT terminator. Other
strains of P. chrysogenum, including mutants of strain Wisconsin 54-1255, having an
improved B-lactam yield, are also suitable. An example of such a strain is CBS 455.95.
Culturing of P. chrysogenum for generation of protoplasts used in
transformation is done in YPD- medium (1% yeast extract, 2% peptone, 2% glucose). It
is well known in the art that the protoplasting and regeneration procedures may differ
slightly depending on the particular strain of P. chrysogenum used and the
transformant selection procedure applied.
S. clavuligerus ATCC 27064 is grown in tryptic soy broth (Difco).
Chromosomal DNA of this strain is used for isolation of the ce/Egene by PCR. The 5'
forward primer 4363: 5'-GAT CAG TGA CAG TTG CAT ATG GAC ACG ACG GTG
CCC ACC TTC AGC CTG-31 (SEQ ID NO. 1) and the 3' reversed primer number 4364:
5'-CCC GGG TCT AGA TCT AGA CTA TGC CTT GGA TGT GCG GCG GAT GTT-3'
(SEQ ID NO. 2) were designed using the published S. clavuligerus Ce/E sequence
(Kovacevlc S, Weigel BJ, Tobin MB, Ingolia TD, Miller JR. J Bacteriol (1989)
171(2):754-60; Ingolia et a/. U.S. Pat. No. 5,070,020).
The obtained PCR product was cut with A/del and Xba\ and the 0.9 kb
fragment was ligated with pMcTNdel also cut with A/del and Xba\ (fragment: 3.9 kb)
resulting in pMcTSE. pMCTNdel is a derivative of pMC-5 (Stanssens et a/. NAR
(1989) 17:4441) and constructed by insertion of a fragment encoding the tac promoter
followed by a ribosome binding sequence (RBS) site and a A/del cloning site (see also
E.P. Pat. No.0,351,029).
First the AT promoter was cloned in front of the expandase gene. The
AT promoter was obtained by PCR using primer 4488: 5-AGA ACG GAT TAG TTA
GTC TGA ATT CAA CAA GAA CGG CCA GAC-3' (SEQ ID NO. 3) and 4489: 5'-GAC
AGA GGA TGT GAA GCA TAT GTG CTG CGG GTC GGA AGA TGG-3' (SEQ ID NO.
4) on chromosomal DNA of P. chrysogenum. The oligo's were based on the P.
chrysogenum penDE gene sequence published by Barredo ef a/., (1989) Gene 83:572-
576 and Diez et a/., Mol. Gen. Genet. (1989) 218:572-576. The product was digested
with EcoR\ and Nde\ resulting in a 1.5 kb fragment. This fragment was ligated with
pBluescript (Stratagene) EcoR\-Xba\ fragment of 3,0 kb and with pMcTSE Ndel-Xbal
fragment of 0.9 kb containing the S. clavuligerus expandase gene. This results in the
plasmid pASE containing the expandase gene in behind the AT promoter.
The penDE (encoding AT) terminator was cloned behind the
expandase gene in the pASE plasmid. Therefore, chromosomal DNA of P.
chrysogenum was used as template for the PCR of the AT terminator using 5' forward
primer 4579: 5'-TTC GAT GTC AGC CTG GAC GGC GAG ACC GCC ACG TTC CAG
GAT TGG ATC GGG GGC AAC TAG GTG AAC ATC CGC CGC ACA TCC AAG GCA
TGA AGG CTC TTC ATG ACG-31 (SEQ ID NO. 6) and 3' reversed primer 4507: 5'-
GGA CTA GTG TCG ACC CTG TCC ATC CTG AAA GAG TTG (SEQ ID NO. 5). The
fragment was digested with Sg/I-Spel and the 0.6 kb product was ligated with the
plasmid pASE digested with Spel and Bgl\ resulting in pASEWA.
Finally, the expandase gene was put behind the P. chrysogenum
IPNS promoter (the IPNS promoter replacing the AT promoter). The IPNS promoter
was amplified using P. chrysogenum chromosomal DNA as a template with 5' oligo
4923: 5'-CGA GGG GAA TTC CTT ATA CTG GGC TG CTG CAT TGG TCT G (SEQ
ID NO. 7) (using the sequence published by: Diez.B., Gutierrez,S., Barredo.J.L, van
Solingen,P., van der Voort,LH. and Martin.J.F. (1990) J. Biol. Chem. 265 (27), 16358-
16365) and 3' oligo 4924: 5'-CCC GGG CAT ATG CAT ATG GGT GTC TAG AAA AAT
AAT GGT GAA AAC (SEQ ID NO. 8) (using the sequence published by Carr LG,
Skatrud PL, Scheetz ME 2nd, Queener SW, Ingolia TD. (1986) Gene 48(2-3):257-66).
pASEWA is digested with EcoRl and A/del resulting in a 4.6 kb fragment with the
expandase gene and the AT terminator was ligated with the 0.9 kb IPNS promoter PCR
product digested with EcoRI-A/del. This yields pISEWA. The ce/E gene with the IPNS
promoter and the AT terminator was obtained from pISEWAn by a Nofi digestion
(fig.3).
The hydroxylase gene (cefF) was isolated from S. clavuligerus (ATCC
27064). Chromosomal DNA of this strain was used for isolation of the ce/Fgene by
PCR introducing a Nde\ site upstream of the gene and a Ate/1 site downstream. This
construct was ligated behind the IPNS promoter and in front of the AT terminator
resulting In pISFWA. For transformation of P. chrysogenum the construct including the
ce/Fgene and the IPNS prmoter and AT terminator was isolated from pISFWA (Fig. 4)
after cutting with A/ofl.
The A. chrysogenum (= Cephalospon'um acremonium)
expandase/hydroxylase gene (cefEF), was obtained from chromosomal DNA by PCR.
The designed forward oligo introduced a Ndel site and the reversed oligo a Nsil site.
After digestion with Ndel and Nsil the gene could be placed in the Penicillium
expression vector resulting in pICEFWA (fig. 5). For transformation of Penicillium, me
ce/EFfrom A. chrysogenum was isolated from pICEFWA with the IPNS promoter and
AT terminator by a Nof/ digestion.
The cmcH gene was obtained from S. clavuligerus by PCR on
chromosomal DNA with the forward primer: AB12586 (5-ACA GAC CAT ATG CTC
GTC GTT GCA TTC AAG -3' (SEQ ID NO. 9)) and the reversed primer: 5'-AB12587
(GAC GGC ATG CAT TCA GGA ACC GGC TAT TCG C-3' (SEQ ID NO. 10)) and was
cut with Ndel, Nsil and ligated with the Ndel, Nsil fragment of pISEWAN (replacing the
expandase gene for the cmcH gene) yielding pIScCTWA (Fig. 6). The cmcH gene
including the IPNS promoter and the AT terminator was isolated from pIScCTWA by a
A/ofl digest.
The AmdS fragment with flanks of HelY was isolated from pHELY-A1
by a digestion with Hind\\l (fig. 7).
The cmcH, with cefEF or cefE and cefF constructs were introduced
by co-transformation with the amdS selection marker in P. chrysogenum ATCC 28089.
The integration of the amdS marker enables the P. chrysogenum transformants to grow
on selection medium containing acetamide as the sole nitrogen source.
Techniques involved in the transfer of DNA to protoplasts of P.
chrysogenum are well known in the art and are described in many references, including
Finkelstein and Ball (eds.), Biotechnology of filamentous fungi, technology and
products, Butterworth-Heinemann (1992); Bennett and Lasure (eds.) More Gene
Manipulations in fungi, Academic Press (1991); Turner, in: POhler (ed), Biotechnology,
second completely revised edition, VHC (1992). The Ca-PEG mediated protoplast
transformation is used as described in EP635574.
Example 2
Fermentative production of adipoyl-7-amino-3-carbamoyloxymethyI-3-cephem-4-
carboxylic acid
The P. chrysogenum transformants obtained according to Example 1
were inoculated at 2x10s conidia/ml into a medium as described for penicillinV
production tests in US20020039758, but instead of potassium phenoxyacetate it was
supplemented with 0.5-10 mg/ml sodium adipate as a side chain precursor (pH before
sterilization 5.5-6.0). The incubation took place for 144 -169 hours at 25°C and 280
rotations per minute.
Filtrates of well grown cultures were analysed by HPLC and NMR for
the production of adipoyl-7-amino-3-carbamoyloxymethy!-3-cephem-4-carboxylic acid.
The NMR spectrum shows the peaks characteristic for the desired compound (See
figure 9.
Example 3
Chemical synthesis of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic
acid
PREPARATION OF PHENYLACETYL-7-AMINO-3-HYDROXYMETHYL-3-CEPHEM-4-
CARBOXYLIC ACID -BENZHYDRYL ESTER [IV]
[IV]
8.5 ml of 20% NaOH solution was added dropwise to a stirred
solution of 7-amino-cephalosporanic acid (5 g, 18.3 mmol) in water (20 ml) at 0°C. After
stirring for 5 min the pH was adjusted to 8.5 with acetic acid and the solution was
diluted with acetone (20 ml). Phenylacetyl chloride (2.9 ml, 22 mmol) in acetone (3 ml)
was then added dropwise with the pH kept between 7.5 and 8.5 by addition of aqueous
NaOH. The solution was then stirred for 1 h at 0°C. The acetone was then removed in
vacua before addition of ethyl acetate (70 ml) and acidification of the aqueous phase to
pH 3 with dilute HCI. The organic phase was separated and the aqueous phase reextracted
with another portion of ethyl acetate. The organic phases were combined,
washed with brine, (MgSO4) and filtered. To this solution was then added a solution of
diphenyldiazomethane (5 g, 25.8 mmol) in ethyl acetate (5 ml) with stirring. The
solution was concentrated to ca. 40ml in vacua and left overnight at 4°C. The resultant
precipitate was collected by filtration and washed with ethyl acetate giving the product
as a white powder (3.83 g, 40%). & ((CD3)2SO, 300 MHz) 3.54 & 3.63 (2H, ABq, J
13.9, PhCtb), 3.65 (2H, s, SCH2), 4.25 (2H, s, CHzOH), 5.15 (1H, d, J4.7, CHCHS),
5.76 (1H, dd, J4.7, 8.2, NHCH), 6.94 (1H, s, CHPh2) 7.2-7.6 (15H, m, PhzCH, PhCH2),
9.17 (1H, d, J 8.2, NHCH). M/z (ES+) 537 (M+Na, 100%).
PREPARATION OF TRICHLOROACETYL- PHENYLACETYL-7-AMINO-3-
CARBAMOYLOXYMETHYL-3-CEPHEM-4-CARBOXYLIC ACID -BENZHYDRYL
ESTER M
Trichloroacetyl isocyanate (0.23 ml, 1.9 mmol) was added to a stirred
solution of [IV] (600 mg, 1.2 mmol) in acetone (20 ml). After stirring for 2 h a white
precipitate was collected by filtration, washed with acetone and dried (811 mg, 99%).
4, ((CD3)2SO, 300 MHz) 3.54 & 3.62 (2H, ABq, J 13.9, PhCHa), 3.66 & 3.76 (2H,
ABq, J18.5, SCHa), 4.90 & 5.02 (2H, ABq, J12.8, CfcbO), 5.20 (1H, d, J4.8, CHCHS),
5.82 (1H, dd, J4.8, 8.1, NHCHCH), 6.96 (1H, s, CHPh2), 7.2-7.6 (15H, m, CHEfe,
£hCH2), 9.17 (1H, d, J 8.1, NHCH), 12.0 (1H, s, CONHCO). M/z (ES-) 702 (M-1,100%).
PREPARATION OF TRICHLOROACETYL-GO PHENYLACETYL-7-AMINO-3-
CARBAMOYLOXYMETHYL-3-CEPHEM-4-CARBOXYLIC ACID RCO [VI]
(Figure Removed) [V] (5.15 g, 7.33 mmol) was dissolved in a cooled (0°C) mixture of
trifluoroacetic acid (35 ml) and anisole (4 ml). After stirring for 2 h the solution was
concentrated in vacua to an oil which was triturated with petroleum ether (40-60°C
fraction) then dissolved in ethyl acetate (30 ml) and decolourised with charcoal. After
filtering, the solution was concentrated in vacua to a yellow oil which was used for the
next step without further purification. S» ((CD3)2SO, 300 MHz) 3.50-3.75 (4H, m,
PhCHa, SCHz), 4.94 (1H, part ABq, J 12.5, CH2O), 5.14 (2H, m, part ABq CHaO,
CHCS), 5.72 (1H, m, NHCHCH), 7.0-7.4 (5H, m, PhCH2), 9.12 (1H, d, J8.2, NHCH),
11.95 (1H, s, CONHCO). M/z (ES-) 536 (M-1, 67%).
PREPARATION OF PHENYLACETYL-7-AMINO-3-CARBAMOYLOXYMETHYL-3-
CEPHEM-4-CARBOXYLIC ACID [VII]
(Figure Removed)The yellow oil from the previous step was carefully dissolved by
addition of 10% NaHCO3 solution until a pH of ca. 9 was reached. The solution was
then stirred overnight. The pH was then lowered to 2 using dilute HCI at which point
precipitation occurred. The precipitate was removed by filtration and washed with ether
to give a pale yellow solid (1.81 g, 63.3% over 2 steps). SH ((CD3)2SO, 300 MHz) 3.4-
3.65 (peaks masked by H2O Peak), 4.63 & 4.91 (2H, ABq, J 12.8, CH2O), 5.10 (1H, d, J
4.5, CHCHS), 5.68 (1H, dd, J4.5, 8.2, NHCHCH), 7.28 (5H, m, PhCH2), 9.11 (1H, d, J
8.2, NHCH). 6H((CD3)2SO + D2O, 300 MHz) 3.52 (4H, m, PhCJi, SCHz), 4.63 & 4.88
(2H, ABq, J 12.9, CJiO), 5.05 (1H, d, J 4.8, CHCHS), 5.65 (1H, d, J4.7, NHCHCH),
7.28 (5H, m, PhCH2). M/z (ES-) 390 (M-1, 20%).
PREPARATION OF 7-AMINO-3-CARBAMOYLOXYMETHYL-3-CEPHEM-4-
CARBOXYLIC ACID [VIII]
[VII] (760 mg, 1.95 mmol) was stirred in potassium phosphate buffer
(20 ml, 0.5 M, pH 7) and the pH was raised to pH 7.8 by addition of aqueous NaOH.
Penicillin amidase on acrylic beads (ca. 375 mg after washing to remove glucose
stabilizing agent) was added and the resultant suspension was stirred for 2.5 h. The
beads were then removed by filtration and the pH of the solution was lowered to 3 by
addition of dilute HCI. The solution was then cooled overnight at 4°C and then filtered
to yield the product as a pale yellow powder (334 mg, 63%). SH ((CD3)2SO + D2O, 300
MHz) 3.36 & 3.53 (2H, ABq, J 18.1, SCH2), 4.60 &. 4.82 (2H, ABq, J12.8, CH2O), 4.74
(1H, d, J4.9, CHCHS), 4.94 (1H, d, J4.9, H2NCHCH).
PREPARATION OF CYCLIC ADIPIC ANHYDRIDE [IX]
[IX]
A mixture of adipic acid (5 g, 34 mmol) and acetic anhydride (15 ml)
was heated at reflux for 4 h. The solution was then concentrated in vacua and the
remaining residue was vacuum distilled to give a colourless oil (exposure to
atmospheric moisture causes polymerisation). 5* (CDCI3, 300 MHz) 2.0 (4H, m,
, 2.76 (4H, t, J6.6,
(Figure Removed)PREPARATION OF ADIPOYL-7-AMINO-3-CARBAMOYLOXYMETHYL-3-CEPHEM-4-
CARBOXYLICACID[X]
[X]
A solution of [VIII] (100 mg, 0.37 mmol) in aqueous acetone (10 ml,
1:1 v/v) was adjusted to pH 8.5 by careful addition of aqueous NaOH. To this solution
was added dropwise at 0°C a solution of [IX] (80 mg, 0.625 mmol) in acetone (2 ml)
with the pH kept between 7.5 and 8.5 by addition of aqueous NaOH. The resultant
solution was stirred at 0°C for 2 h before removal of the acetone in vacua and
adjustment of the pH to 2 by addition of aqueous HCI. The solution was extracted with
cyclohexanone (2 x 20 ml) and the combined organic phases concentrated to a few ml.
The concentrated cyclohexanone solution was poured into cyclohexane (200 ml) giving
a precipitate that was collected by filtration. (61 mg, 41%). 5* ((CD3)2SO, 300 MHz)
1.50 (4H, m, CHjCtkCHzCHz), 2.22 (4H, m, CHbCHzCHaCtb), 3.45 & 3.59 (2H, ABq, J
18.1, SCH2), 4.62 & 4.90 (2H, ABq, J 12.9, CH2O), 5.10 (1H, d, J4.8, CHCHS), 5.67
(1H, dd, J4.8, 8.2, NHCHCH), 8.82 (1H, d, J8.2, NHCH). IWz (ES-) 801 (2M-1, 17%),
400 (M-1, 35%) (Figure 10).
Example 4
In vitro production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic
acid using byconversion
Preparation of cell-free extract
The c/ncHgene of Streptomyces clavuligerusvtas expressed in
co//XL1-Blue under the control of the lac promoter using plasmid pGK105 (Figure 11).
For expression of the cmcHgene, 5 ml of chloramphenicol-containing LB growth
medium was inoculated with a single colony of XL1-Blue:pGK105 and grown at 37°C
for 16 hours with shaking at 250 rpm. This culture was used as a 1% inoculum for a
flask containing 100 ml of sterile LB medium supplemented with chloramphenicol which
was then incubated at 27°C with shaking at 250 rpm and good aeration until the OD600
reached 0.4-0.6. IPTG was then added to the culture to a final concentration of 0.3 mM
and growth was continued for a further 16 hours. Cells were harvested by
centrifugation .(4000 g, 20 min, 4°C) and the cell pellet was resuspended in 5 ml buffer
(20 mM Tris.HCI, 200 mM NaCI, 1 mM EDTA, pH 7) and frozen either at -80°C for 1
hour or at -20°C overnight. After thawing in an ice-water bath, the cell suspension was
sonicated (MSE Soniprep 150). 15-20 Cycles of 10 s sonication each followed by 10
sec. of cooling time were sufficient for complete lysis as monitored by release of
soluble protein using Bradford assay. Cell debris, insoluble protein and unlyzed cells
were pelleted by centrifugation (4000 g, 60 min, 4°C). The supernatant was treated with
potassium clavulanate (5 mgrnl"1) for 1 hour at 27 °C to inactivate any chromosomally
encoded p-lactamases. Excess clavulanate was removed by gel filtration over
Sephadex G-25® using PD-10 desalting columns and the resulting enzyme solution
was used immediately or stored at -20°C until required.
Carbamoyl transferase assay
Assays (100 |j,l) were performed at 28°C for four hours and contained
adipoyl-deacetylcephalosporanic acid (1-5 mM), MgSO4 (0.8 mM), MnCI2 (1 mM),
imidazole (100 mM), ATP (5.4 mM), carbamoyl phosphate (9.8 mM), pH 6.75-7. The
amount of protein used ranged from 10 ug to 250 ug. The enzyme solution was thawed
on ice before adding to the reaction mixture, which had been allowed to reach 28°C in
a heating block. The reaction was terminated by the addition of ice-cold methanol (100
ul) and mixed before centrifuging (10000 g, 5 min, 4°C) to pellet the precipitated
protein. Soluble protein that had been denatured by heat-treating at 100°C for 5 min
was used in a control reaction, which was always performed alongside normal assays.
HPLC/MS analysis
Separation of reaction components was achieved on a Spherisorb
analytical C18 column (250 x 4.6 mm) at RT using a Gilson HPLC system with a mobile
phase consisting of 0.1 M sodium dihydrogen phosphate as buffer A and 0.05 M
sodium dihydrogen phosphate/50% acetonitrile as buffer B, a flow rate of 1 ml/min"1
and monitoring at 254 nm. Using these conditions authentic standards of the
cephalosporin starting material and product had retention times of 19 min and 22.5 min
respectively. Product peaks from several analytical runs were pooled, acidified to pH
1.8 and extracted with cyclohexanone. The organic phase was then back-extracted
with water with the pH of the Diphasic extraction mixture adjusted to 7 in between
periodic shaking. The aqueous phase was lyophilised, redissolved in water and
analysed by electrospray (negative ion mode) ionisation MS and the carbamoylated
cephalosporin product identified from its characteristic [M - Hf (Figure 12).
Conclusion
From this experiment it can be concluded that adipoyl-7-amino-3-
carbamoyloxymethyl-3-cephem-4-carboxylic acid can be produced using byconversion
Example 5
Extracellular export of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic
acid
The samples as prepared according to Example 2 were analysed in
more detail by separating the biomass from the filtrate. After 168 hours of growth at 25
degrees Celsius and 280 rotations per minute, the filtrates of well-grown cultures were
separated from biomass via filtration and analysed by NMR. The biomass was washed
twice with ice cold physiological salt (0.9 mM NaCI), frozen in liquid nitrogen, freeze
dried, resuspended in water, and also analysed by NMR.
Besides adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-
carboxylic acid the strains also produce intermediates, respectively IPN, 6APA,
adSAPA, ad7ADCA and adAHCA (=adADAC). However, adipoyl-7-amino-3-
carbamoyloxymethyl-3-cephem-4-carboxylic acid is the only p-lactam exclusively
secreted.
The results of these experiments are summarized in Figure 13 (A and
B)
Example 6
Fermentative production of phenoxyacetoyl-7-amino-3-carbamoyloxymethyl-3-cephem-
4-carboxylic acid
The P. chrysogenum transformants obtained according to Example 1
were inoculated in production test medium as described in example 2, but in stead of
sodium acetate it was supplemented with 5 mg/ml potassium phenoxyacetate as a side
chain precursor for production tests (pH before filtration 6.0). The cultivation time was
168 hours at 25 degrees Celsius and 280 rotations per minute.
Filtrates of well-grown cultures were separated from biomass and
analysed by NMR. The biomass was washed twice with ice cold physiological salt (0.9
mM NaCI), frozen in liquid nitrogen, freeze dried, resuspended in water, and also
analysed by NMR. Phenoxyacetoyl -7-amino-3-carbamoyloxymethyl-3-cephem-4-
carboxylic acid was observed in the mycelial fractions.
Example 7
Fermentative production of transhydromuconoyl-7-amino-3-carbamoyloxymethyl-3-
cephem-4-carboxylic acid
The P. chrysogenum transformants obtained according to Example 1
were inoculated in production test medium as described in example 2, but in stead of
sodium acetate it was supplemented with 4 mg/ml transhydromuconic acid as a side
chain precursor for production tests (pH before filtration 6.0). The cultivation time was
168 hours at 25 degrees celsius and 280 rotations per minute.
Filtrates of well-grown cultures were separated from biomass and
analysed by NMR. The biomass was washed twice with ice cold physiological salt (0.9
mM NaCI), frozen in liquid nitrogen, freeze dried, resuspended in water, and also
analysed by NMR. Transhydromuconoyl -7-amino-3-carbamoyloxymethyl-3-cephem-4-
carboxylic acid was observed in the mycelial fractio'ns.
Example 8
Fermentative production of aminoadipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-
carboxylic acid
The P. chrysogenum transformants obtained according to Example 1
were inoculated in production test medium as described in example 2, but without any
additional side chain precursor. The cultivation time was 168 hours at 25 degrees
celsius and 280 rotations per minute.
Filtrates of well-grown cultures were separated from biomass and
analysed by NMR. The biomass was washed twice with ice cold physiological salt (0.9
mM NaCI), frozen in liquid nitrogen, freeze dried, resuspended in water, and also
analysed by NMR. Aminoadipoyl -7-amino-3-carbamoyloxymethyl-3-cephem-4-
carboxylic acid was observed in both fractions.
Example 9
Bioactivity of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid
Theadipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic
acid prepared according to Example 3 was used to evaluate its activity versus different
bacteria. Bacteria used were: Escherichia coli ESS [xxxref komt nogxx], Micrococcus
luteus DSM 348 [Andrade, A.C., Van Nistelrooy, J.G.M., Peery, R.B., Skatrud, P.L., De
Waard, M.A., 2000, The role of ABC transporters from Aspergillus nldulans in
protection against cytotoxic agents and in antibiotic production] and Bacillus subtilis
ATCC 6633 [xxxref komt nogxx] The bacteria were grown overnight in liquid 2xTY
[xxxref komt nogxx] at 37 degrees and 280 rotations per minute and subsequently
diluted 1000-fold in fresh medium. Two ml deep-well mlcrotiterplates were inoculated
with 1 ml of the diluted bacterial cultures and different concentrations of adipoyl-7-
amino-3-carbamoyIoxymethyl-3-cephem-4-carboxylic acid (adipoyl-ACCA or Ad-ACCA)
were added to individual wells wells. As control several other active and less-active blactams
were used: 6-APA, Adipoyl-6-APA, 7-ADCA, adipoyl-7-ADCA, 7-ACA,
cephalosporinC and cefuroxime. The microtiterplates were incubated for 2 days at 25
degrees Celsius and 550 rotations per minute.
The results for growth inhibition by adipoyl-7-ADCA, adipoyl-ACCA
and cefuroxime are summarized in Figures 14 (B. subtilis), 15 (£ coli) and 16 (M.
luteus).
The minimal inhibitory concentrations of adipoyl-7-amino-3-
carbamoyloxymethyl-3-cephem-4-carboxylic acid for B. subtilis ATCC 6633, E. coli
ESS and M. luteus DSM 348 were 4, 7 and 7 uM, respectively.



We claim;
1. A ceph-3-em compound characterized by formula [I]:


(Formula Removed)
or a salt or ester thereof,
wherein R is selected from the group consisting of
a) HOOC-X-CO-
wherein X is defined as (CH2)4
or wherein X is defined as (CH2)p-A-(CH2)q, wherein
p and q each individually are 0, 1, 2, 3 or 4, and
A is CH=CH, C=C, CHB, C=O, O, S, NH, the nitrogen optionally being substituted or
the sulfur optionally being oxidized, and B is hydrogen, halogen, C1-3 alkoxy, hydroxyl,
or optionally substituted methyl, with the proviso that p+q should be 2 or 3, when A is
CH=CH or C=C, or p+q should be 3 or 4, when A is CHB, C=0, O, S or NH
or wherein X is (CH2)m-CH=A-(CH2)n or (CH2)m-C=C-(CH2)m wherein
m and n each individually are 0, 1, 2 or 3 and m+n = 2 or 3, and
A is CH or N,
or wherein X is (CH2)p-CH=CH-CH=C-(CH2)q, wherein
p and q each individually are 0 or 1 and p+q=0 or
b) (carboxymethylthio)propionyl
c) (carboxyethylthio)propionyl
and wherein R' is selected from the group consisting of
d) OH
e) 0-(alkyl 1-6C) wherein the alkyl can be straight or branched and
f) 0-C(alkyl l-6C)-0-{alkyl 1-6C) wherein the alkyl groups can be straight or branched.

2. Compound as claimed in claim 1 or a salt or ester thereof, wherein the group R' is
OH and wherein the group R is selected from adipoyl, phenoxyacetyl and
tetrazoleacetyl.
3. Adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid or a salt or
ester thereof.
4. A bioprocess for the fermentative production of a ceph-3-em compound as claimed
in claims 1 to 3 characterized by formula [I]:
(Formula Removed)
or a salt thereof,
wherein R is selected from the group consisting
a. HOOC-X-CO-
wherein X is defined as (CH2)4
or wherein X is defined as (CH2)p-A-(CH2)q, wherein
p and q each individually are 0, 1, 2, 3 or 4, and
A is CH=CH, C=C, CHB, C=0, O, S, NH, the nitrogen optionally being
substituted or the sulfur optionally being oxidized, and B is hydrogen,
halogen, C1-3 alkoxy, hydroxyl, or optionally substituted methyl,
with the proviso that p+q should be 2 or 3, when A is CH=CH or C=C, or
p+q should be 3 or 4, when A is CHB, C=0, O, S or NH
or wherein X is (CH2)m-CH=A-(CH2)n or (CH2)m-C=C-(CH2)n, wherein
m and n each individually are 0, 1, 2 or 3 and m+n = 2 or 3, and
A is CH or N,
or wherein X is (CH2)p-CH=CH-CH=C-(CH2)p, wherein
p and q each individually are 0 or 1 and p+q=0 or
b. (carboxymethylthio)propionyl

c. (carboxyethylthio)propionyl
d. Y-CH2-CO-
wherein Y is phenyl, phenoxy or tetrazolyl
and wherein R' is selected from the group consisting of
e. OH
f. O-(alkyl 1-6C) wherein the alkyl can be straight or branched and
g. 0-C(alkyl l-6C)-0-{alkyl 1-6C) wherein the alkyl groups can be straight or
branched.
comprising the steps of
A) maintaining in a culture medium capable of sustaining its growth, a strain of P, chrysogenum which produces isopenicillin N and adding to said culture medium a feedstock comprising any one or more of the side chain precursors selected from the group consisting of
• 3'-carboxymethylthiopropionic acid or a salt or ester thereof,
• 3,3'-thiodipropionic acid or a salt or ester thereof
• Y-CH2-COOH or a salt or ester thereof wherein Y is phenyl, phenoxy or tetrazolyl
• a compound of the general formula HOOC-X-COOH or a salt or ester thereof,
wherein X is defined as (CH2)4
or wherein X is defined as (CH2)p-A-(CH2)q, wherein
p and q each individually are 0, 1, 2, 3 or 4, and
A is CH=CH, C=C, CHB, C=0, O, S, NH, the nitrogen optionally being substituted or
the sulfur optionally being oxidized, and B is hydrogen, halogen, C1-3 alkoxy, hydroxyl,
or optionally substituted methyl,
with the proviso that p+q should be 2 or 3, when A is CH=CH or C=C,
or p+q should be 3 or 4, when A is CHB, C=0, O, S or NH
or wherein X is (CH2)m-CH=A-(CH2)n or (CH2)m-C=C-(CH2)„, wherein
m and n each individually are 0, 1, 2 or 3 and m+n=2 or 3, and
A is CH or N,
or wherein X is (CH2)p-CH=CH-CH=C-(CH2)q, wherein
p and q each individually are 0 or 1 and p+q=0 or 1

which are capable of being "assimilated" and utilized by said strain of P. chrysogenum to produce the corresponding acyl-6-aminopenicillanic acid (acyl-6-APA), whereby said acyl-6-APA is produced;
B) carrying out the following enzymatic conversion by in situ expression of the corresponding gene:
i) the acyl-6-APA is in situ ring-expanded to form the corresponding acyl-7-amino-desacetoxycephalosporanic acid (adipoyl-7-ADCA) by expandase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the expandase enzyme capable of accepting said acyl-6-APA as a substrate, whereupon as a result of its expression, said acyl-6-APA produced by said strain is also thereafter in situ-ring-expanded to form the corresponding acyl-7-ADCA;
ii) the 3-methyl side chain of said acyl-7-amino-desalkylcephalosporanic acid (acyl-7-ADAC) by hydroxylase enzyme, wherein said strain of P-chrysogenum has been transformed by DNA encoding the hydroxylase enzyme capable of accepting said acyl-7-ADCA as a substrate, whereupon as a result of its expression, said acyl-7-ADCA produced by said strain is also thereafter in situ hydroxylated to form the corresponding acyl-7-ADAC;
iii) the 3-hydroxmethyl side chain of said acyl-7-ADAC is in situ O-carbamoylated to yield the compound according to formula [I] by O-carbamoyl transferase enzyme, wherein said strain of P. chrysogenum has been transformed by DNA encoding the O-carbamoyl transferase enzyme capable of accepting said acyl-7-ADAC as a substrate, whereupon as a result of its expression, said acyl-7-ADAC produced by said strain is also thereafter in situ carbamoylated to form a compound according to the present invention.
5. A process for the production of a ceph-3-em antibiotic comprising the conversion of
the compound as defined in claim 1.
6. A process for the production of a 3-carbamoyloxymethyl-3-cephem antiotic
comprising the conversion of the compound as defined in claim 1.

7. A process as claimed in claim 6 wherein the 3-carbamoyloxymethyl-3-cephem
antiobiotic is selected from the group consisting of
a. Cefuroxime,
b. Cefroxitine
c. Cefcapene pivoxil
8. A process for the production of a 7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid or a salt or ester thereof comprising the conversion of the compound as defined in claim 1.
9. Micro-organism of the species P. chrysogenum capable of producing isopenicillin N and suitable for the production of the compounds as claimed in claims 1 to 3 which has been provided with DNA fragments encoding:
a. an expandase enzyme
b. a hydroxylase enzyme
c. a O-carbamoyl transferase enzyme.
10. Micro-organism of the species P. chrysogenum capable of producing isopenicillin N
and suitable for the production of the compounds as claimed in claims 1 to 3 which
has been provided with DNA fragments encoding:
a. a combined expandase/hydroxylase enzyme
b. a O-carbamoyl transferase enzyme.
11. Pharmaceutical preparation containing a compound as claimed in claims 1-3.


Documents:

5202-DELNP-2005-Abstract-(30-10-2008).pdf

5202-delnp-2005-abstract.pdf

5202-delnp-2005-Assignment-(26-07-2012).pdf

5202-DELNP-2005-Claims-(30-10-2008).pdf

5202-delnp-2005-claims.pdf

5202-delnp-2005-Correspondence Others-(26-07-2012).pdf

5202-DELNP-2005-Correspondence-Others-(30-10-2008).pdf

5202-delnp-2005-correspondence-others.pdf

5202-DELNP-2005-Description (Complete)-(30-10-2008).pdf

5202-delnp-2005-description (complete).pdf

5202-DELNP-2005-Drawings-(30-10-2008).pdf

5202-delnp-2005-drawings.pdf

5202-DELNP-2005-Form-1-(30-10-2008).pdf

5202-delnp-2005-form-1.pdf

5202-delnp-2005-Form-16-(26-07-2012).pdf

5202-delnp-2005-form-18.pdf

5202-DELNP-2005-Form-2-(30-10-2008).pdf

5202-delnp-2005-form-2.pdf

5202-DELNP-2005-Form-3-(30-10-2008).pdf

5202-delnp-2005-form-3.pdf

5202-delnp-2005-form-5.pdf

5202-delnp-2005-GPA-(26-07-2012).pdf

5202-DELNP-2005-GPA-(30-10-2008).pdf

5202-delnp-2005-gpa.pdf

5202-delnp-2005-pct-105.pdf

5202-delnp-2005-pct-210.pdf

5202-delnp-2005-pct-301.pdf

5202-delnp-2005-pct-304.pdf

5202-delnp-2005-pct-409.pdf

5202-delnp-2005-pct-416.pdf

5202-DELNP-2005-Petition-137-(30-10-2008).pdf

5202-DELNP-2005-Petition-138-(30-10-2008).pdf

abstract.jpg


Patent Number 232946
Indian Patent Application Number 5202/DELNP/2005
PG Journal Number 13/2009
Publication Date 27-Mar-2009
Grant Date 24-Mar-2009
Date of Filing 11-Nov-2005
Name of Patentee DSM IP ASSETS B.V.
Applicant Address HET OVERLOON 1, 6411 TE HEERLEN, THE NETHERLANDS
Inventors:
# Inventor's Name Inventor's Address
1 MARCO ALEXANDER VAN DEN BERG VOORSTRAAT 31B, 2685 EH, POELDIJK, THE NETHERLANDS
2 ROELOF ARY LANS BOVENBERG KRALINGSE PLASLAAN 10, 3062 DA ROTTERDAM, THE NETHERLANDS
3 ERIK DE VROOM DE MEIJ VAN STREEFKERKSTRAAT 65, 2313 JM LEIDEN, THE NETHERLANDS
4 ROELAND CHRISTIAAN VOLLINGA WEIDEBLOEMENLAAN 153, 3448 HC WOERDEN, THE NETHERLANDS
5 LOURINE MADELEINE RAAMSDONK KNOBBELZWAANSINGEL 15, 2496 LN DEN HAAN, THE NETHERLANDS
6 JOHN DAVID SUTHERLAND 7 NEWSTEAD CLOSE, POYNTON, SK12 1ES CHESHIRE, UNITED KINGDOM
PCT International Classification Number C07D 501/00
PCT International Application Number PCT/NL2004/000367
PCT International Filing date 2004-05-24
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 03076636.4 2003-05-28 EUROPEAN UNION