Queen's University at Kingston

Saul Wolfe

Susan E Jensen

Donald Westlake

Chia-Cheng Shaw

A process for producing unnatural penicillins and cephalosporin derivatives thereof in which peptide analogs of ACV in which the L-.alpha.-aminodipyl moiety is replaced by L-S-carboxymethyl cysteine or other obvious substituents, are reacted with cyclase, epimerase, ring expansion and hydroxylase enzymes isolated from a cell free extract of a prokaryotic organism such as S. clavuligerus. The product depends upon the presence or absence of co-factors such as ferrous ion, .alpha.-ketoglutarate, and ascorbate. In an alternative embodiment, a penicillin analog having the formula: ##STR1## may be reduced with L-cysteine to produce an analog of isopenicillin N which may be reacted with the enzyme reagent to produce the desired penicillin or cephalosporin.

Biosynthesis of unnatural cephalosporins

Enzymes extracted from a prokaryotic .beta.-lactam producing microorganism are immobilized on a support for producing desacetoxycephalosporin and analogs thereof from L-.alpha.-aminoadipyl-L-cysteinyl-D-valine and analogs thereof. The enzymes are an epimerase, a cyclase and a ring expansion enzyme extracted preferably from S. clavuligerus, S. cattleya or S. lipmanii. The support is preferably a diethylaminoethyl ion exchange resin.

Enzyme immobilization for producing cephalosporin antibiotics

A process is disclosed for converting a peptide precursor of the ACV type, in which the valine moiety may be replaced by any readily available amino acid, to an unnatural cephalosporin of the type ##STR1## where R.sub.1 is H, lower alkyl or functionalized carboxylic group and R.sub.2 is H or lower alkyl, in which the precursor is reacted with cyclase, epimerase and a ring expansion enzyme isolated from a cell-free extract of a prokaryotic organism such as Streptomyces clavuligerus. The three enzymes may be immobilized on a suitable support medium and the conversion may be carried out in a continuous mode.

Cyclase, epimerase and a ring expansion enzyme are isolated separately from a cell free extract of a prokaryotic beta-lactam producing organism to provide three separate and stable enzyme reagents for commercial production of cephalosporins from peptide precursors. Isolation is carried out by using ammonium sulfate, gel filtration and ion exchange chromatography. The enzymes may be immobilized on a suitable support and the production of cephalosporins may be carried out continuously.

Stable epimerase reagent, cyclase reagent and ring expansion reagent for

A process for preparing compounds of the formula ##STR1## in which R is hydrogen or methoxy,

Intermediates for process for 1-oxacephem derivatives

The enzymes, cyclase, epimerase and a ring expansion enzyme, are isolated separately from a cell-free extract of a prokaryotic beta-lactam producing organism. The isolated enzymes are used to produce unnatural cephalosporins from polypeptide precursors. Isolation is carried out by adding ammonium sulfate to 40% saturation to the cell-free extract to precipitate contaminating proteins, adding ammonium sulfate to 70% saturation to the resultant supernatant to precipitate the enzymes, suspending the precipitated enzymes in a buffer, separating epimerase from the suspension by gel filtration, and separating cyclase and the ring expansion enzyme from each other by ion exchange chromatography.

Isolation of cyclase, epimerase and a ring expansion enzyme for

Process for 1-oxacephem derivatives

The invention provides a novel process for opening the ring of 2-R-6-(1'-R.sup.1 -oxycarbonyl-2'-methyl-prop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0]h ept-2-en-7-one by treatment with a sulfonic acid salt thereby forming R.sup.1 2-(2'R-R-oxy-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate sulfonic acid salts which can provide oxapenicillins by two different routes depending on the substituent desired on the carboxamido group of the 1-oxapenicillins. In the alternate processes, new intermediates are also provided.

Preparation of 1-oxapenicillins and 4-acyloxy azetidinone intermediates

The present invention relates to a novel group of compounds which are broadly referred to as 1-oxacephems and processes for preparing same.

4-Chloroazetidinone

The invention provides a novel process for opening the ring of 2-R-6-(1'-R.sup.1 -oxycarbonyl-2'-methyl-prop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0]h ept-2-en-7-one by treatment with a sulfonic acid salt thereby forming R.sup.1 2-(2'R-R-oxy-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate sulphonic acid salts which can provide oxapenicillins by two different routes depending on the substituent desired on the carboxamido group of the 1-oxapenicillins. In the alternate processes, new intermediates are also provided.

Preparation of 1-oxapenicillins and intermediates therefor

The title compounds, which can casually be called 1-oxapenicillins, are produced by total synthesis. Thus 1-oxabenzylpenicillin is produced either by phenylacetylation of 2,2-dimethyl-3R-carboxy-6S-amino-1-oxa-4-aza-5R-bicyclo[3,2,0]heptan-7-one or by hydrolysis and rearrangement of 2-benzyl-6-(1'-methoxycarbonyl-2'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S, 5R-bicyclo[3,2,0]hept-2-en-7-one.

2,2-Dimethyl-3R-carboxy-6S-

The present invention relates to novel 1-oxacephems of the formula ##STR1## wherein R.sup.9 is hydrogen and R.sup.8 is hydrogen or

1-Oxacephems

Chlorinated esters of anhydrobenzylpenicillin are reacted with alumina or silica gel to produce fused oxazoline azetidinones. One product may be named methyl 2-(3-benzyl-7-oxo-4-oxa-2,6-diazabicyclo[3,2,0]hept-2-en-6-yl)-3-methylbut -2-enoate or 2-benzyl-6-(1'-methoxycarbonyl-2'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S,5 R-bicyclo[3,2,0]hept-2-en-7-one.

Production of fused oxazoline azetidinones

The title compounds, which can also be called oxapenicillins G and V, are produced by chemical synthesis and then, if desired, are deacylated.

2,2-Dimethyl-3R-carboxy-6S-phenyl(or

The title compound is produced by chemical synthesis and, if desired, converted as by N-phenylacetylation to a new .beta.-lactam antibiotic such as oxapenicillin G.

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Kingston, Canada

Saul Wolfe