Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
  • C12P35/06—Cephalosporin C; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring

Definitions

• the invention relates to a process for preparing 3-cephalosporin C derivatives which are used in the preparation of ⁇ -lactam antibiotics.
• the invention relates to an enzymatic process for the preparation of 3-thiolated derivatives of 3-acetoxy-methyl-7-amino-ceph-3-em-carboxylic acid (3-thiolated -7-ACA) using ⁇ -ketoacid intermediates.
• the ⁇ -ketoacids or ⁇ -oxoacids are important biopharmaceutical compounds.
• Oxoacids of essential amino acids are gaining importance as nutraceuticals (Pszcola, DE, Food Technol. 52, 30, 1998) as well as therapeutic agents for treating nitrogen accumulation disorders (Schaefer et al, Kidney Int. Suppl 27,
• the transformation can be carried out by a D- amino acid transaminase from Bacillus licheniformis ATCC 9945, which converts cephalosporin C with ⁇ -ketoacids into ⁇ -ketoadipyl-7-ACA and the corresponding D- ⁇ -amino acid, as described in DE 3447023 (Hoechst).
• This conversion is a transamination, the amino group of cephalosporin C being converted non-oxidatively into the keto group, without the release of hydrogen peroxide.
• there is a low level of activity of this enzyme as described in EP 0315786.
• the first stage consists of using a D-amino acid oxidase (E.C. 1.4.3.3, hereinbelow indicated as DAAO) from different sources (Trigonopsis variabilis, GB 1,272,769; Rhodotorula gracilis, EP 0,517,200; or Fusarium solan M-0718, EP 0,364,275).
• DAAO D-amino acid oxidase
• DAAO oxidises the lateral D-5-amido-carboxypentanoyl chain of cephalosporin C in the presence of molecular oxygen, to produce 7 ⁇ -(5- carboxy-5-oxopent-amido)-ceph-3-em-carboxylic acid (or ⁇ -ketoadipyl-7- aminocephalo-sporanic acid, hereinbelow indicated as ⁇ -ketoadipyl-7-ACA) and hydrogen peroxide, which chemically decarboxylate the ⁇ -ketoadipyl-7-ACA to 7 ⁇ -(4-carboxy butanamido)-ceph-3-em-4-carboxylic acid (or glutaryl-7- aminocephalo-sporanic acid, hereinbelow indicated as GL-7-ACA).
• E.C. 3.5.1.3 a Pseudomonas type microorganism (US 3,960,662, EP 0496993) over expressed in E. coli, which deacylates the GL- 7-ACA into 7-amino-ceph-3-em-4-carboxylic acid (or 7-amino cephalosporanic acid, hereinbelow indicated as 7-ACA).
• This procedure can be defined as an enzymatic-chemical-enzymatic (ECE) process, since the isolated GL-7-ACA comes from a bioconversion of solubilised cephalosporin C, then GL-7-ACA is reacted with the heterocyclic thiols and finally the 3-heterocyclic thio-derivative is enzymated with GL-7-ACA acylase.
• ECE enzymatic-chemical-enzymatic
• R is a heterocyclic group comprising at least a nitrogen atom.
• the compound of formula III is enzymatically converted into a compound of formula IV by an immobilised enzyme system.
• the enzyme system comprises co-immobilised D-Amino acid oxidase and catalase.
• the enzymatic conversion is carried out in the presence of molecular oxygen, at a pressure of 1 to 5 bar absolute, a pH of from 6.5 to 8.0 and at a temperature of from 15 to 30°C for a period of from 30 mins to 180 mins.
• the process comprises the step of separating the enzyme system from the reaction mixture, preferably by filtration.
• the process includes the step of purifying the compound of formula IV.
• the compound is purified using an adsorption column.
• the enzymes are co-immobilised using a suitable cross-linker agent in a suitable solid support.
• the enzymes may be in the form of crystals of a size suitable for use as a biocatalyst.
• the enzymatic processes are carried out while maintaining the enzyme in dispersion in an aqueous substrate solution.
• the or each enzymatic process is carried out in a column.
• the process includes the step of recovering the enzyme for reuse.
• the compound of formula IV is used without purification in a continuous process for obtaining any useful derivative.
• R group in compounds of formula III and IV is a heterocyclic group comprising at least one nitrogen atom and optionally a sulphur or oxygen atom.
• R is a heterocyclic group selected from any one or more of the group comprising thienyl, diazolyl, tetrazolyl, thiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzo thiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably 5 -methyl- 1,3, 4-thiadiazol-2- yl, 1 -methyl- lH-tetrazol-5-yl or l,2,5,6-tetrahydro-2-methyl-5,6-dioxo-l,2,4- triazin-3-yl.
• the invention provides a 3-thiolated- ⁇ -ketoadipyl-7-aminocephalosporanic acid derivative of formula IV whenever prepared by a process of the invention.
• the invention provides a compound of the Formula:-
• R is l,2,5,6-tetrahydro-2-methyl-5,6-dioxo-l,2,4- triazin-3-yl.
• the invention provides a compound of the Formula:-
• R is 1 -methyl- lH-tetrazol-5-yl.
• the invention also provides use of a compound of formula IV as an intermediate in a process for preparing cephalosporin C antibiotics.
• the invention also provides use of an intermediate compound of the formula:-
• R is 5-methyl-l,3,4-thiadiazol-2-yl.
• the invention further provides a process for preparing cephalosporanic acid derivatives of the invention comprising the step of:
• R is a heterocyclic group comp ⁇ smg at least one nitrogen atom and Ri and R 2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
• the compound of formula IV is enzymatically converted to form a compound of formula I using Glutaryl-7-ACA acylase, most preferably the enzymation takes place at a temperature of approximately 20°C and at a pH of between 6.5 and 8.0.
• the enzyme is immobilised using a suitable cross-linker agent in a suitable solid support.
• the enzyme is in the form of crystals of a size suitable for use as a biocatalyst.
• the enzymation is carried out while maintaining the enzyme in dispersion in an aqueous substrate solution.
• the enzymatic process is carried out in a column.
• the process includes the step of recovering the enzyme for reuse.
• the invention also provides use of a compound of formula I as an intermediate in a process for preparing cephalosporin C derivatives.
• the invention further provides a process for preparing 3-thiolated cephalosporanic acid derivatives comprising the steps of;-
• R is a heterocyclic group comprising at least one nitrogen atom and Ri and R 2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
• the compound of formula III is enzymatically converted into a compound of formula I in one step by an immobilised enzyme system.
• the enzyme system comprises a combination of co-immobilised D-amino acid oxidase/catalase in the presence of immobilised Glutaryl-7-ACA acylase.
• the enzymation takes place at a temperature of approximately 20°C and at a pH of between 6.5 and 8.0.
• the enzymes are co-immobilised using a suitable cross-linker agent in a suitable solid support.
• the enzymes are in the form of crystals of a size suitable for use as a biocatalyst.
• the enzymatic processes are carried out while maintaining the enzyme in dispersion in an aqueous substrate solution.
• the or each enzymatic process is carried out in a column.
• the process includes the step of recovering the enzyme for reuse.
• the compound of formula III is used without purification in a continuous process for obtaining any useful derivative.
• the invention also provides a process for preparing cephalosporanic acid derivatives comprising the steps of:- reacting cephalosporin C with a thiol compound of the general formula II
• R is a heterocyclic group comprising at least one nitrogen atom, to form a 3-thiolated cephalosporin compound of formula III
• the excess thiol is removed by adsorption on an anion exchange resin.
• the anion exchange resin is a microporous resin having a cross-linked acrylic copolymer structure.
• the anion exchange resin comprises an 8% cross-linking containing functional thialkyl benzyl ammonium group.
• the resin may be in the chloride, hydroxy, phosphate or acetate cycle.
• the excess thiol is removed by crystallisation.
• crystallisation is carried out at an acidic pH.
• the excess thiol is removed by crystallisation followed by adsorption on an anion exchange resin.
• the cephalosporin C is in an aqueous medium.
• the cephalosporin C is in the form of a concentrated cephalosporin C solution.
• the reaction is carried out at a pH of between 5.5 and 8.0, at a temperature of from 60°C to 80°C, for a period of from 1 to 8 hours. Most preferably the reaction is carried out at a pH of approximately 6.0 and at a temperature of approximately 65°C.
• the thiol compound is present in an amount of between 1 and 5 mol/mol of cephalosporin C.
• R is a heterocyclic group comprising at least one nitrogen atom and optionally a sulphur or oxygen atom.
• R is a heterocyclic group selected from any one or more of thienyl, diazolyl, thiazolyl, tetrazolyl, thiadiazolyl, triazinyl, oxazolyl, oxadiazolyl, pyridyl, pirimidinyl, benzothiazolyl, benzimidazolyl, benzoxazolyl, or any derivative thereof, preferably 5-methyl-l,3,4-thiadiazol-2-yl, l-methyl-tetrazol-5-yl or 1,2,5,6- tetrahydro-2-methyl-5,6-dioxo-l,2,4-triazin-3-yl.
• the invention provides a compound of formula III
• R is a heterocyclic group comprising at least one nitrogen atom
• the invention provides a compound of the formula:-
• R is 5-methyl-l,3,4-thiadiazol-2-yl.
• the invention further provides a compound of the formula:-
• R is l,2,5,6-tetrahydro-2-methyl-5,6-dioxo-l,2,4- triazin-3-yl.
• the invention also provides use of a compound of formula III as an intermediate in a process for preparing cephalosporin C derivatives.
• One embodiment of the invention provides a process for preparing cephalosporanic acid derivatives comprising the steps of :-
• R is a heterocyclic group comprising at least a nitrogen atom.
• R is a heterocyclic group comprising at least one nitrogen atom and Ri and R 2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
• R is a heterocyclic group comprising at least one nitrogen atom and Ri and R 2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
• a compound of formula IV is enzymatically converted to form a compound of formula I with Glutaryl-7-ACA acylase.
• compounds of formula I, III and IV are in a solid form or in the form of a non-toxic salt thereof.
• the invention provides a process for the preparation of cephalosporin C antibiotics and derivatives thereof comprising forming a compound of formula III, IV and I as hereinbefore defined and subsequent enzymation of the compound.
• the antibiotic may be any one or more of cefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiam and ceftriaxone.
• 3-thiolated cephalosporanic C derivatives may be enzymated into ⁇ -ketoacid derivatives in the presence of a co-immobilised D- amino acid oxidase/catalase system. These ⁇ -ketoacid derivatives have been shown to be stable when isolated. A new improved process to obtain 3-thiolated -7-ACA derivatives, both in one step or in two consecutive enzymatic steps is thereby provided.
• the present invention relates to an improved and more efficient process for preparing compounds of formula I from cephalosporin C.
• R is a heterocyclic group comprising at least one nitrogen atom and Ri and R 2 are both hydrogen atoms or one of them is a hydrogen atom and the other is an acyl donor.
• the process involves the formation of new stable ⁇ -ketoadipyl-7-ACA derivative intermediates. Alternatively the process may be carried out in a single one pot reaction without the formation of intermediates.
• cephalosporin C are prepared from cephalosporin C.
• the cephalosporin C solution may be in a purified or crude form.
• the cephalosporin C is in the form of any non-toxic salt of cephalosporin C.
• the reaction of nucleophilic substitution in the 3 'position is carried out in an aqueous medium, dissolving the heterocyclic thiol and any non-toxic cephalosporin C salt in water by addition of a basic compound which form a water soluble salt, such as alkali metal hydroxide, ammonium hydroxide or preferably alkali metal carbonate or bicarbonate.
• a basic compound which form a water soluble salt such as alkali metal hydroxide, ammonium hydroxide or preferably alkali metal carbonate or bicarbonate.
• any commercially available salt of cephalosporin C and of the heterocyclic thiols can be used in the process of this invention without changing the fundamentals of the process.
• both reactants are mixed together in the same reactor, before or after heating the solution to a temperature from about 65°C to 80°C at a pH value of between 5.5 and 7.0.
• the temperature and pH are maintained preferably at approximately 65°C and 6.0 respectively, for a period of time of approximately 1 hour to 4 hours.
• the heterocyclic thiol/cephalosporin C molar ratio is an important variable in the yield of the reaction and has to be optimised for each heterocyclic thiol used.
• Molar ratios are between 1.0 and 4.0, preferably at a molar ratio of approximately 4.
• cephalosporin C remains quite stable with low ⁇ -lactam ring degradation, compared to a cephalosporin C solution without the thiol, which is completely degraded within 40 min at 80°C.
• the reaction mixture is cooled to a temperature from about 2°C to about 10°C, with or without acidification at a pH of from pH 3.0 to 5.5, preferably approximately 5.2, with strong mineral acids, such as hydrogen halides or oxy acids.
• This acidification step gives in some cases, crystallisation of the heterocyclic thiol, with the concomitant possibility of reuse for a new reaction.
• microporous resin offers certain advantages. They are less fragile, require less care in handling and possess higher loading capacities. As they have no discrete pores solute ions diffuse through the particle to interact with exchange sites. The total exchange capacity of the mentioned resin is in the order of 1,4 meq/mL.
• Amberlite IRA-400 has less binding capacity for 3 '-heterocyclic thiomethyl derivatives of cephalosporin C than for the same derivatives of glutaryl-7-ACA and 7-ACA.
• the 3 '-heterocyclic thiomethyl derivative of glutaryl-7-ACA produced with MMTD binds at a level of 76.3 % to the column.
• the same result is found with 3 '-heterocyclic thiomethyl derivative of 7-ACA, with MMTD, which binds at a level of 92.7 % to the column.
• the removal of heterocyclic thiols by the process of the invention is particularly advantageous on an industrial scale as the eluate of the column can be used for enzymation without isolation of the modified cephalosporin C and represents a new concept in the field of cephalosporin intermediates wherein the impurities are bound to the column and the ⁇ -lactam derivative is simply eluted by water.
• the column is typically regenerated with a 1.5 N solution of a strong mineral acid, such as hydrogen halide containing variable amounts of an organic solvent, preferably 10-20 % acetonitrile.
• a strong regeneration using 3 N HC1 and 40 % acetonitrile may be carried out.
• regeneration with 1.5M HC1 and 1.0 N NaOH is also possible.
• the thiol is concentrated and reused.
• the column is rinsed with deionised water to remove excess regenerant before the next cycle.
• the first bed volume of the rinse should be performed at the flow rate used for regeneration. The remainder is run at the adsorption flow rate.
• R is a heterocyclic group comprising at least a nitrogen atom with or without a sulphur or oxygen.
• a further industrial advantage of the invention is the easy transfer of the chemical solution comprising a compound of formula III, after chromatography in a strong anionic exchange resin (Amberlite ® IRA-400, manufactured by Rohm and Haas) to an enzymatic reactor containing the co-immobilised enzymes. This enables the process to be conducted continuously with a single liquid stream from cephalosporin C to compound IV.
• the enzymatic stage may be carried out in different ways:
• D-AAO in this invention is obtained from T ⁇ gonopsis variabilis CBS 4091 obtained from the Spanish collection of microorganisms (CECT, Valencia, Spain). This yeast is grown under the conditions to induce D-AAO (Kubicek et al, J. Appl Biochem. 7; 104, 1985) and the enzyme was purified by ammonium sulfate fractionation between 30%-55% as described by Szwjcer et al (Biotechnol Lett. 7, 1, 1985). Amino acid oxidases may also be sourced from Rodotorula gracilis.
• Catalase from Micrococcus lisodeikticus is obtained from a commercial source (Fluka, Madrid, Spain), but it may also be sourced from Aspergillas niger.
• Eupergit two classes are commercialised (Rohm Pharma) C and C250L, the latter type is particularly suitable for binding high molecular weight enzymes, since its contents in oxirane groups are at least 0.36% compared with the 0.93% in Eupergit C.
• This C250L type show outstanding properties when employed in industrial biocatalytic processes.
• the morphology of the carrier i.e., its narrow particle size distribution (200 ⁇ m) and high mechanical stability accounts for their good properties in stirred-tank reactors. It is not mechanically destroyed in stirred systems and filtration at the end of the reaction cycle is quick and very easy to perform. Changes in the pH and ionic strength have no effect on swelling of the matrix.
• this Eupergit C250L has never been used to immobilise D-amino acid oxidase and catalase.
• One unit of D-AAO is defined as the amount of enzyme that consumes a ⁇ mol of O 2 per minute using cephalosporin C as substrate at pH 8.0 and 25°C.
• One unit of catalase is defined as the amount of enzyme that decomposes 1 ⁇ mol of hydrogen peroxide per minute at pH
• the enzymatic conversion of the compound III into compound IV is carried out in an aqueous solution of compound III containing from about 0.0016 to 0.004 moles and with less than 0.2 mg/ml of the heterocyclic thiol.
• This solution is obtained after passing the solution comprising 3-thiolated cephalosporin C (compound III) and remaining heterocyclic thiol used in the nucleophilic displacement of 3 acetoxy group of cephalosporin C through a column of a strong anion exchanger, such as Amberlite ® IRA-400 (Rohm and Hass).
• the pH of the eluate is adjusted to pH about 6.5 to 8.0, preferably to pH 6.75, due to the instability of cephalosporanic compounds at basic pH values.
• the solution comprising compound III as described above is fed into a bioreactor, containing wet Eupergit C250L with co-immobilised D- AAO/catalase, usually D-AAO from 20-40 U/g, and catalase, usually from 10- 30 kU/g.
• the reaction temperature can be fixed from 15°C to 35°C, and is normally fixed at 20°C.
• the molecular oxygen, needed for the oxidative deamination, is blown into solution by a bottom diffuser at a flow rate from 0.01 to 1 volume/volume of solution/minute, preferably at 0.1 wm under a suitable mechanical stirring of about 400 rpm.
• This bioreactor design is preferred versus a percolation column containing the immobilised enzymes to avoid the diffusional problems of the molecular oxygen, which reduce the yield of compound IV.
• the pH is titrated to pH 6.75 by dosing a concentrated organic or inorganic base, preferably 3 M ammonia, by means of an autotitrator.
• the conversion is controlled by HPLC and when the conversion of compound III is greater than 97%, the reaction is stopped and the solution filtered off.
• the time required for such conversion is of the order 0.5 to 3 hours, depending on the operating conditions, but usually approximately 1 hour.
• Isolation of compound IV when required, is carried out by decreasing the pH of the above solution to a pH of about 4.5 to 6.0, preferably 5.0 with the same base used during the enzymatic reaction, and loading into a column packed with the adsorptive resin Amberlite XAD-2.
• the elution of compound IV is carried out with water at a flow rate of 2-3 bed volumes per hour. Fractions containing the compound IV with a HPLC purity higher than 90-95% are pooled and lyophilised.
• the process of the invention for preparing compounds of formula I from cephalosporin C may also be carried out in a single one pot reaction.
• filtrate from an anion exchange column comprising compounds of formula III is enzymatically converted into compounds of formula I by an immobilised enzyme system comprising D-AAO and catalase in the presence of glutaryl-7- ACA acylase.
• Compounds of formula I have been prepared in this way with a HPLC of approximately 95%. The process is easy and efficient to carry out.
• 3-thiolated-7-ACA derivatives are easily and economically prepared.
• These compounds may by subsequent enzymation with penicillin G acylase for example, be used for the preparation of semisynthetic ⁇ -lactam antibiotics.
• the ⁇ -lactam antibiotics may include any one or more of cefazolin, cefazedone, cefoperazone, cefamandol, cefatriazine, cefotiam and ceftriaxone.
• Examples 1 to 5 illustrates the preparation of 3-thiolated-7-ACA derivatives of formula III from cephalosporin C.
• Examples 6 to 8 illustrate the enzymatic process for the preparation of a 3- thiolated ⁇ -ketoadipyl-7-ACA derivatives of formula IV from 3-thiolated cephalosporin C derivatives of formula III.
• Examples 9 to 11 illustrate the enzymatic process for the preparation of 3- thiolated-7-ACA derivatives (TXA) of formula I from 3-thiolated derivatives of formula III via the formation of stable ⁇ -ketoadipyl-7-ACA derivatives of formula IV.
• Examples 12 to 14 illustrate the enzymatic process for the preparation of 3- thiolated-7-ACA derivatives of formula I from 3-thiolated derivatives of formula III in a single step (one pot).
• EXAMPLE 1 Preparation of 7- ⁇ -(5-amino-5-carboxypentanamido)-3-(5-methyl- l,3,4-thiadiazole-2-yl thiomethyl)-3-cephem-4-carboxylic acid (TDC).
• MMTD 2-mercapto-5-methyl-l,3,4-thiadiazole
• a solution of concentrated sodium cephalosporin C (98 % purity by HPLC) was prepared by dissolving 33.23 g of sodium cephalosporin C (75 % free acid, 0.06 moles) in 200 ml of water.
• the concentrated cephalosporin C solution was added and the mixture was stirred at approximately 65°C for 240 minutes, controlling the reaction kinetic until the level of cephalosporin C was below 2 %. The following reaction kinetics were found:
• the reaction mixture was then cooled to about 4°C, where the crystallisation of the excess of MMTD begins.
• the pH was acidified with stirring (150 rpm) to a pH 5.2 with 37 % hydrochloride acid and left under slow stirring (50 rpm) for 60 minutes for the completion of crystallisation.
• the precipitated MMTD was filtered and dried at 35°C under vacuum. 23 g of recovered MMTD was obtained (purity 99 % by HPLC) with a recovery yield of about 95 %.
• the column is regenerated with 1 L of 1.5 M HCl containing 10 % acetonitrile and rinsed free of the excess regeneration by washing with 2 litres of deionised water.
• the resin can be subjected to a strong regeneration using 1 litre of 3 M HCl with 40 % acetonitrile.
• regeneration with 1.5 M HCl and 1.0 N NaOH is also possible.
• the TDC solution at pH 5.0 was loaded onto a Amberlite XAD-2 adsorption column and the column was washed with water. After washing, the resin was eluted with water, and 25 ml portions were pooled.
• EXAMPLE 2 Comparative Example: Preparation of 7- ⁇ -(5-amino-5- carboxypentanamido)-3-(5-methyl-l,3,4-thiadiazole-2-ylthiomethyl)-3-cephem-4- carboxylic acid on different columns.
• the TDC derivative was prepared as described in Example 1 and the filtrate containing it was loaded onto different types of resins. The following data was obtained after washing with 100 ml of water in the first cycle of column usage:
• the glutaryl-7-ACA derivative (TDG) and 7-ACA derivative (7-TDA) were prepared as in Example 1 using glutaryl-7-ACA and 7-ACA as starting material. The following data was obtained from the filtrate of the Amberlite IRA-400.
• TDG and TDA appear to remain bound to the Amberlite IRA-400 as well as the MMTD.
• EXAMPLE 4 Preparation of 7- ⁇ -(5-amino-5-carboxypentanamido)-3-[(l- methyl-lH-tetrazol-5-yl)-thiomethyl]-cephalosporanic acid (TZC).
• MMTZ 5-mercapto-l-methyltetrazole
• a solution of concentrated sodium cephalosporin C was prepared by dissolving 33.23 g of sodium cephalosporin C (75 % free acid, 0.06 moles purity 98 % by HPLC) in 200 ml of water.
• the concentrated cephalosporin C solution was added and the mixture was stirred at about 70°C for 120 minutes, controlling the reaction kinetic until the level of cephalosporin C was below 3 %.
• the reaction mixture was cooled at about 4°C, but crystallisation of the excess of MMTZ did not start, even when the pH was decreased.
• the solution containing 0.04 moles of the TZC derivative from MMTZ and 0.19 moles of MMTZ was adjusted to pH 7.25 with 3 M ammonia and loaded onto an Amberlite IRA-400 column in chloride cycle (bed volume equal to 150 ml) covered with deionised water at flow rate 20 ml/min. After the first pass through the column the remaining MMTZ was higher than 13 % of the initial (0.032 moles).
• MMTZ concentration was 0.0013 moles, which is less than 1 % of the original MMTZ after chemical reaction. With this low level of MMTZ, enzymation of the derivative is possible without poisoning the enzyme.
• EXAMPLE 5 Preparation of 7- ⁇ -(5-amino-5-carboxypentanamido)-3-[(l,2,5,6- terahydro-2-methyl-5,6-dioxo-l,2,4-triazin-3-yl)-thiomethyl]-cephalosporanic acid (TTC).
• TTZ 2,5-dihydro-3-mercapto-2-methyl-5,6-dioxo-l,2,4-triazine
• cephalosporin C was prepared by dissolving 33.23 g of sodium cephalosporin C (75 % free acid, 0.06 moles, purity 98 % by HPLC) in 200 ml of water. When the TTZ was dissolved, the concentrated cephalosporin C solution was added and the mixture was stirred at approximately 75°C for 75 minutes, controlling the reaction kinetic until the level of cephalosporin C was below 2 %.
• the reaction mixture was cooled at approximately 4°C, but crystallisation of the excess of TTZ does not start, even when the pH was decreased.
• the solution containing the 0.036 moles of TTC and 0.19 moles of TTZ was adjusted to pH 7.25 with 3 M ammonia and loaded onto an Amberlite IRA-400 column in chloride cycle (bed volume equal to 209 ml) covered with deionised water at flow rate 20 ml/min. After the first column the remaining TTZ was 0.015 moles.
• the column was washed with deionised water (ca 120 ml) until 60 % recovery of loaded TTC with a 90% purity by HPLC.
• the pH of the effluent was about 5.4 and was neutralised to pH 7.0 with 3 M ammonia. The remaining
• TTZ concentration was 0.00096 moles, which is less than 1 % of the original TTZ after chemical reaction. With this level of TTZ, enzymation of the derivative is possible without poisoning the enzyme.
• EXAMPLE 6 Preparation of 7 ⁇ -(5-carboxy-5-oxopentamide)-3-[(5-methyl- l,3,4-thiadiazole-2-yl)-thiomethyl] cephalosporanic acid (TDK).
• Amberlite ® IRA-400 containing 0.0035 moles of 7 ⁇ -(5-amino-5-carboxypentamido)-3-[(5-methyl- l,3,4-thiadiazole-2-yl)-thiomethyl] cephalosporanic acid (TDC) with 94.3 % purity (HPLC) and less than 0.2 mg/mL of 2-mercapto-5-methyl-l,3,4
• the TDC solution was fed into a 0.125 litre stirred glass vessel with 30.76 g of wet Eupergit C250L with a co-immobilised D-amino acid oxidase/catalase system (11.77 U of DAAO/g and 15 kU of catalase/g).
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 6.75 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Nucleosil 120 3-C18 125 x 8 x 4 mm.
• the mobile phase was 20 mM acetate ammonium pH 5.5 containing 4 % acetonitrile at 1 ml/min with a 260 nm detection.
• the TDC appeared at 7.0 minutes, the TDK at 8.5 min and the 3-thiolated glutaryl-7- ACA intermediate (TDG) at 11.5 min.
• EXAMPLE 7 Preparation of 7 ⁇ -(5carboxy-5-oxopentanamide)-3-[(l-methyl- lH-tetrazol-5-yl)-thiomethyl]-cephalosporanic acid (TZK).
• Amberlite ® IRA-400 containing 0.0039 moles of 7-(5'-amidoadipamido)-3-[(l-methyl-lH-tetrazol-5- yl)-thiomethyl] -cephalosporanic acid (TZC) with 90.1 % purity (HPLC) and less than 0.2 mg/ml of 5-mercapto-l-methyltetrazole (MMTZ) was adjusted to pH 6.75 with 3 M am
• the TZC solution was fed into a 0.125 litre stirred glass vessel with 30.76 g of wet Eupergit C250L with a coimmobilised D-amino acid oxidase/catalase system (11.77 U of DAAO/g and 15 kU of catalase/g).
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 6.75 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC on a reverse phase column Nucleosil 120 3 - C18 125 x 8 x 4 mm.
• the mobile phase was 20 mM ammonium acetate pH 5.5 containing 4% acetonitrile at 1 ml/min with a 260 nm detection.
• the TZC appeared at 3.0 minutes, the TZK at 3.6 min and the 3-thiolated glutaryl-7- ACA intermediate (TZG) at 4.6 min.
• the resulting solution was adjusted to pH 5.0 with 3 M ammonia and passed through a column packed with 40 g of the adsorptive resin
• EXAMPLE 8 Preparation of 7 ⁇ -(5carboxy-5-oxopentanamide)-3-[(l,2,5,6- tetrahydro-2-methyl-5,6-dioxo-l,2,4-triazin-3-yl)-thiomethyl]-cephalosporanic acid (TTK).
• the TTC solution was fed into a 0.125 litre stirred glass vessel with 30.76 g of wet Eupergit C250L with a co-immobilised D-amino acid oxidase/catalase system (11.77 U of DAAO/g and 15 kU of catalase/g).
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 6.75 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a column Eclipse ® XDB-C8 5 ⁇ m 4.6 x 150 mm.
• the mobile phase was 35 % methanol in 10 mM TBHS (tetrabutylammonium hydrogen sulfate) and 15 mM potassium dihydrogen phosphate at 1 ml/min with 260 nm.
• the TTC appeared at 2.6 minutes, the 3- thiolated glutaryl-7-ACA intermediate (TTG) at 5.5 min and the TTK at 6.6 min.
• the resulting solution was adjusted to pH 5.0 with 3 M ammonia and passed through a column packed with 40 g of the adsorptive resin Amberlite XAD-2 (68.7 ml of bed volume). Elution was carried out with water at a flow rate of 200 ml/h (about 3 bed volumes per hour). Fractions of 25 ml containing the TTK with a purity >90 % (HPLC) were pooled and then lyophilised to obtain the target product as solid to further analyse it. After the elution process, the adsorbent surface is reactivated by applying 2 bed volumes of regeneration solution (25% methanol in water at 3 bed volumes per hour). Before the column can be used again, this solution is removed from the column.
• EXAMPLE 9 Synthesis of 7-amino-3-[(5-methyl-l,3,4-thiadiazol-2-yl)- thiomethyl] -cephalosporanic acid (TDA).
• Amberlite ® IRA-400 containing 0.0011 moles of 7 ⁇ -(5-amino-5-carboxypentamido)-3-[(5-methyl- l,3,4-thiadiazole-2-yl)-thiomethyl] cephalosporanic acid (here below indicated as TDC) with 94.01 % purity (HPLC) and less than 0.2 mg/m
• the TDC solution was fed into a 0.125 litre stirred glass vessel with 16 g of wet Eupergit C250L with a co-immobilised D-amino acid oxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g).
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 6.75 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Eclipse XDB-C8 150 mm x 4.6 mm ID x 5 ⁇ m; the mobile phase was 10 mM tetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogen phosphate, pH 6.5 containing 35 % methanol at 1 ml/min with a 260 nm detection.
• the TDC appeared at 3.0 min, the TDK at 10.9 min and the TDG at 8.1 min, respectively.
• Representative samples of the reaction mixture except for the enzyme were taken and the result obtained are reported as percentage of total ⁇ -lactams in the following table:
• the reaction was stopped and the reaction solution, containing TDK with a HPLC purity of 88.27 %, was filtered off, and adjusted to pH 7.25 with 3 M ammonia.
• the TDK solution was fed into a 0.125 litre stirred glass vessel with 23 g of wet
• Glutaryl-7-ACA Acylase (87 U/g). The conversion was performed at 20 °C, 400 rpm at 1 bar absolute pressure. The pH was titrated to pH 7.25 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC under the above-mentioned conditions.
• the reaction solution contained TDA with a HPLC purity of 88.59%.
• EXAMPLE 10 Synthesis of 7-amino-3-[(l-methyl-lH-tetrazol-5-yl)-thiomethyl]- cephalosporanic acid (TZA).
• the TZC solution was fed into a 0.125 litre stirred glass vessel with 16 g of wet Eupergit C250L with a coimmobilised D-amino acid oxidase/catalase system
• the conversion was performed at 20 °C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 6.75 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Eclipse XDB-C8 150 mm x 4.6 mm ID x 5 ⁇ m; the mobile phase was 10 mM tetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogen phosphate, pH 6.5 containing 35 % methanol at 1 ml/min with a 260 nm detection.
• the TZC appeared at 2.1 minutes, the TZK at 5.0 min and the TZG at 4.3 min, respectively. Representative samples of the reaction mixture except for the enzyme were taken and the result obtained are reported as percentage of total ⁇ -lactams in the following table:
• the reaction was stopped and the reaction solution, containing TZK with a HPLC purity of 82.42 %, was filtered off, and adjusted to pH 7.25 with 3 M ammonia.
• the TZK solution was fed into a 0.125 litre stirred glass vessel with 23 g of wet
• Glutaryl-7-ACA Acylase (87 U/g). The conversion was performed at 20 °C, 400 rpm at 1 bar absolute pressure. The pH was titrated to pH 7.25 with 3 M ammonia by an autotitrator.
• the reaction solution contained TZA with a HPLC purity of 80.77 %.
• EXAMPLE 11 Synthesis of 7-amino-3-[(l,2,5,6-tetrahydro-2-methyl-5,6-dioxo- l,2,4-triazin-3-yl)-thiomethyl] -cephalosporanic acid (TTA).
• TTC 7 ⁇ -(5-amino-5-carboxypentamido)-3-[(l,2,5,6- tetrahydro-2-methyl-5,6-dioxo-l,2,4-triazin-3-yl)-
• the TTC solution was fed into a 0.125 litre stirred glass vessel with 16 g of wet
• Eupergit C250L with a coimmobilized D-amino acid oxidase/catalase system 25 U of DAAO/g and 30 kU of catalase/g.
• the conversion was performed at 20 °C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 6.75 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Eclipse XDB-C8 150 mm x 4.6 mm ID x 5 ⁇ m; the mobile phase was 10 mM tetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogen phosphate, pH 6.5 containing 35 % methanol at 1 ml/min with a 260 nm detection.
• the TTC appeared at 2.4 minutes, the TTK at 6.1 min and the TTG at 5.5 min, respectively.
• Representative samples of the reaction mixture except for the enzyme were taken and the result obtained are reported as percentage of total ⁇ -lactams in the following table:
• the reaction was stopped and the reaction solution, containing TTK with a HPLC purity of 90.0 %, was filtered off, and adjusted to pH 7.25 with 3 M ammonia.
• the TTK solution was fed into a 0.125 litre stirred glass vessel with 23 g of wet
• Glutaryl-7-ACA Acylase (87 U/g ). The conversion was performed at 20 °C, 400 rpm at 1 bar absolute pressure. The pH was titrated to pH 7.25 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC under the above-mentioned conditions.
• Amberlite ® IRA-400 containing 0.0011 moles of 7 ⁇ -(5-amino-5-carboxypentamido)-3-[(5-methyl- 1, 3, 4-thiadiazole-2-yl)-thio methyl] cephalosporanic acid (hereinbelow indicated as TDC) with 95.41 % purity (HPLC) and less than 0.2 mg/ml of 2-mercapto
• the TDC solution was fed into a 0.125 litre stirred glass vessel with 16 g of wet Eupergit C250L with a co-immobilised D-amino acid oxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g) and 23 g of wet Glutaryl-7-ACA Acylase (87 U/g).
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 7.25 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Eclipse XDB-C8 150 mm x 4.6 mm ID x 5 ⁇ ; the mobile phase was 10 mM tetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogen phosphate, pH 6.5 containing 35 % methanol at 1 ml/min with a 260 nm detection.
• the TDC appeared at 3.0 min, the TDK at 10.9 min, the TDG at 8.1 min and the TDA at 4.1 min, respectively.
• the reaction solution contained TDA with a HPLC purity of 95.13 %.
• EXAMPLE 13 Synthesis of 7-amino-3-[(l-methyl-lH-tetrazol-5-yl)-thiomethyl]- cephalosporanic acid (TZA) in a single step.
• Amberlite ® IRA-400 containing 0.00195 moles of 7 ⁇ -(5-amino-5-carboxypentamido)-3-[(l-methyl-lH- tetrazol-5-yl)-thiomethyl] cephalosporanic acid (hereinbelow indicated as TZC) with 93.55 % purity (HPLC) and less than 0.2 mg/ml of 5-mer
• the TZC solution was fed into a 0.125 litre stirred glass vessel with 16 g of wet Eupergit C250L with a co-immobilised D-amino acid oxidase/catalase system (25 U of DAAO/g and 30 kU of catalase/g) and 23 g of wet Glutaryl-7-ACA Acylase (87 U/g).
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 7.25 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Eclipse XDB-C8 150 mm x 4.6 mm ID x 5 ⁇ ; the mobile phase was 10 mM tetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogen phosphate, pH 6.5 containing 35 % methanol at 1 ml/min with a 260 nm detection.
• the TZC appeared at 2.1 minutes, the TZK at 5.0 min, the TZG at 4.3 min, and the TZA at 2.5 min, respectively.
• the reaction solution contained TZA with a HPLC purity of 78.17 %.
• EXAMPLE 14 Synthesis of 7-amino-3-[(l,2,5,6-tetrahydro-2-methyl-5,6-dioxo- l,2,4-triazin-3-yl)-thiomethyl]-cephalosporanic acid (TTA) in a single step.
• the TTC solution was fed into a 0.125 litre stirred glass vessel with 16 g of wet Eupergit C250L with a co-immobilised D-amino acid oxidase/catalase system
• the conversion was performed at 20°C, 400 rpm and with an oxygen flow through a bottom diffuser of 0.1 vol/vol/min at 1 bar absolute pressure.
• the pH was titrated to pH 7.25 with 3 M ammonia by an autotitrator.
• the conversion was controlled by HPLC in a reverse phase column Eclipse XDB-C8 150 mm x 4.6 mm ID x 5 ⁇ ; the mobile phase was 10 mM tetrabutylammonium hydrogen sulfate, 15 mM potassium dihydrogen phosphate, pH 6.5 containing 35 % methanol at 1 ml/min with a 260 nm detection.
• the TTC appeared at 2.4 minutes, the TTK at 6.1 min, the TTG at 5.5 min and the TTA at 2.9, respectively.
• the reaction solution contained TTA with a HPLC purity of 88.69 %.

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