Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-4-aza-bicyclo
Reexamination Certificate
2000-04-18
2001-09-11
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-4-aza-bicyclo
C435S041000, C435S050000, C435S177000, C435S178000, C435S180000
Reexamination Certificate
active
06287799
ABSTRACT:
The invention relates to a process for the preparation of a &bgr;-lactam antibiotic in which a &bgr;-lactam nucleus is subjected to an enzymatic acylation reaction with the aid of an acylation agent at a molar ratio of acylation agent/&bgr;-lactam nucleus of less than 2.5.
A similar process is disclosed in for example WO-A-96/23897.
The yield of &bgr;-lactam antibiotic to be achieved in the enzymatic acylation reaction according to the prior art per amount of &bgr;-lactam nucleus employed and per amount of acylation agent employed is in general relatively low inasmuch as the &bgr;-lactam nuclei and/or the &bgr;-lactam antibiotics often are relatively instable, whilst the reaction time is relatively long because of the usually low solubility of the reactants. Moreover, at the relatively low acylation agent to &bgr;-lactam nucleus ratio mentioned, only a relatively low yield of &bgr;-lactam antibiotic can be achieved per amount of &bgr;-lactam nucleus employed.
The invention provides a process in which in an enzymatic acylation reaction a shorter reaction time is achieved and a higher yield of &bgr;-lactam antibiotic is achieved per amount of &bgr;-lactam nucleus and/or per amount of acylation agent employed, with the ratio of the amount of acylation agent employed to &bgr;-lactam nucleus being relatively low.
This is achieved according to the invention in that the acylation agent and/or the &bgr;-lactam nucleus are/is supersaturated in the reaction mixture during at least part of the acylation reaction.
The applicant has found that it is possible to achieve a high degree of supersaturation of the &bgr;-lactam nucleus and/or the acylation agent in the reaction mixture and that, especially surprisingly, the supersaturation can be kept stable for hours. This allows the concentration of the &bgr;-lactam nucleus and/or the dissolved acylation agent to be strongly increased, so that the reaction proceeds more rapidly, with less degradation of the &bgr;-lactam antibiotic and/or the reactants and also with a higher yield of &bgr;-lactam antibiotic per amount of &bgr;-lactam nucleus employed and/or per amount of acylation agent employed. This also results in a higher production capacity.
In addition, it is known from literature, for example WO-A-92/01061, that a high yield of &bgr;-lactam antibiotic per amount of &bgr;-lactam nucleus can be obtained by applying a high molecular ratio between the acylation agent and the &bgr;-lactam nucleus. However, a drawback of applying a high molecular ratio between the acylation agent and the &bgr;-lactam nucleus is that large amounts of acylation agent are lost as a result of hydrolysis of the acylation agent (and possibly the &bgr;- lactam antibiotic). Consequently, a low synthesis/hydrolysis ratio (S/H), the molar ratio between synthesis product (&bgr;-lactam antibiotic) and hydrolysis product, is realized. Moreover, it has been found that working up the &bgr;-lactam antibiotic is often hampered by a relatively large amount of hydrolyzed acylation agent relative to &bgr;-lactam antibiotic being present in the reaction mixture obtained after the enzymatic acylation reaction, as a result of which a smaller amount of &bgr;-lactam antibiotic can be isolated.
For the purposes of the present invention, the yield of &bgr;-lactam antibiotic per amount of reactant (&bgr;-lactam nucleus or acylation agent) to be achieved in the acylation reaction means the (molar) amount of &bgr;-lactam antibiotic formed in the acylation reaction per (molar) amount of reactant employed.
For the purposes of the present invention, the solubility of a compound in a mixture means the dissolved concentration of the compound in the presence of all other components of the mixture, expressed in mmol/litre or mass %. The solubility is measured by dissolving the compound at constant pH and temperature and in the presence of all components of mixture. Thereafter, the solubility can be calculated from the amount of the compound dissolved on reaching equilibrium (saturated solution).
For the purposes of the present invention, a compound is supersaturated in a mixture when the dissolved concentration of that compound in the mixture is greater than the solubility. The supersaturation factor means the ratio between the two aforementioned solubilities (supersaturated divided by saturated). The supersaturation factor to be achieved and the time during which supersaturation is maintained depend on a number of factors such as the nature and concentration of the compound, the nature and concentrations of the other components in the mixture, the pH and the temperature. The supersaturation factor to be obtained depends largely on the compound involved and is preferably larger than 2, more particularly larger than 5.
The concentration of the dissolved &bgr;-lactam nucleus is expressed as the amount of dissolved &bgr;-lactam nucleus in moles per kg of liquid reaction mixture; the total concentration of dissolved and undissolved &bgr;-lactam nucleus is expressed as the amount of &bgr;-lactam nucleus in moles per kg of the total reaction mixture; the total reaction mixture may contain, besides the solution, a plurality of solids, for example &bgr;-lactam nucleus, &bgr;-lactam antibiotic, (hydrolyzed) acylation agent and immobilized enzyme. Similar definitions are applicable for the acylation agent and the &bgr;-lactam antibiotic.
A mixture in which the &bgr;-lactam nucleus or the acylation agent, respectively, is supersaturated can be obtained by means of for example a pH shift. To in that end, if necessary, a concentrated mixture can first be prepared as a slurry or a solution by dissolving &bgr;-lactam nucleus or acylation agent, respectively, present in solid form with the aid of for example a pH increase or a pH decrease, or a pH decrease, respectively. It is preferred for the &bgr;-lactam nucleus and/or the acylation agent to be dissolved in the mixture obtained. However, it is also possible for a portion of the &bgr;-lactam nucleus and/or the acylation agent to be still present in solid form. Subsequently, this slurry or solution can be subjected to a pH decrease or a pH increase, or a pH increase, respectively. In this way, a slurry or solution is obtained in which the &bgr;-lactam nucleus or acylation agent is supersaturated.
Any solid &bgr;-lactam nucleus present can be dissolved by for example decreasing the pH until a pH lower than 3, preferably lower than 2, in particular lower than 1 is reached; or by increasing the pH to a pH higher than 6, preferably higher than 7, in particular higher than 8. In practice, the final pH is preferably chosen such that the &bgr;-lactam nucleus goes only just completely into solution so that as concentrated a solution as possible is obtained. In practice, the concentrated solution will usually have a concentration of the &bgr;-lactam nucleus of at least 5 wt. %. The final pH will usually be lower than 10 and greater than 0.
Subsequently, a supersaturated solution can be obtained from a solution, whose pH may or may not have been decreased or increased, by increasing or decreasing the pH to a value between for example 3.0 and 9.0, preferably between 4.0 and 8.5, in particular between 4.5 and 8.0.
Any solid acylation agent present can be dissolved by for example decreasing the pH until a value lower than 8.0, preferably lower than 6.5, in particular lower than 5.0 is reached; preferably, the final pH is chosen such that the acylation agent is only just completely dissolved and thus as concentrated a solution as possible is obtained. In practice, the concentrated solution will have a concentration of the acylating agent of at least 5 wt. %. The final pH will usually be greater than 1.
A mixture supersaturated with for example the acylation agent can be obtained from a mixture, preferably a solution, whose pH may optionally have been reduced, by increasing the pH to a value greater than for example 4.5, preferably greater than 5.5, in particular greater than 6.0.
Another manner of obtaining a mixture in which the &bgr;-lactam nucleus and/or the acylation age
Moody Harold Monro
Smeets Johanna Christina Maria
Van Dooren Theodorus Johannes Godfried Marie
Bierman, Muserlian and Lucas
DSM N.V.
Ozga Brett
Prats Francisco
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