Inovações na síntese enzimática de amoxicilina

Abstract

Penicillin G acylase (PGA, E.C.3.5.1.11) from Escherichia coli is an enzyme of great industrial importance, widely used for the hydrolysis of penicillin G, producing the 6-aminopenicillanic acid (6-APA), which is a key molecule for the synthesis of semi-synthetic penicillins. Among them, amoxicillin has a broad spectrum of activity against a variety of bacteriological infections. Industrially, amoxicillin is produced by chemical processes, which require drastic reaction conditions, several steps of protection and deprotection of reactive groups in order to prevent non-selective hydrolytic reactions, use of organochloride solvents with non-recyclable waste generation, which are toxic and harmful to the environment. The enzymatic synthesis is a more attractive alternative from the environmental point of view and economic. The tendency of the pharmaceutical industry is the development of enzymatic methods to produce these β-lactam semi-synthetic antibiotics, including amoxicillin. Nevertheless, a major obstacle to its industrial implementation is the limited yield, as a consequence of undesirable hydrolytic side-reactions, which lead to the formation of the by-product (p-hydroxyphenylglycine, POHPG) throughout the course of the reaction. This drawback can be partially avoided by reducing the water activity (aw) in the medium. For this purpose, ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability, non-flammability, easy recycling, and negligible vapor pressure. Within this context, this work researched the development of an integrated green process for the recovery, reuse and recycle of the by-product (POHPG) of the kinetically controlled enzymatic synthesis of amoxicillin, employing PGA immobilized on Sepabeads® in a totally aqueous medium reaction (sodium phosphate buffer 100 mM, pH 6.5), and assessed the catalytic activity of this biocatalyst in the presence of different ILs as cosolvents for these synthetic reactions, in terms of selectivity (synthesis/hydrolysis, S/H ratio) and conversion of the substrate 6-aminopenicillanic acid (6-APA). The recovery of the by-product (POHPG) of the kinetically controlled enzymatic synthesis of amoxicillin in a totally aqueous reaction medium was done efficiently, achieving a final purity of 99% for the POHPG, which was successfully reused for the production of the substrate p-hydroxyphenylglycine ethyl ester (POHPGEE), achieving a conversion of 93%. Then, POHPGEE was recycled to the reactor (without any further purification) for another batch of enzymatic synthesis of amoxicillin, following the characteristic profile that is expected for these synthetic reactions. This integrated green process generated sodium chloride (NaCl) as waste, which is an inert and harmless salt. Moreover, the assessment of the use of ILs as cosolvents for the reactions of kinetically controlled enzymatic synthesis of amoxicillin presented promising results. An increase of 400% in the selectivity was observed for the reactions carried out in the presence of 1-butyl-3-methylimidazolium hexafluorophosphate (BMI.PF6), as cosolvent at a concentration of 75% (VIL/VWATER) in relation to the standard reaction performed in totally aqueous medium. Similarly, this figure reached more than 350% for reactions conducted in 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMI.NTf2) at the same volume fraction, while for 1-butyl-3-methylimidazolium tetrafluoroborate (BMI.BF4) there was only a slight increase in selectivity (about 57%). The highest conversion of 6-APA was achieved using BMI.NTf2 as cosolvent at a concentration of 71% (VIL/VWATER), representing an increase of more than 36% compared to standard aqueous reaction. No deactivation of the enzyme was observed after the reactions in any of the ILs, and the physical integrity of the biocatalyst particles was entirely maintained. The results of this work collaborated for the advance in the study of the enzymatic synthesis of semi-synthetic penicillins through the use of technologies more green .