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Ethylene glycol has the ability to act as a reactant when employed as a co-solvent for the enzymatic synthesis of ampicillin from (R)-phenylglycine methyl ester and 6-aminopenicillanic acid. The side reaction positively affects yield because its product, (R)-phenylglycine hydroxyethyl ester, is an intermediate for ampicillin synthesis., (© 2013 Wiley Periodicals, Inc.)

Semi-synthetic β-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the β24 position. βPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, βPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. βPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

• Crystal purity in continuous enzymatic ampicillin production and crystallization. • Multiple steady states in well-mixed reactive crystallizer. • Well-mixed and plug flow reactive crystallizer series gives high yield and purity. • Developed process purity robust to parameter and operational variability. Continuous reactive crystallization of β-lactam antibiotics can ease shortage and contamination concerns for an important class of medicines. With a model of continuous reactive crystallization catalyzed by penicillin G acylase (PGA), a process is developed to ensure purity of the antibiotic product. Guaranteeing the solid-phase purity of ampicillin and cephalexin is important because PGA produces a slightly soluble byproduct, phenylglycine, which can contaminate the antibiotic crystals. In a single well-mixed reactive crystallizer, productivity is optimized and nearly two-fold greater than the non-crystallizing maximum productivity. Process robustness is essential because multiple steady states, only one of which produces pure ampicillin crystals, exist in the well-mixed reactive crystallizer. By combining well-mixed and plug-flow reactive crystallizers in series, crystallization of phenylglycine can be suppressed while high yield and comparable productivity are still achieved. The reactive crystallizer series demonstrates high purity with uncertainty in model parameter values and variability in operating conditions. [ABSTRACT FROM AUTHOR]

The enzyme penicillin G acylase (PGA) catalyzes the condensation of phenylglycine methyl ester (PGME) with 6-aminopenicillanic acid (6-APA) to form ampicillin. We improved the selectivity of ampicillin synthesis with PGA by running simultaneous reaction and crystallization. However, the enzyme also catalyzes two undesirable side reactions: the hydrolysis of PGME to phenylglycine and the hydrolysis of ampicillin to phenylglycine and 6-APA. We demonstrate that a fifty percent improvement in selectivity for ampicillin over phenylglycine is achieved by combining reaction and crystallization in batch at pH value of 6 with saturated 6-APA and equimolar PGME. The enhancement in selectivity is mainly attributed to the decreased rates of enzymatic ampicillin hydrolysis; however, the course of the pH value during the reaction also has an effect on enzyme activity that improved selectivity. In addition to showing experimental results, we developed a new kinetic process model that predicts the observed improvement. The new model accounts for the solubility limits of different species as functions of pH value as well as the large change in pH value at high conversion. Previous work does not account for changes in activity with conversion. The pH-dependent activity for the specific enzyme used in this system, Assemblase® from DSM-Sinochem, is well-realized by the model and generalization to other PGAs is possible within the model framework; the selectivity parameters α, β 0 , and γ for Assemblase® are compared to PGA from E. coli as evidence. [ABSTRACT FROM AUTHOR]

Crystallization kinetics of ampicillin were estimated from induction-times and seeded and unseeded experiments. The concentration of ampicillin in solution was monitored by polarimetry and refractometry, while crystals were analyzed by focused beam refractive measurements and light microscopy. Variations of solute concentration with time, along with measured induction times were used to estimate parameters in kinetic expressions for primary nucleation, secondary nucleation, and crystal growth rate. Experimental data were obtained in runs involving different rates of change of pH, mass of seed crystals, and initial supersaturation. These data were used in parameter-estimation routines consisting of a stochastic minimization to localize a set of parameters spanning all the experiments, followed by a deterministic minimization to refine the parameters. The result was a set of parameters that fit a range of conditions of seeded and unseeded crystallizations and gave good estimates of desupersaturation rates. [ABSTRACT FROM AUTHOR]

Crystallization kinetics of ampicillin were estimated from induction-times and seeded and unseeded experiments. The concentration of ampicillin in solution was monitored by polarimetry and refractometry, while crystals were analyzed by focused beam refractive measurements and light microscopy. Variations of solute concentration with time, along with measured induction times were used to estimate parameters in kinetic expressions for primary nucleation, secondary nucleation, and crystal growth rate. Experimental data were obtained in runs involving different rates of change of pH, mass of seed crystals, and initial supersaturation. These data were used in parameter-estimation routines consisting of a stochastic minimization to localize a set of parameters spanning all the experiments, followed by a deterministic minimization to refine the parameters. The result was a set of parameters that fit a range of conditions of seeded and unseeded crystallizations and gave good estimates of desupersaturation rates. [ABSTRACT FROM AUTHOR]

Abstract: α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25°C and pH 6.8, with kinetic parameters of k cat of 72.5s−1 and K M of 1.1mM. The synthesis parameters α, β o, and γ for ampicillin, determined here first for this class of proteins, are α =0.25, β o =42.8M−1, and γ =0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307. [ABSTRACT FROM AUTHOR]

Prediction and control of crystal size distributions, a prerequisite for production of consistent crystalline material in the pharmaceutical industry, requires knowledge of potential non-idealities of crystal growth. Ampicillin is one such medicine consumed in crystal form (ampicillin trihydrate). Typically it is assumed that all crystals of the same chemical and geometric type grow at the same rate, however a distribution of growth rates is often observed experimentally. In this study, ampicillin produced enzymatically is crystallized and a distribution of growth rates is observed as individual crystals are monitored by microscopy. Most studies of growth rate dispersion use complex flow apparatuses to maintain a constant supersaturation or imprecise measurements of size distributions to reconstruct growth rate dispersions. In this study, the controllable enzyme reaction enables the same information to be gathered from fewer, less complicated experiments. The growth rates of individual ampicillin trihydrate crystals were found to be normally distributed, with each crystal having an intrinsic growth rate that is constant in time. Differences in the individual crystals, such as different number and arrangement of dislocations and surface morphology, best explain the observed growth rates. There is a critical supersaturation below which growth is not observed, thought to be caused by reactants adsorbing to the crystal surface and pinning advancing growth steps. The distribution of critical supersaturation also suggests that individual crystals' surface morphologies cause a distribution of growth rates. [ABSTRACT FROM AUTHOR]

Abstract: α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25°C, an observed half-life of 5min at 30°C, and a “T-50” value, the temperature at which the half-life is 30min, of 27°C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a “T-50” value of 34°C (7°C improvement) and 1.3-fold activity compared to wild-type. [Copyright &y& Elsevier]