Dorel T. Guranda

Penicillin acylase-catalyzed resolution of amines in aqueous organic solvents

Abstract Penicillin acylase from Alcaligenes faecalis catalyzes the enantioselective acylation of amines with phenylacetamide in a kinetically controlled reaction in water at pH 11. Addition of cosolvent to the reaction mixture significantly improved the enantioselectivity in most cases. Penicillin acylase from E. coli also catalyzed the phenylacetylation of amines, but an order of magnitude less efficiently than with the enzyme of A. faecalis . Amine resolution via kinetically controlled acylation competes effectively with hydrolysis of N -acylated compounds and constitutes a synthetically useful alternative to existing lipase-based methods.

KINETIC STUDY OF PENICILLIN ACYLASE FROM ALCALIGENES FAECALIS

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, K m=0.0042 mM) and colorimetric (6‐nitro‐3‐phenylacetamidobenzoic acid, K m=0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (K i=0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower (K S′=54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6‐nitro‐3‐phenylacetamidobenzoic acid (k cat=95 s−1 and k cat/K m=2.1×10−7 M−1 s−1) and benzylpenicillin (k cat=54 s−1 and k cat/K m=1.3×10−7 M−1 s−1) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.

Highly efficient and enantioselective enzymatic acylation of amines in aqueous medium

Abstract A new strategy based on the unique catalytic properties, stability and enantioselectivity of the relatively unknown penicillin acylase from Alcaligenes faecalis has been developed for the effective and enantioselective acylation of amines in aqueous medium. In contrast to lipase-catalyzed acylations in organic solvents, the penicillin acylase-catalyzed acylation of amines in aqueous solution is a rapid and chemoselective process leading to a product which can subsequently be deacylated by the same enzyme, imposing secondary enantiocontrol and leading to effective resolution.

Aliphatic amidase from Rhodococcus rhodochrous M8 is related to the nitrilase/cyanide hydratase family

A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.

pH Stability of penicillin acylase from Escherichia coli.

The inactivation kinetics of penicillin acylase from Escherichia coli have been investigated over a wide pH range at 25 and 50°C. The enzyme was very stable in neutral solutions and quickly lost its catalytic activity in acidic and alkaline solutions. In all cases, the inactivation proceeded according to first order reaction kinetics. Analysis of the pH dependence of enzyme stability provides evidence that stable penicillin acylase conformation is maintained by salt bridges. Destruction of the salt bridges due to protonation/deprotonation of the amino acid residues forming these ion pairs causes inactivation by formation of the unstable “acidic” EH43+, EH32+, EH2+ and “alkaline” E−enzyme forms. At temperatures above 35°C penicillin acylase apparently undergoes a conformational change that is accompanied by destruction of one of these salt bridges and change in the catalytic properties.

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.

Cloning of penicillin acylase from Escherichia coli: Catalytic properties of recombinant enzymes

The gene of penicillin acylase (PA) from Escherichia coli has been cloned from a PA producer strain that is an analogue of strain ATCC 11105. Optimization of the cultivation conditions made it possible to obtain up to 130 mg of active enzyme per liter of culture broth. A number of single, double, and triple mutants were obtained by the method of site-specific mutagenesis using PCR. As a result of isolation and purification procedures, homogeneous preparations of the wild-type enzyme and its mutants were obtained. Studies showed that (1) the obtained enzymes have the correctly folded structure; (2) complexing agents and metal cations do not inhibit their catalytic activity; (3) mutant PAs, like the wild type, are efficiently inactivated by phenylmethylsulfonyl fluoride (PMSF), which makes it possible to titrate their active sites; and (4) the obtained mutants are characterized by a greater specificity constant in the reaction of hydrolysis of a colorimetric substrate; however, they are inferior to the wild type in the synthesis of ampicillin by acyl transfer.

A New N-Acyl Derivative of (S)-Cysteine for Quantitative Determination of Enantiomers of Amino Compounds by HPLC with a Precolumn Modification with o-Phthalaldehyde

N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enantiomers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was compared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctionalized amines and amino alcohols. After the NAC-modification, the resulting diastereomeric isoindoles were difficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols. The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition, allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the amino component and the thiol reagent favored the diastereomer separation. This method was used for determination of the absolute concentration of individual enantiomers of amines in the course of stereoselective enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of penicillin acylase.

Placental transfer of tyrosine kinase inhibitors used for chronic myeloid leukemia treatment

Abstract Both favorable pregnancy outcomes and fetal abnormalities have been associated with the use of tyrosine kinase inhibitors (TKIs) during pregnancy. The placental transfer of TKIs in humans is poorly understood. We observed women with chronic myeloid leukemia who used imatinib or nilotinib during the late pregnancy stages. The newborns had no birth abnormalities. We evaluated the drug concentrations in maternal blood, umbilical cord blood, and placental samples collected during labor. We found limited placental transfer of the TKIs. The fetal/maternal concentration ratio ranged from 0.5 to 0.58 for nilotinib and from 0.05 to 0.22 for imatinib. The placental/maternal ratio was higher for imatinib than for nilotinib. Theoretical pharmacokinetic modeling of passive placental crossing was insufficient to predict the in vivo data because the calculated fetal/maternal ratio was close to 1 for both drugs. We propose that active placental transport contributes to fetal protection against TKI exposure during pregnancy.