Hartmut H. Meyer

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones.

The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.

Directed evolution of an esterase: screening of enzyme libraries based on ph-Indicators and a growth assay

In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).

Factors affecting the lipase catalyzed transesterification reactions of 3-hydroxy esters in organic solvents.

Abstract Chiral resolutions of racemic 3-hydroxy esters were performed in organic phases with lipases from Pseudomonas cepacia , Chromobacterium viscosum and Porcine pancreas. The reaction conditions have been optimized with 3-hydroxy octanoic acid methyl ester. Different organic solvents have been tested showing a tendentious correlation with the hydrophobicity of the solvents expressed as log P. The reaction time was shortened six fold by using irreversible acylating agents. We have found solvent type, lipase type and acylating agent acting as tools for changing the enantioselectivity. Lipase from Pseudomonas cepacia was lyophilized at different pH and the influence of the amount of water added was investigated, resulting in the highest activity at the pH optimum and a denaturation of the lipase above 1 % water (w/w lipase ). The water activity was measured on-line with a humidity sensor. Water activities greater than 0.4 led to a decrease in enantioselectivity and reaction rate. In the optimized system the resolutions of other 3-hydroxy esters were tested. Aliphatic compounds reacted with lower enantioselectivity, only the substrates could be isolated in high enantiomeric purity. In contrast, aromatic 3-hydroxy esters were acylated by lipases with high stereoselectivity. A model of the active site of lipase from Pseudomonas sp . explained these experimental observations.

Chemoenzymatic route to β-blockers via 3-hydroxy esters

Abstract Enantiomerically pure precursors of β-blockers (propranolol, alprenolol and 1-(isopropylamino)-3- p -methoxy-phenoxy-2-propanol) were synthesized. Key step is the lipase-catalyzed kinetic resolution of rac -3-hydroxy esters either by O-acylation using vinyl acetate or by hydrolysis of the ester group. Both approaches were highly enantioselective (> 95 %ee) with E-values > 150 using lipase from Pseudomonas cepacia . The formal synthesis of (−)-(S)-propranolol was developed in subsequent steps.

Lipase-catalyzed kinetic resolution of 3-hydroxy esters in organic solvents and supercritical carbon dioxide

Abstract The lipase-catalyzed kinetic resolution of different 3-hydroxy esters in organic solvents and supercritical carbon dioxide (SCCO2) was studied. Similar enantiomeric excesses (ee, up to 99%) as found for transesterifications in organic solvents were achieved also in SCCO2. The influence of various reaction parameters was investigated for the resolution of 3-hydroxy octanoic acid methyl esters with lipase from Pseudomonas cepacia in SCCO2. Optimum conditions have been found at 110 bar, 40°C, and in the presence of 1.5 g molecular sieve by using vinyl acetate as acyl donor. The addition of different cosolvents had only a small effect on the reaction. Immobilization of the lipase on VA-epoxy resulted in similar optical purities as found for the crude lipase, but at halved reaction times. The effect of supercritical conditions on the stability of the lipase was also investigated.

The enantioselective hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester in supercritical carbon dioxide using lipases.

A new experimental high-pressure-unit was constructed for the enantioselective enzymatic hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester (a precursor for biological interesting substances) in a biphasic buffer/SCCO(2)-system. One objective is to take advantage of the solubility differences of the substrate and the produced acid. Thus the different solubilities of the substrates and the products in the different phases were studied regarding to an overall process integration. One ester enantiomer is preferably hydrolyzed, the other remains in the supercritical phase. And the produced acid enantiomer is concentrated in the buffer phase. The decrease in pressure is followed by an extraction process of the remaining substrate-enantiomer, in consequence it will be possible to combine an enzymatic reaction with a separation step. The catalysis was optimized in regard to enantioselectivity, enantiomeric excess, conversion and reaction time. A high enantioselectivity is achieved for the aromatic substrate using the lipase of Pseudomonas cepacia. The results show that this unconventional reaction system offers tremendous advantages for enzyme process development.

Total synthesis of the piperidinol alkaloid (−)-(2R,3R,6S)-cassine

Abstract A highly efficient, flexible and diastereoselective route to all-cis-2,6-disubstituted 3-piperidinols has been accomplished. The key component of the synthesis is a chiral β-hydroxyester, which was obtained by lipase catalyzed kinetic resolution. Based on this starting material, diastereoselective alkylation of its dianion, Curtius rearrangement to a 2-oxazolidinone, Grignard reaction to introduce the side-chain and conversion of the aliphatic 2-oxazolidinone into a 3-piperidinol by imine cyclization lead to the exemplary total synthesis of naturally occurring (−)-(2R,3R,6S)-cassine.

Biosensors for enantioselective analysis

Abstract The development of enantioselective enzyme field effect transistors (EnFETs) for the detection of β-hydroxy acid esters and aromatic amino esters and their application in flow-injection analysis is described. The EnFETs were produced by immobilising the enzyme on the pH-sensitive gate surface of a pH-FET. The characteristics of the sensor signals and their dependence on certain process parameters, such as pH, buffer capacity and flow conditions in the medium being measured, were examined. Due to the short response time, the EnFETs are ideal for application in flow-injection analysis. The use of enantioselective EnFETs in on-line process monitoring is described.

GC–MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK(L) 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC-MS method for the analysis of S-nitrosothiols and their (15)N- or (2)H-(15)N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S(15)NACET or d(3)-S(15)NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO(2)](-)) for SNACET and m/z 47 (i.e., [(15)NO(2)](-)) for S(15)NACET/d(3)-S(15)NACET for compound X, and m/z 157 for SNACET and m/z 160 for d(3)-S(15)NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S(15)NACET/d(3)-S(15)NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0-10microM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200nM in total. Oral administration of S(15)NACET to rabbits (40-63micromol/kg body weight) resulted in formation of ALB-S(15)NO, [(15)N]nitrite and [(15)N]nitrate in plasma.

Properties of unusual phospholipids IV: Chemoenzymatic synthesis of phospholipids bearing acetylenic fatty acids

Abstract 14-Octadecynoic ( 1 ) and 4-octadecynoic ( 2 ) acid were synthesized in high yields (56% & 57%) and incorporated into glycerophosphatidylcholine at sn 1- and sn 2-position. The head group of these phosphatidylcholines was exchanged using phospholipase D (PLD) in a biphasic system. PLD from Streptomyces antibioticus, Streptomyces sp. , peanut and cabbage were studied in this transphosphatidylation reaction. Under optimized conditions (40 °C, organic solvent: buffer ratio 1:1.5 (v/v), pH 5.6), the conversion of phosphatidylcholine to the corresponding phospholipids with the head groups glycerol, ethanolamine and L -serine reached 99% at 40°C in 1 h. The quantitative analysis of the transphosphatidylation reactions was performed by TLC-FID.