In order to obtain enantiomerically pure sigma1 receptor ligands with a 2-benzopyran scaffold an Oxa-Pictet-Spengler reaction with the enantiomerically pure 2-phenylethanol derivatives (R)-4 and (S)-4 was envisaged. The kinetic resolution of racemic alcohol (+/-)-4 using Amano Lipase PS-C II and isopropenyl acetate in tert-butyl methyl ether led to the (R)-configured alcohol (R)-4 in 42% yield with an enantiomeric excess of 99.6%. The (S)-configured alcohol (S)-4 was obtained by Amano Lipase PS-C II catalyzed hydrolysis of enantiomerically enriched acetate (S)-5 (76.9% ee) and provided (S)-4 in 26% yield and 99.7% ee. The absolute configuration of alcohol (R)-4 was determined by exciton coupled CD spectroscopy of the bis(bromobenzoate) (R)-7. The next important step for the synthesis of 2-benzopyrans 2 and 3 was the Oxa-Pictet-Spengler reaction of the enantiomerically pure alcohols (R)-4 and (S)-4 with piperidone ketal 8 and chloropropionaldehyde acetal 12. The conformationally restricted spirocyclic 2-benzopyrans 2 revealed higher sigma1 affinity than the more flexible aminoethyl derivatives 3. The (R)- and (R,R)-configured enantiomers (R)-2 and (R,R)-3 represent the eutomers of this class of compounds with eudismic ratios of 4.8 (2b) and 4.5 (2c). High sigma1/sigma2 selectivity (>49) was found for the most potent sigma1 ligands (R)-2b, (R)-2c, (R)-2d, and (S)-2d (Ki(sigma1) 9-15nM).
The hydrolase fold is one of the most versatile structures in the protein realm according to the diversity of sequences adopting such a three-dimensional architecture. In the present study, we clarified the crystal structure of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 refined to 2.1 A resolution, determined its main biochemical characteristics and also carried out an analysis of its associative behaviour in solution. We found that the versatility of a canonical alpha/beta hydrolase fold, the basic framework of the crystal structure of Cest-2923, also extends to its oligomeric behaviour in solution. Thus, we discovered that Cest-2923 exhibits a pH-dependent pleomorphic behaviour in solution involving monomers, canonical dimers and tetramers. Although, at neutral pH, the system is mainly shifted to dimeric species, under acidic conditions, tetrameric species predominate. Despite these tetramers resulting from the association of canonical dimers, as is commonly found in many other carboxylesterases from the hormone-sensitive lipase family, they can be defined as 'noncanonical' because they represent a different association mode. We identified this same type of tetramer in the closest relative of Cest-2923 that has been structurally characterized: the sugar hydrolase YeeB from Lactococcus lactis. The observed associative behaviour is consistent with the different crystallographic results for Cest-2923 from structural genomics consortia. Finally, the presence of sulfate or acetate molecules (depending on the crystal form analysed) in the close vicinity of the nucleophile Ser116 allows us to identify interactions with the putative oxyanion hole and deduce the existence of hydrolytic activity within Cest-2923 crystals. STRUCTURED DIGITAL ABSTRACT: Cest-2923 and Cest-2923 bind by x-ray crystallography (1, 2) Cest-2923 and Cest-2923 bind by cosedimentation in solution (1, 2) DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 4BZW for Cest-2923 from native crystals not soaked with substrates (P63 22 space group); 4C01 for Cest-2923 from crystals soaked with phenyl acetate (C2 space group); 4BZZ for Cest-2923 from crystals soaked with isopropenyl acetate (P622 space group).
Title: Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media Mine Y, Fukunaga K, Yoshimoto M, Nakao K, Sugimura Y Ref: Biotechnol Lett, 25:1863, 2003 : PubMed
Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, D- and L-2-(3-[bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl]-propiony lamino)-pentanedioic acid didodecyl ester (D- and L-BIG2C12CA); D- and L-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (D- and L-2C12GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19-48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the L-BIG2C12CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68-78 fold.
