Chemoenzymatic synthesis and binding affinity of novel (R)- and (S)-3-aminomethyl-1-tetralones, potential atypical antipsychotics

A series of (R)- and (S)-3-aminomethyl-1-tetralones, conformationally constrained analogues of haloperidol, have been obtained by enzymatic resolution of the corresponding racemic 3-hydroxymethyl-1-tetralones using Pseudomonas fluorescens lipase. Their binding affinities at dopamine D(2) and serotonin 5-HT(2A) and 5-HT(2C) receptors were determined showing in some cases an atypical antipsychotic profile with Meltzer's ratio higher than 1.30.     

Novel Chiral Phosphoramidite Monomers from (R)- and (S)-1,3-Butanediol: Synthesis of Modified Oligonucleotides and Binding Affinity Studies

Abstract

Two optically active phosphoramidite monomers for modified oligodeoxyribonucleotides were prepared. These monomers were then introduced into dodecanucleotides in the middle of the sequences. The modified dodecanucleotides were characterized by various analytical methods including MALDI-TOF mass spectrometry and Tm values were obtained to appraise the binding affinity, by measuring change in UV absorbance at 260 nm.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

Novel (2R,3R)-2,3-butanediol dehydrogenase from potential industrial strain Paenibacillus polymyxa ATCC 12321

A (2R,3R)-2,3-butanediol dehydrogenase (BDH99::67) from Paenibacillus polymyxa ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of meso- and (2S,3S)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which meso- and (2S,3S)-2,3-butanediol dehydrogenases belong.     

Synthesis of (R)-1,3-butanediol by enantioselective oxidation using whole recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4% from a racemate of 15% and an optical purity of 95% ee.     

Stereoselective features of (R)- and (S)-atenolol: Clinical pharmacological,pharmacokinetic, and radioligand binding studies

In a randomized, double-blind, cross-over study in 12 healthy volunteers, the effects of single oral doses of 100 mg rac-atenolol were compared during exercise to those of equal amounts of the optically pure enantiomers, i.e., 50 mg (R)- and 50 mg (S)-atenolol. The mean rate pressure product decreased with rac-atenolol (?37%; P < 0.01) and half-dosed (S)-atenolol (?35%; P < 0.01) to the same extent, whereas (R)-atenolol caused no effect. Radioligand binding studies in beta-adrenergic receptors of the guinea pig heart yielded a eudismic ratio of 46 for (S)- to (R)-atenolol. The mean AUCs, maximal plasma concentrations, and plasma half-lives of the enantiomers were similar regardless of whether they were administered as optically pure enantiomers or as racemic mixture. On the other hand, the AUC of (R)-atenolol was 1.08-fold greater (P < 0.01) than that of the (S)-enantiomer. The reason for this finding remains unclear. We conclude that only (S)-atenolol, but not (R)-atenolol, contributes to the beta-blocking effect of currently used rac-atenolol since the same effect can be elicited with the (S)-enantiomer alone. © 1993 Wiley-Liss, Inc.     

A novel versatile phosphoramidite building block for the synthesis of 5'- and 3'-hydrazide modified oligonucleotides

We introduce a novel versatile phosphoramidite building block for the modification of oligonucleotides (ONs) with acyl hydrazides on the 5'- or 3'-terminus, or both. The reaction of these hydrazide functionalized ONs with 4-methoxyphenylaldehyde is demonstrated for solution derivatization. Hydrazides are considered nowadays as promising reactants, which show enhanced reactivity at neutral and slightly acidic conditions and higher stability of yielding products as compared to the aliphatic amines, which are broadly used for ONs derivatization. Our method to introduce hydrazides into ONs employs a phosphoramidite modifier designed to split, during ammonia or lithium hydroxide treatment, into two hydrazides via beta-elimination of a central bis-2-carbonylethoxysulfone unit. It allows the creation of ONs derivatized with a hydrazide moiety at the 5'-, 3'- and both 5'- and 3'-termini, as well as two different hydrazide containing ONs at the same time, viz. in one sequence on the same solid support In latter case one can, for example, synthesize two hydrazide containing ONs, where one is 5'-modified and second one is 3'-modified.     

(R)-oxynitrilase-catalysed synthesis of chiral silicon-containing aliphatic (R)-ketone-cyanohydrins

Optically active 2-trimethylsilyl-2-hydroxyl-ethylcyanide was prepared by enzymatic enantioselective transcyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system at 35°C and pH 5. (R)-Oxynitrilase from apple seed meal was the best among all the enzymes explored and diisopropyl ether was the most suitable organic phase. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon analogue. The substrate conversion and product enantiomeric excess of 2-trimethylsilyl-2-hydroxyl-ethylcyanide were >99% and >99%, respectively.     

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C₅-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M _r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M _r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - M _r relative molecular mass - MTPA-Cl -methoxy--trifluoromethylphenyl acetic acid chloride - PEIC 1-phenylethylisocyanate     

A novel enantioselective synthesis of (S)-(-)- and (R)-(+)-nornicotine via alkylation of a chiral 2-hydroxy-3-pinanone ketimine template

An asymmetric synthesis of the optically pure isomers of the minor tobacco alkaloid and CNS nicotine metabolite, nornicotine, has been achieved with moderately high optical purity. The synthetic pathway involves alkylation of a chiral ketimine, prepared from either 1R,2R,5R-(+)- or 1S,2S,5S-(-)-2-hydroxy-3-pinanone and 3-(aminomethyl)pyridine with 3-bromopropan-1-ol. After cleavage of the respective C-alkylated ketimines with NH2OH.HCl, and treatment of the resulting amino alcohols with HBr, followed by base-catalyzed intramolecular ring closure, (S)-(-)-nornicotine and (R)-(+)-nornicotine were obtained with ee values of 91% and 81%, respectively.     

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales.     

Microbial synthesis of (R)- and (S)-3,4-dimethoxyamphetamines through stereoselective transamination

Two soil isolates, Arthrobacter sp. KNK168 and Pseudomonas sp. KNK425, aminated 3,4-dimethoxyphenylacetone in presence of sec-butylamine as an amino donor to yield 3,4-dimethoxyamphetamine (DMA) with different enantioselectivities. The former gave (R)-DMA (>99% e.e.) and the latter the (S)-isomer (>99% e.e.).     

Total synthesis of (R,R,R)- and (S,S,S)-schweinfurthin F: differences of bioactivity in the enantiomeric series

Total synthesis of the (R,R,R)- and (S,S,S)-enantiomers of the natural product schweinfurthin F has been completed. Comparisons of spectral data and optical rotations with those reported for the natural product, as well as a variety of bioassay data, allow assignment of the natural material as the (R,R,R)-isomer.     

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.     

Synthesis and hybridization properties of oligonucleotides containing (2S,3R)-9-(2,3,4-trihydroxybutyl)adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A(C2) and A(C3), are described. The ON containing A(C2) involves the 3'-->4' and 3-->5' phosphodiester linkages in the strand, whereas that containing A(C3) possesses the 3'-->4' and 2'-->5' phosphodiester linkages. It was found that incorporation of the analogs, A(C2) or A(C3), into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A(C2) is greater than that of A(C3) in the ON/RNA duplexes.     

A straightforward stereoselective synthesis of meso-, (S,S)- and (R,R)-2,6-diaminopimelic acids from cis-1,4-diacetoxycyclohept-2-ene

A straightforward synthesis of meso-2,6-diaminopimelic acid (DAP) meso-1 was developed from 1,4-diacetoxycyclohept-2-ene (2) via an oxidative ring cleavage. Subsequently, an enantio-divergent synthesis of (S,S)- and (R,R)-1 was performed using a homochiral monoacetate 7 available from 2 by enzymatic desymmetrization.     

Optimization of CRF1R binding affinity of 2-(2,4,6-trichlorophenyl)-4-trifluoromethyl-5-aminomethylthiazoles through rapid and selective parallel synthesis

An efficient approach was developed to synthesize 2-(2,4,6-trichlorophenylamino)-4-trifluoromethyl-5-aminomethylthiazoles, corticotropin-releasing factor type 1 receptor (CRF(1)R) antagonists, by monoalkylation of amines with chloromethyl intermediate 5. The effect of variations in aminomethyl side chain of 6 on binding affinity is discussed.     

(2R,3S)-(+)- and (2S,3R)-(-)-Halofuginone lactate: synthesis, absolute configuration, and activity against Cryptosporidium parvum

The trans-enantiomers of the commercially important anti-protozoal compound Halofuginone have been prepared and characterized, and the absolute configuration was assigned by X-ray crystallography. The activity of both enantiomers against Cryptosporidium parvum was determined in vitro and related to acute toxicity in vivo. It was shown that both the activity and the toxicity are properties of the (2R,3S)-enantiomer. We conclude that with respect to broadening the therapeutic window there is no advantage in application of one enantiomer over the application of the racemic mixture in the treatment of C. parvum infections.     

Enhancement of (R,R)-2,3-butanediol production from xylose by Paenibacillus polymyxa at elevated temperatures

Conversion of xylose to (R,R)-2,3-butanediol by Paenibacillus polymyxa in anaerobic batch and continuous cultures was increased by 39% and 52%, respectively, by increasing the growth temperatures from 30 to 39 °C. There was no effect of temperature when glucose was used as substrate. 39 mM (R,R)-2,3-butanediol, 65 mM ethanol, and 47 mM acetate were obtained from 100 mM xylose after 24 h batch culture at 39 °C. With 100 mM glucose and 100 mM xylose used together in a batch culture at 39 °C, all xylose was consumed after 24 h and 82 mM (R,R)-2,3-butanediol, 124 mM ethanol and 33 mM acetate were produced.