Preparation of arylzinc reagents and their use in the rhodium-catalyzed asymmetric 1,4-addition for the synthesis of 2-aryl-4-piperidones

A protocol for the catalytic asymmetric synthesis of 2-aryl-4-piperidones with high enantiomeric excess (ee) (typically > or = 99% ee) has been described here. The preparation of arylzinc reagents, which are used as nucleophiles in catalysis, is also included. The whole protocol can be completed in 10-20 h, starting from the preparation of the arylzinc reagents, depending on the reaction time of the rhodium-catalyzed process. A detailed protocol is described using the preparation of 4-fluorophenylzinc chloride and its addition to benzyl 3,4-dihydro-4-oxo-1(2H)-pyridinecarboxylate in the presence of [RhCl((R)-binap)]2 as an example.     

Stereoisomeric flavour compounds LXVII. 2-, 3-, and 4-Alkyl-branched acids,part 1: General approach to the synthesis of the enantiopure acids

A general approach to the synthesis of 2-, 3-, and 4-alkyl-branched acids of high enantiomeric purity is described. The enantiopure 2-alkyl-branched acids are prepared via liquid chromatographic resolution of diastereomeric phenylglycinol amides and their absolute configuration is deduced from the ¹H-NMR data of the separated diastereomers. Chain elongation methods, by Arndt–Eistert synthesis, via 2-alkylated alkyl carbonitrile or by malonic ester synthesis, are used to prepare 3- and 4-alkyl-branched acids of high configurational purity and known absolute configuration starting from the enantiomeric 2-alkyl-branched acids. © 1994 Wiley-Liss, Inc.     

Separation of enantiomeric amines and acids using chiral ion-pair chromatography on porous graphitic carbon

The application of porous graphitic carbon as adsorbing phase for direct separation of enantiomeric acids and amines using chiral ion-pair chromatography is described. The enantiomeric amines were separated as diastereomeric ion pairs with N-benzyloxycarbonylglycyl-L -proline, N-benzyloxycarbonylglycylglycyl-L -proline, or captopril as the chiral counterion. High enantioselectivities were obtained for amines having a hydrogen bonding function in the vicinity of the asymmetrical carbon atom. Quinine was the chiral counterion used to separate the enantiomeric acids. The strongly UV-absorbing quinine improved detection of solutes having low UV-absorbing properties, e.g., (R,S)-2-chloropropionic acid, by “indirect detection.” Retention and stereoselectivity of enanticmeric acids were regulated by the quinine concentration and by the addition of carboxylic acids as well as polar modifiers, e.g., methanol and 2-propanol, to the mobile phase. © 1992 Wiley-Liss, Inc.     

Determination of the enantiomeric excess of chiral carboxylic acids by 31P NMR with phosphorylated derivatizing agents from C2-symmetrical diamines containing the (S)-alpha-phenylethyl group

The use of P(III) and P(V) organophosphorus derivatizing agents prepared from C(2) symmetrical (1R,2R)- and (1S,2S)-trans-N,N'-bis-[(S)-alpha-phenylethyl]-cyclohexane-1,2-diamines 1 and 2, as well as (1R,2R)- and (1S,2S)-trans-N,N'-bis-[(S)-alpha-phenylethyl]-4-cyclohexene-1,2-diamines 3 and 4 for the determination of enantiomeric composition of chiral carboxylic acids by (31)P NMR, is described.     

Selective synthesis of 3-hydroxy acids from Meldrum's acids using SmI2-H2O

The single-step synthesis of 3-hydroxy carboxylic acids from readily available Meldrum's acids involves a selective monoreduction using a SmI(2)-H(2)O complex to give products in high crude purity, and it represents a considerable advancement over other methods for the synthesis of 3-hydroxy acids. The protocol includes a detailed guide to the preparation of a single electron-reducing SmI(2)-H(2)O complex and describes two representative examples of the methodology: monoreduction of a fully saturated Meldrum's acid (5-(4-bromobenzyl)-2,2-dimethyl-1,3-dioxane-4,6-dione) and tandem conjugate reduction-selective monoreduction of α,β-unsaturated Meldrum's acid (5-(4-methoxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione). The protocol for selective monoreduction of Meldrum's acids takes ～6 h to complete.     

A New Enzymatic Reaction: Enzyme Catalyzed Ammonolysis of Carboxylic Esters

A new enzymatic reaction of carboxylic esters and ammonia (ammonolysis) was studied. This reaction provides a synthetically useful and mild alternative for the synthesis of amides. Several lipases and one esterase acted as catalyst. Ammonolysis of esters of chiral carboxylic acids gave higher ee values than hydrolysis under comparable reaction conditions. Furthermore, consecutive enzymatic esterification and ammonolysis provided a convenient one-pot synthesis of carboxylic amides from carboxylic acids.     

Direct measurement of enantiomeric ratios of enzymatic resolution by chiral high-performance liquid chromatography

(R)- and (S)-Methyl 2-(phenoxy)propionate and their acids could be separated simultaneously by a Chiralcel OD or OK column, while (R)- and (S)-methyl 2-(4-chlorophenoxy)propionate and their acids were separated concurrently only by an OK column. This is a novel and facile way to measure the enantiomeric excesses of the remaining substrate and product in the reaction of enzymatic resolution; enantiomeric ratios could then be calculated.     

Preparation of (25R)- and (25S)-26-functionalized steroids as tools for biosynthetic studies of cholic acids

A new synthesis of both epimeric forms of 26-cholestanoic acids and 26-alcohols containing a 3beta-hydroxy-Delta(5)- or a Delta(4)-3-keto-functionality in ring A is described starting from stigmasterol or (20S)-3beta-acetoxy-pregn-5-en-20-carboxylic acid. The obtained compounds are useful as standards for studies of cholic acids. Construction of the side chain was achieved by linkage of steroidal 23-iodides to sulfones prepared from (2R)- and (2S)-3-hydroxy-2-methylpropanoates. Oxidation of intermediate 26-alcohols into the corresponding carboxylic acids ensuring preservation of stereochemistry at C-25 and functional groups in the cyclic part was achieved with sodium chlorite catalyzed by TEMPO and bleach.     

Kinetic and Thermodynamic Approach to Design of Processes for Enzymatic Synthesis of Betalactams

An optimal way to design an enzymatic process for the production of betalactam antibiotics based on thermodynamic and kinetic studies is described. The study was performed on model reactions involving synthesis of cephalosporin-acids (cephalotin, cefazolin, cefoxitin) using immobilised cephalosporin-acid synthetase from Escherichia coli as biocatalyst, and aminocephalosporins (cephalexin) using immobilised cells of Xanthomonas rubrilineans containing the aminocephalosporin synthetase. The possibility of direct synthesis of cephalotin and cefoxitin was shown, the main equilibrium parameters were determined and the operation conditions were evaluated. The maximum key amino acid conversion to product of approximately 90% for cefoxitin and cephalotin was achieved using initial concentrations of the corresponding key amino acids of 0.05 λM and, respectively, 2-fold and 4-fold molar excess of the carboxylic acids. Cefazolin and cephalexin production by enzymatic synthesis with using of corresponding biocatalyst with a mechanism of action involving the acylenzyme intermediate was shown possible. The kinetic parameters of the process were estimated and the relationship between the maximum antibiotic yield and the initial concentrations of the substrate and nucleophile in the kinetically controlled synthesis was determined. The technologies for cefazolin and cephalexin enzymatic synthesis were designed and the cefazolin technology was optimised. Maximum yields of cefazolin and cephalexin of more than 90% were predicted by the kinetic model using 4-6-fold molar excess of the acylating agents and maximum yields of approximately 85% were achieved in experiments.     

Kinetic and Thermodynamic Approach to Design of Processes for Enzymatic Synthesis of Betalactams

An optimal way to design an enzymatic process for the production of betalactam antibiotics based on thermodynamic and kinetic studies is described. The study was performed on model reactions involving synthesis of cephalosporin-acids (cephalotin, cefazolin, cefoxitin) using immobilised cephalosporin-acid synthetase from Escherichia coli as biocatalyst, and aminocephalosporins (cephalexin) using immobilised cells of Xanthomonas rubrilineans containing the aminocephalosporin synthetase. The possibility of direct synthesis of cephalotin and cefoxitin was shown, the main equilibrium parameters were determined and the operation conditions were evaluated. The maximum key amino acid conversion to product of approximately 90% for cefoxitin and cephalotin was achieved using initial concentrations of the corresponding key amino acids of 0.05 &#117 M and, respectively, 2-fold and 4-fold molar excess of the carboxylic acids. Cefazolin and cephalexin production by enzymatic synthesis with using of corresponding biocatalyst with a mechanism of action involving the acylenzyme intermediate was shown possible. The kinetic parameters of the process were estimated and the relationship between the maximum antibiotic yield and the initial concentrations of the substrate and nucleophile in the kinetically controlled synthesis was determined. The technologies for cefazolin and cephalexin enzymatic synthesis were designed and the cefazolin technology was optimised. Maximum yields of cefazolin and cephalexin of more than 90% were predicted by the kinetic model using 4-6-fold molar excess of the acylating agents and maximum yields of approximately 85% were achieved in experiments.     

Stereospecific reduction of ethyl 2' -ketopantothenate to ethyl d-(+)-pantothenate with microbial cells as a catalyst

A novel enzymatic process for the synthesis of D-(+)-pantothenic acid through the asymmetric reduction of the 2' -ketopantothenate ester is described. Candida macedoniensis AKU 4588 was found to convert ethyl 2' -ketopantothenate (80 mg/ml) almost specifically to ethyl D-(+)-pantothenate (>98% enantiomeric excess), with a molar yield of 97.2%.     

Total synthesis of 12,13-desoxyepothilone B (Epothilone D)

A highly convergent total synthesis of 12,13-desoxyepothilone B (4, Epothilone D) is described involving the coupling of vinyl iodide (5) and olefin (6). Key steps in the synthesis are the introduction of chirality at C15 via highly enantioselective lipase-mediated enzymatic resolution, diastereoselective alkylation at C8, highly diastereoselective Evans aldol reaction to establish C6-C7, and Mukaiyama aldol reaction to introduce chiral center C3. Palladium catalyzed Suzuki coupling of (5) and (6) provided the methyl ester (27), which was converted to 12,13-desoxyepothilone B (4).     

Production of a doubly chiral compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone,by two-step enzymatic asymmetric reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD(+), and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Direct ester condensation catalyzed by bulky diarylammonium pentafluorobenzenesulfonates

A protocol for ester condensation between equimolar amounts of carboxylic acids and alcohols catalyzed by bulky diarylammonium pentafluorobenzenesulfonate is described. We also present procedures for the synthesis of N-(2,6-diisopropylphenyl)-N-mesitylammonium pentafluorobenzenesulfonate. The present ester condensation proceeds well under mild conditions even without the removal of generated water. The synthesis of N-(2,6-diisopropylphenyl)-N-mesitylammonium pentafluorobenzenesulfonate will take approximately 5 days. The ester condensation reactions will take approximately 6 h to 3 days.     

One-pot biocatalyzed preparation of substituted amides as intermediates of pharmaceuticals

A lipase-catalyzed procedure is described for the one-pot conversion of carboxylic acids into substituted amides via in-situ formation of the ethyl ester and subsequent aminolysis. The procedure was optimized for the preparation of tetrahydro-N-[3-(methylamino)-propyl]-2-furancarboxamide, an intermediate in the synthesis of Alfuzosin, a reducing agent of symptoms associated with benign prostatic hypertrophy. This methodology proved to be general and can be applied to open-chain, cyclic, hydroxy-, amino-, dicarboxylic, various chain lengths, and unsaturated acids. Moreover, the enzyme shows a regioselective behavior in relation to primary and secondary amino groups. The procedure involved the treatment of the corresponding carboxylic acid with ethyl alcohol in presence of immobilized Candida antarctica lipase followed by addition of amine. The amide is obtained in good yields and regioselective way.     

(-)-(S)-flunoxaprofen and (-)-(S)-naproxen isocyanate: two new fluorescent chiral derivatizing agents for an enantiospecific determination of primary and secondary amines

The synthesis and analytical testing of two new fluorescent chiral derivatizing agents (-)-(S)-flunoxaprofen and (-)-(S)-naproxen isocyanate, is described. In a few simple steps the free carboxylic acids [(S)-flunoxaprofen and (S)-naproxen] are activated with ethyl chloroformate/sodium azide and transformed to the corresponding isocyanates. The crystalline reaction products display high enantiomeric and chemical purity and stability. The direction of the optical rotation of both substances is inverse to that of the corresponding carboxylic acids. At ambient temperature the reagents swiftly react with primary and secondary amines, yielding highly fluorescent ureas. The applicability of the two reagents for the resolution of racemic amines was tested with a number of pharmaceuticals (antiarrhythmics, beta-adrenergic antagonists, calcium channel blockers, centrally acting antidepressants). The diastereoisomeric derivatives were efficiently resolved and separated from side-products by means of normal and reversed-phase high-performance liquid chromatography (HPLC). The use and sufficient sensitivity of the two reagents for pharmacokinetic studies were demonstrated with a determination of plasma levels of propranolol enantiomers after oral administration of the racemic drug [80 mg (R,S)-propranolol-HCl] to two volunteers.     

Stereospecific Reduction of Ethyl 2′ -Ketopantothenate to Ethyl D-(+)-Pantothenate with Microbial Cells as a Catalyst

A novel enzymatic process for the synthesis of D-(+)-pantothenic acid through the asymmetric reduction of the 2′ -ketopantothenate ester is described. Candida macedoniensis AKU 4588 was found to convert ethyl 2′ -ketopantothenate (80 mg/ml) almost specifically to ethyl D-(+)-pantothenate (>98% enantiomeric excess), with a molar yield of 97.2%.     

Solid-supported and pegylated H-Pro-Pro-Asp-NHR as catalysts for asymmetric aldol reactions

H-Pro-Pro-Asp-NH2 is a highly active and selective catalyst for asymmetric aldol reactions. Here, the versatility of H-Pro-Pro-Asp-NH2 has been further improved by immobilization on a solid support and functionalization with a short polyethylene glycol linker at the C-terminus. The development, synthesis, and the catalytic properties in aldol reactions of H-Pro-Pro-Asp-resin and H-Pro-Pro-Asp-Ahx-NH(CH2CH2O)3CH3 are described. For the solid-supported catalyst, TentaGel with a loading of 0.1-0.2 mmol g(-1) proved to be the optimal support. The solid-supported catalyst can be recycled at least three times without a significant drop in the catalytic activity or selectivity. Using the pegylated catalyst H-Pro-Pro-Asp-Ahx-NH(CH2CH2O)3CH3, only 0.5 mol % are necessary to obtain aldol products in up to 96% yield and 91% enantiomeric excess. In all cases, enantioselectivities are comparable to those obtained with the parent catalyst H-Pro-Pro-Asp-NH2. Thus, immobilization of H-Pro-Pro-Asp-NH2 on Tentagel as well as pegylation led to catalysts with selectivities comparable to the nonmodified catalyst, exhibiting additional distinct advantages such as facile reusability, ease of handling, higher solubility, and thereby greater versatility. handling, higher solubility, and thereby greater versatility.     

Hydrolysis and Esterification with Lipase from Candida Cylindracea. Influence of the Reaction Conditions and Acid Moiety on the Enantiomeric Excess

The enantiomeric ratio for hydrolysis and synthesis of 1-phenyl ethanol esters of straight chain aliphatic carboxylic acids catalyzed by Candida cylindracea lipase was determined. A distinct maximum in enantiomeric ratio was observed for valeric and caproic acid in the hydrolytic direction. No significant maximum could be determined in the esterification reaction. Even though the enzyme provided larger enantiomeric ratios in the synthetic direction the enantiomeric excess of the alcohol was not higher. The enantiomeric excess was depressed by racemization reactions in the esterification as the reaction approached thermodynamic equilibrium at an insufficient conversion. While choosing the optimal chain length of the acyl donor is important in hydrolytic reactions it seems to be of greater value to raise the equilibrium conversion in the esterification reactions.     

Non-radioactive labeling and detection of nucleic acids. IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin)

The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2'-deoxyuridine-5'-triphosphate. This results in digoxigenin-modified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[11]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic labeling of DNA with digoxigenin-haptens by Klenow enzyme-catalysed random-primed synthesis. In addition the synthesis of N-[4-azidobenzoyl]-N'-[(3-O-digoxigeninyl)methylcarbonyl)]-1 ,8-diamino- 3,6-dioxaoctane (photodigoxigenin), a photoactivatable analog of digoxigenin, is described which can be applied for photolabeling of DNA and RNA with digoxigenin-haptens leaving the nucleic acid molecules intact.