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ChemInform Abstract: Polyfunctional (R)-2-Hydroxycarboxylic Acids by Reduction of 2-Oxo Acids with Hydrogen Gas or Formate and Resting Cells of Proteus vulgaris.
Abstract
Various ()-2-hydroxy acids such as ()-2-hydroxy-3-enoic-, 3,5-dienoic-, 4-oxo-, ()-3-hydroxy and some others were prepared on a scale up to 0.12 mol by biocatalytic reduction of the corresponding 2-oxo acids with P. vulgaris and hydrogen gas and/or formate as electron donors. With the exception of the 2-hydroxy-4-oxo acids it could be proved that the enantiomeric excess is > 97%. For the 4-oxo derivatives this enantiomeric excess can be assumed. The yields of isolated products are high because they were isolated from rather small amounts of biocatalyst and low buffer concentrations, product concentrations in the range of 0.1–0.24 M were obtained. For 1 mmol of product formation in 15–20 h about 20–40 mg (dry weight) of P. vulgaris cells are necessary.
... 20 Although there is sufficient literature evidence available for synthesis of R, -unsaturated keto esters, the present set of conditions afforded compounds 38, 39, and 75-84 in better yields and are scalable. [21][22][23][24][25][26][27][28] As dihydropyrazole analogues 4-24 contained a chiral center at position 5, the above synthesis provides a target compound in the form of racemic mixture. To further explore the stereochemical (chiral) requirements/consequences for binding to the CB1 receptor, the key compound 9 was synthesized as a chiraly pure moiety by introducing resolution at suitable stage. ...
A number of analogues of diaryl dihydropyrazole-3-carboxamides have been synthesized. Their activities were evaluated for appetite suppression and body weight reduction in animal models. Depending on the chemical modification of the selected dihydropyrazole scaffold, the lead compounds--the bisulfate salt of (+/-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 26 and the bisulfate salt of (-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 30--showed significant body weight reduction in vivo, which is attributed to their CB1 antagonistic activity and exhibited a favorable pharmacokinetic profile. The molecular modeling studies also showed interactions of two isomers of (+/-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 9 with CB1 receptor in the homology model similar to those of N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant) 1 and 4S-(-)-3-(4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (SLV-319) 2.
Six optically active α-hydroxyl-β,γ-unsaturated acid esters 1a to 1f were synthesised, and they are significant moieties of the cerebrosides. The chiral intermediate alkynol 4 prepared by catalytic asymmetric addition had 99% ee, and which was converted into the target compounds 1a to 1f with high enantiomeric purity.
A highly enantioselective preparation (92–99% enantiomeric excess, 100% conversion) of various 4-aryl-2-hydroxy but-3-enoic carboxylic acid esters from the corresponding 4-phenyl-2-keto but-3-enoic acid esters mediated by plant cell cultures of Daucus carota under mild and environmentally benign conditions in aqueous medium is described.
Deracemisation of racemic (3E)-alkyl-4-(hetero-2-yl)-2-hydroxybut-3-enoates using Candida parapsilosis ATCC 7330 resulted in the formation of one enantiomer in high enantiomeric excess [up to >99% ee] and isolated yields [up to 79%]. The absolute configuration of the enantiomerically pure (3E)-ethyl-4-(thiophene-2-yl)-2-hydroxybut-3-enoate as determined by 1H NMR of the Mosher esters was found to be (S).
A convenient and practical method for the selective reduction of C=O bond of a wide spectrum of alpha-keto beta,gamma-unsaturated esters via transfer hydrogenation reaction under the catalysis of Ru(p-cymene)(TsDPEN) (TsDPEN: monotosylated 1,2-diphenylethylene- 1,2-diamine) was developed.
Rhodococcus erythropolis NCIMB 11540 was found to have a highly active nitrile hydratase/amidase enzyme system present which accepts the nitrile function of α-hydroxynitriles (cyanohydrins) as substrates. This biocatalytic hydrolysis using whole bacterial cells leads to α-hydroxy carboxylic acids which are much valued chiral building blocks in organic synthesis. Employing enantiopure cyanohydrins, which are easy available using (R)- or (S)-hydroxynitrile lyases, the products were obtained in high yield without racemization, decomposition or side reactions. Herein, the application of this biotransformation for preparative scale applications is described. To clarify the substrate acceptance of the nitrile hydrolyzing enzymes of R. erythropolis NCIMB 11540, several selected model compounds were subjected to biocatalytic hydrolysis. Reaction conditions were optimized to enable preparative scale conversions. In this manner, (R)-2-chloromandelic acid and (R)-2-hydroxy-4-phenylbutyric acid, two important pharmaceutical intermediates, were prepared in a gram scale. The substrate concentrations used were 9.3 and 13g/l, respectively. The process yielded both acids in high optical (ee>99 and 98%) and chemical (98%) yield after short reaction times (3 and 1.5h).
The application of various artificial electron mediators in combination with redox enzymes present in anaerobically grown cells is described. Examples include viologens of different redox potential, cobalt complexes, anthraquinones, phenoxazines, phenazines and others. The regeneration of the reduced or oxidised mediators by various methods is discussed, with hydrogen gas, carbon monoxide, formate or a cathode as electron donors, and dimethylsulphoxide, quinones, oxygen or an anode as electron acceptors. The enzymes used, usually in the form of resting cells or crude cell extracts of clostridia or Proteus species are mostly reversible. Examples for preparative reductions as well as for dehydrogenations are given.
(S)-Cyclohexyl lactic acid is a component of the selective E-selectin inhibitor 2 ((S)-cHexLact-2-O-(3-Galβ(1→3)ddGlc(4→1)αFuc). We describe the evaluation of various synthetic routes to this building block: (A) diazotation of phenylalanine followed by phenyl ring hydrogenation; (B) phenyl ring hydrogenation of phenyl alanine followed by diazotation; (C) acidic hydrolysis of the cyanohydrin derived from phenylacetaldehyde, enantiomeric resolution of the resulting, racemic phenyl lactic acid via diasteromeric salt formation and phenyl ring hydrogenation; (D) enantioselective dihydroxylation of a cinnamate ester, followed by hydrogenation of the benzylic hydroxy group and the aromatic nucleus; (E) enantioselective biocatalytic reduction of phenylpyruvic acid, followed by phenyl ring hydrogenation. The development of (2R)-2-O-(4-nitrophenyl)sulfonyl-cyclohexyl lactic acid p-bromobenzylester 21 as a buidling block with improved crystallinity and stability is also described.
Mandelate racemase (EC 5.1.2.2) is one of the few biochemically well-characterized racemases. The remarkable stability of this cofactor-independent enzyme and its broad substrate tolerance make it an ideal candidate for the racemization of non-natural -hydroxycarboxylic acids under physiological reaction conditions to be applied in deracemization protocols in connection with a kinetic resolution step. This review summarizes all aspects of mandelate racemase relevant for the application of this enzyme in preparative-scale biotransformations with special emphasis on its substrate tolerance. Collection and evaluation of substrate structure-activity data led to a set of general guidelines, which were used as basis for the construction of a general substrate model, which allows a quick estimation of the expected activity for a given substrate.
(R,R)-Bis(diphenylphosphino)-1,3-diphenylpropane as a versatile ligand for enantioselective hydrogenationsSynthesis of both enantiomers of 1-Phenylethanol by reduction of acetophenone with Geotrichum candidum IFO 5767Titanocene-catalyzed reduction of ketones in the presence of water. A convenient procedure for the synthesis of alcohols via free-radical chemistryXyl-tetraPHEMP: a highly efficient biaryl ligand in the [diphosphine RuCl2 diamine]-catalyzed hydrogenation of simple aromatic ketonesN-Arenesulfonyl- and N-Alkylsulfamoyl-1,2-diphenylethylenediamine ligands for ruthenium-catalyzed asymmetric transfer hydrogenation of activated ketonesThe synthesis and application of BrXuPHOS: a novel monodentate phosphorus ligand for the asymmetric hydrogenation of ketonesIn Situ formation of ligand and catalyst: application in ruthenium-catalyzed enantioselective reduction of ketonesSynphos and Difluorphos as ligands for ruthenium-catalyzed hydrogenation of alkenes and ketonesAn arene ruthenium complex with polymerizable side chains for the synthesis of immobilized catalystsSelective reduction of carbonyl group in β, γ-unsaturated α-alpha-ketoesters by transfer hydrogenation with Ru-(p-cymene) (TsDPEN)Preparation of polymer-supported Ru-TsDPEN catalysts and their use for the enantioselective synthesis of (S)-fluoxetinePolymer-supported chiral sulfonamide-catalyzed reduction of β-keto nitriles: a practical synthesis of (R)-Fluoxetine
Lipases such as Candida rugosa lipase and Pseudomonas cepacia (Amano Ps) lipase immobilized in gelatin gels (gelozymes) exhibit very high enantioselectivities (E>200) during alcoholysis of racemic 2-acetoxy-4-phenyl-(E)-but-3-enenitrile with n-butanol in hexane and diisopropyl ether. C. rugosa lipase in n-hexane shows selectivity towards the (R)-ester while P. cepacia (Amano Ps) lipase shows selectivity towards the (S)-ester producing the corresponding cyanohydrins. After decomposition of the cyanohydrin by treatment with 1M imidazole solution to cinnamaldehyde and its removal by bisulfite treatment, (R)- or (S)-enantiomer of 2-acetoxy-4-phenyl-(E)-but-3-enenitrile can be obtained in high yields (90%) and high enantiomeric excess (>99%).
Biocatalytic racemization of aliphatic, (aryl)aliphatic and aromatic alpha-hydroxycarboxylic acids was achieved via a reversible oxidation-reduction sequence using a pair of stereo-complementary Prelog- and anti-Prelog D- and L-alpha-hydroxyisocaproate dehydrogenases from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008, resp., overexpressed in Escherichia coli. The mild reaction conditions ensured essential 'clean' isomerization, undesired 'over-oxidation' of the substrate forming the alpha-ketoacid could be suppressed by exclusion of O(2) and adjustment of the NAD(+)/NADH-ratio.
An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2‐oxocar‐boxylates at the expense of reduced artificial redox mediators to (2 R )‐hydroxycarboxylates has been purified to a specific activity of up to 1800 μmol · min ⁻¹ · mg ⁻¹ for the reduction of phenylpyruvate. The membrane‐bound non‐pyridine nucleotide‐dependent enzyme appears in the form of various oligomers of the 80‐kDa monomer.
The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on ⁹⁹ Mo‐labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis.
Permanganate oxidation delivers 0.7 mol pterin‐6‐carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron‐sulphur clusters.
Proteus vulgaris and P. mirabilis were grown anaerobically on glucose in the absence or presence of dimethylsulphoxide (DMSO) as electron acceptor or on (S)- or (RS)-lactate in the presence of nitrogen (N)-oxides, sulphur (S)-oxides or pyruvate. During growth on glucose the main fermentation product was ethanol and in the presence of DMSO it was (R)-lactate. Growth on (RS)-lactate led to acetate and (R)-lactate and growth on (S)-lactate produced almost only acetate. Depending on the growth medium, in crude extracts of P. vulgaris the activities of (R)-2-hydroxycarboxylate viologen oxidoreductase (HVOR), measured as (R)-lactate dehydrogenase, and DMSO reductase were 0.5–8.0 and 0.3–1.1 units (U)/mg protein, respectively. Addition of nitrate to the growth medium diminished both enzyme activities to P. mirabilis showed also high HVOR activity when grown on (RS)-lactate in the presence of DMSO. Also, Clostridium homopropionicum contained 1.8 U/mg of a pyridine-nucleotide-independent reversible (R)-lactate dehydrogenase when tested with the electron acceptor 1,1-carbomoylmethylviologen (NH2CO-MV). None of the organisms studied were significantly active with (S)-lactate and NH2CO-MV. The possible physiological role of the HVOR may be as a dissimilatory (R)-lactate dehydrogenase.
For the stereoselective reduction of 2-oxo acids by hydrogen gas or formate to d-2-hydroxy acids, anaerobically grown Proteus vulgaris cells were immobilized in alginate, -carrageenan, chitosan, polyurethane and polyacrylamide acylhydrazide. With the exception of the last matrix, immobilization led to a decrease in the apparent activity, probably caused by diffusional limitations. Chitosan or polyurethane-entrapped cells kept their initial catalytic activity for over more than 600 h in a continuous working period. In both matrices the cells could be partially reactivated by incubation of the immobilisates in growth medium. Polyurethane-immobilized cells (and also cell membranes) were repeatedly usable. After 30 batch operations, 30–40% of the initial reduction rate was still detectable. Chitosan-immobilized cells did not lose any activity during 17 months of storage at 4 C under exclusion of oxygen.
Enantioselective bioreduction of alkyl 2-oxo-4-arylbutanoates and 2-oxo-4-arylbut-3-enoates mediated by Candida parapsilosis ATCC 7330 resulted in the formation of the corresponding (S)-2-hydroxy compounds in high enantiomeric excesses (93–99%) and good isolated yields (58–71%). The absolute configuration of enantiomerically pure ethyl 2-hydroxy-4-(p-methylphenyl)but-3-enoate obtained by the reduction of the corresponding keto ester was assigned by 1H NMR using Mosher’s method.
Proteus vulgaris or P. mirabilis dehydrogenate (R)-lactate with high space time yield and high productivity numbers. Twenty grammes (dry weight) resting cells of P. mirabilis per litre converted a 0.65 M solution of (R)-lactate in 1 h to 94% pyruvate, which corresponds to a space time yield of 294 mol l−1 d−1 or about 25.8 kg l−1 d−1. In most cases anthraquinone-2,6-disulphonate was used as an effective redox mediator. The reduced quinone was regenerated by one of the following enzymic reductions: dimethylsulphoxide to dimethylsulphide, which leaves the system; N-oxides to amines; fumarate to succinate; and nitrate to nitrogen. For the latter mediator regeneration it was necessary to apply Paracoccus denitrificans together with Proteus species. The bioconversion of (R)-lactate to pyruvate can be conducted without using a buffer.Also the electrochemical reoxidation of anthraquinone-2,6-disulphonate is possible.Solutions of 0.24 M (R)-lactate can be dehydrogenated with a yield of 99% by stoichiometric quinone concentrations.
New and high regioselective method of the synthesis of 2,7-diaryl-4,7-dihydropyrazolo[1,5-a]pyrimidine-5-carboxylic acids by reaction of 3-aryl-5-aminopyrazoles with arylidenpyruvic acid at room temperature under ultrasonication was developed and discussed.
The synthesis of the C1-C11 fragment 33 of bafilomycin A(1) was achieved. Intermediate ketone 16 was prepared in six steps from 4-oxopimelate 13. Desymmetrization of this ketone using Koga's chiral base followed by TMSCl quench furnished silyl enol ether 17 with excellent enantioselectivity. Further elaboration led to C5-C11 aldehyde 24, which was coupled with sulfone 3 to give lactone 25 in very good yield. The subsequent reductive elimination created the E-trisubstituted C4-C5 olefin with a 13:1 selectivity. The E C2-C3 double bond was then installed by methanol elimination, and compound 33 was obtained after a few functional group manipulations and a Negishi methyl zirconation.
The concise synthesis of a potent thrombin inhibitor was accomplished by a mild lactone aminolysis between an orthogonally protected bis-benzylic amine and a diastereomerically pure lactone. The lactone was synthesized by the condensation of l-proline methyl ester with an enantiomerically pure hydroxy acid, which in turn was synthesized by a highly stereoselective (>500:1 er) and productive (100,000:1, S/C) enzymatic reduction of an alpha-ketoester. In addition, a second route to the enantiomerically pure lactone was accomplished by a diastereoselective ketoamide reduction.
The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.
Addition of ethoxalyl chloride (ClCOCOOEt) to terminal alkynes at 60 degrees C in the presence of a rhodium(I)-phosphine complex catalyst chosen from a wide range affords 4-chloro-2-oxo-3-alkenoates regio- and stereoselectively. Functional groups such as chloro, cyano, alkoxy, siloxy, and hydroxy are tolerated. The oxidative addition of ethoxalyl chloride to [RhCl(CO)(PR(3))(2)] proceeds readily at 60 degrees C or room temperature and gives [RhCl(2)(COCOOEt)(CO)(PR(3))(2)] (PR(3) = PPh(2)Me, PPhMe(2), PMe(3)) complexes in high yields. The structure of [RhCl(2)(COCOOEt)(CO)(PPh(2)Me)(2)] was confirmed by X-ray crystallography. Thermolysis of these ethoxalyl complexes has revealed that those ligated by more electron-donating phosphines are fairly stable against decarbonylation and reductive elimination. [RhCl(2)(COCOOEt)(CO)(PPh(2)Me)(2)] reacts with 1-octyne at 60 degrees C to form ethyl 4-chloro-2-oxo-3-decenoate. The catalysis is therefore proposed to proceed by oxidative addition of ethoxalyl chloride, insertion of an alkyne into the Cl--Rh bond of the resulting intermediate, and reductive elimination of alkenyl-COCOOEt.
Structure-guided redesign of the active site of almond (R)-PaHNL5 for increased enantioselectivity resulted in four improved muteins. In particular, mutation V360I gave enhanced conversion rates and enantioselectivities higher than 96% ee for two structurally related substrates 1 and 2. Chiral building blocks for the synthesis of pharmaceutically active "prils" (example shown) can thus be produced on a large scale. (Chemical Equation Presented).
4-(5-methyl-3-phenyl-1H-pyrazole-4-yl)-benzenesulfonamide, the pyrazole isostere of valdecoxib was synthesised. The title compound was prepared by a six-step procedure starting from commercially available deoxybenzoin 1. The pyrazole isostere 13 showed slight anti-inflammatory and analgesic activities.
Asymmetric reduction of 3-chloro-2-oxoalkanoates with baker's yeast gave optically active 3-chloro-2-hydroxyalkanoates with ≥95% e.e. in most cases, which were converted to optically active 2,3-epoxyalcohols with ≥78.0% e.e.
Various ( )-2-hydroxy acids such as ( )-2-hydroxy-3-enoic-, 3,5-dienoic-, 4-oxo-, ( )-3-hydroxy and some others were prepared on a scale up to 0.12 mol by biocatalytic reduction of the corresponding 2-oxo acids with P. vulgaris and hydrogen gas and/or formate as electron donors. With the exception of the 2-hydroxy-4-oxo acids it could be proved that the enantiomeric excess is > 97%. For the 4-oxo derivatives this enantiomeric excess can be assumed. The yields of isolated products are high because they were isolated from rather small amounts of biocatalyst and low buffer concentrations, product concentrations in the range of 0.1–0.24 M were obtained. For 1 mmol of product formation in 15–20 h about 20–40 mg (dry weight) of P. vulgaris cells are necessary.
“Biocatalytic enantiomer resolution of 2‐hydroxycarboxylic acids”, rac ‐1 , has been achieved with the (nonisolated) 2‐oxoacid reductase from Proteus vulgaris . This enzyme is usually employed to reduce 2‐oxomonocarboxylic acids to ( R )‐2‐hydroxycarboxylic acids (> 99% ee) with methyl‐ or benzylviologen as electron mediator. The use of carbamoylmethylviologen has allowed, for the first time, the reverse reaction to be carried out. Only the ( R ) enantiomer of rac ‐ 1 undergoes dehydrogenation; the ( S )‐2‐hydroxycarboxylic acid remains unchanged. The mediator can be regenerated.
The L-lactate dehydrogenase of Bacillus stearothermophilus (BSLDH) is a stable, thermophilic oxidoreductase. It has been selected as a model of enzymes with considerable future promise in asymmetric synthesis in that it has been cloned to ensure a plentiful and inexpensive supply and because of the potential for tailoring its specificity to accept unnatural substrate structures via the site-directed mutagenesis techniques of molecular biology. In this study, the specificity of BSLDH toward representative α-keto acids possessing straight- and branched-chain alkyl, cycloalkyl, or aromatic side chains has been evaluated. The results show that substrates that are sterically bulky in the region of the α-keto group to be reduced are poorly accepted by the enzyme. Graphics analyses indicated that the low activities of these hindered substrates might be partly due to a bad interaction of the active site residue Gln102 with large or branched substituents adjacent to the α-keto group. Accordingly, Gln102 has been replaced by the smaller Asn residue by site-directed mutagenesis in an attempt to expand the active site volume available to receive substrates larger than the natural pyruvate. However, the kinetic data show that bulky α-keto acids are only marginally better accommodated by the Gln102 → Asn mutant than by the wild-type enzyme.
The rate of reduction of 2-oxocarboxylates by the 2-oxocarboxylate reductase of Proteus vulgaris and P. mirabilis shows its maximum already at reduced methylviologen concentrations of about 0.05 mM. Oxidized methylviologen is a positive, rate increasing effector. Exploiting this behavior leads to exceptionally high productivities especially with Proteus vulgaris, for the preparation of many different (2R)-hydroxycarboxylates in an electrochemical cell.Whole cells of Proteus vulgaris can use hydrogen and/or formate as electron donors with different viologens. Formate is more effective than hydrogen gas as electron donor. Surprisingly, the combination of H2 and formate leads to rates which are at least the sum or even twice the sum of the reduction rates observed with H2 or formate only. The presence of tetrachloromethane or heptane in shaken suspensions causes a rate enhancement with H2 and/or formate as electron donors.The growth of the cells has been optimized with respect to formate dehydrogenase and 2-oxocarboxylate reductase activity. The stability of both enzymes is sufficient for preparative work.
The resolution of 2-hydroxy acid enantiomers by the principle of ligand exchange chromatography on chemically modified silica
gel is described. The phases were synthezised by fixing L-amino acids via 2-glycidoxypropyl-trimethoxysilane to silica gel.
The highest enantioselectivity for 2-hydroxy acids was obtained by using L-hydroxyproline as fixed ligand and Cu(II) as complexing
agent. A number of aromatic as well as aliphatic 2-hydroxy acids could be resolved on this sorbent.