Enantioselective Hydrolysis of d,l‐Menthyl Benzoate to L‐(–)‐Menthol by Recombinant Candida rugosa Lipase LIP1

The stereoselective synthesis of L ‐menthol is an attractive process in the flavor and fragrance industry. One promising way to obtain optically pure menthol is the enantioselective hydrolysis of menthol esters under enzymatic catalysis. We developed an effective and highly enantioselective method for the synthesis of L ‐(−)‐menthol (>99% EE) by hydrolyzing the key industrial starting compound, d, l ‐menthyl benzoate. The enzyme of choice was the lipase from Candida rugosa (CRL). While commercially available preparations of this lipase showed only minor selectivity (E=15), excellent enantiomeric purity (E>100) was achieved using the heterologously expressed isoenzyme LIP1.     

Enhancement of Gluconobacter oxydans catalyzing D‐lactic acid production by coupling of synthesis and separation

BACKGROUND: Gluconobacter oxydans was applied for enantioselective oxidation of racemic‐1,2‐propanediol. Accumulation of product in the biotransformation reactor inhibited the activity and enantioselectivity of oxidative enzymes, and it also led to overproduction of lactaldehyde as an intermediate of incomplete oxidation. It was important to improve the enzymatic activity and ee value of the chiral product. RESULTS: The coupling of biotransformation and separation was studied with an in situ product removal method. Anion exchange resin was applied to remove D ‐lactic acid on line. The ee value achieved was 95% and product yield increased by 50%, compared with the control result without coupling system. CONCLUSION: Coupling of biotransformation and separation effectively decreased the free D ‐lactic acid concentration in the system. Product inhibition of enzyme activity and enantioselectivity was reduced, and the ee value and product yield were improved. Copyright © 2009 Society of Chemical Industry     

Simple and Highly Convenient Two‐Step Practical Procedure for the Synthesis of Optically Pure Methyl D‐erythro‐2,3‐Dihydroxybutanoate

A synthesis of enantiopure methyl D ‐erythro‐2,3‐dihydroxybutanoate has been realized using two simple and consecutive reactions on D ‐erythronolactone as the starting material. The two reactions are lactone ring opening with hydrobromic acid in methanol and subsequent reductive debromination.     

D-(-)-苯甘氨酸衍生物拆分DL-扁桃酸的研究

描述了用D-(-)-苯甘氨酸丁酯及其盐酸盐为拆分剂,在水或醇的反应体系中,与扁桃酸反应,制备R-(-)-扁桃酸和S-(+)-扁桃酸的过程;其中R-(-)-扁桃酸有98.9％的收率和32％的光学纯度;而S-(+)-扁桃酸有90.1％的收率和66.73％的光学纯度。另外也描述了苯甘氨酸丁酯及其盐酸盐的一般合成方法。     

Cyclopropyl Building Blocks for Organic Synthesis,Part 100. Advanced Syntheses of Cyclopropylideneacetates –Versatile Multifunctional Building Blocks for Organic Synthesis

A well reproducible and inexpensive preparation of the cyclopropylideneacetates 2 – 4 has been developed. The key intermediate 2‐(1′‐mesyloxycyclopropyl)acetic acid ( 8 ), produced either from methyl phenylacetate ( 1 ) or 3,3‐dimethoxypropionate ( 5 ‐Me) and 3,3‐diethoxypropionate ( 5 ‐Et) in a sequence of Kulinkovich reductive cyclopropanation, mesylation and oxidative cleavage or cleavage and oxidation, respectively, was either converted to the benzyl ester 11b , or chlorinated (brominated) via the in situ formed acid chloride. The α‐chloro‐ 12a and α‐bromo ester 12b were dehydromesylated by treatment with triethylamine to furnish methyl 2‐chloro‐2‐cyclopropylideneacetate ( 3 ‐Me) and the 2‐bromo analogue 4 ‐Me with an overall yield of 68% (65%, 68%) and 52% (49%, 51%) respectively, starting from 1 ( 5 ‐Me, 5 ‐Et). The parent benzyl cyclopropylideneacetate 2 ‐Bn was obtained by dehydromesylation of 11b with potassium t‐butoxide in t‐butyl methyl ether with an overall yield of 60% (57%, 9%) from 1 ( 5 ‐Me, 5 ‐Et).     

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

A recombinant Escherichia coli strain was constructed which efficiently expressed the enantioselective nitrilase from Alcaligenes faecalis DSMZ 30030 as a hisitidine‐tagged enzyme variant under the control of a rhamnose inducible promoter. The enzyme was purified from cell extracts and used for the preparation of cross‐linked enzyme aggregates (CLEAs). The conditions for the preparation of the CLEAs were optimized using various organic solvents and cross‐linking agents and a procedure was developed which combined a precipitation with 85 % (v/v) isopropyl alcohol and a cross‐linking with 30 mM glutaraldehyde. Thus, about 80 % of the initial nitrilase activity could be incorporated into the CLEAs. The hydrolysis of racemic mandelonitrile to (R)‐mandelic acid was compared between the soluble nitrilase preparations and their CLEAs (nit‐CLEAs). The nitrilase activity in the CLEAs was at 30 °C and 60 °C about 5 times more stable than in the soluble preparations. The CLEAs could be reused 5 times with only about 10 % reduction in activity. The enantioselectivity of the nitrilase for the formation of (R)‐mandelic acid from racemic mandelonitrile decreased for both preparations with increasing temperatures (10 °C to 50 °C), but this effect was significantly less pronounced for the CLEAs. A detailed analysis of solvent effects on nitrilase enantioselectivity allowed thermodynamic insights into contributions from free energy component (activation enthalpy and entropy) to chiral preference of nitrilase in such non conventional media.     

Efficient Biosynthesis of (R)‐ or (S)‐2‐Hydroxybutyrate from l‐Threonine through a Synthetic Biology Approach

An efficient multi‐enzyme cascade reaction for the synthesis of (R)‐ or (S)‐2‐hydroxybutyric acid [(R)‐ or (S)‐2‐HB] from l ‐threonine was developed by using recombinant Escherichia coli cells expressing separately or co‐expressing l ‐threonine deaminase from Escherichia coli K‐12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l ‐lactate dehydrogenase (l ‐LDH) from Oryctolagus cuniculus or d ‐lactate dehydrogenase (d ‐LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l ‐threonine were completely transformed to (R)‐ or (S)‐2‐HB in optically pure form (>99% ee) with high isolated yields. This one‐pot multi‐enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds.

     

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate,D‐Fructose 6‐Phosphate and Analogues

One‐pot multienzymatic reactions have been performed for the synthesis of 1‐deoxy‐D ‐fructose 6‐phosphate, 1,2‐dideoxy‐D ‐arabino‐hept‐3‐ulose 7‐phosphate, D ‐fructose 6‐phosphate and D ‐arabinose 5‐phosphate. The whole synthetic strategy is based on an aldol addition reaction catalysed by fructose‐6‐phosphate aldolase (FSA) as a key step of a three or four enzymes‐catalysed cascade reaction. The four known donors for FSA – dihydroxyacetone (DHA), hydroxyacetone (HA), 1‐hydroxy‐2‐butanone (HB) and glycolaldehyde (GA) – were used with D ‐glyceraldehyde 3‐phosphate as acceptor substrate. The target phosphorylated sugars were obtained in good to excellent yields and high purity.     

Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi‐Enzyme System

Mutant α‐amino‐ε‐caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D ‐amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co‐expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)‐phenylalanine and non‐natural (R)‐phenylalanine derivatives (4‐OH, 4‐F, 3‐F, and 2‐F‐Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)‐phenylalanine with 84% yield and 99% ee from (RS)‐phenylalaninamide (400 mM) in 22 h. (R)‐Tyrosine and 4‐fluoro‐(R)‐phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co‐expresed two genes encoding mutant ACL racemase and L ‐amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)‐phenylalanine derivatives. Moreover, 2‐aminophenylpropionitrile was converted to (R)‐phenylalanine by DKR using a combination of the non‐stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.     

Catalytic Hydrogenation of α‐Iminophosphonates as a Method for the Synthesis of Racemic and Optically Active α‐Aminophosphonates

It was shown that the catalytic hydrogenation of α‐iminophosphonates by molecular hydrogen can serve as a convenient method for the synthesis of racemic and optically active α‐aminophosphonates. Up to 94% ee was achieved in the rhodium‐catalyzed enantioselective hydrogenation using chiral ligand (R)‐BINAP.     

扁桃酸的光学拆分

描述了用D-(-)-苯甘氨酸丁酯及其盐酸盐为拆分剂,在水或醇的反应体系中,与扁桃酸反应,制备R-(-)-扁桃酸和 S-(+)-扁桃酸的过程;其中 R-(-)-扁桃酸有 98.9%的收率和32 %的光学纯度;而S-(+)-扁桃酸有90.1%的收率和 66.73%的光学纯度。另外也描述了苯甘氨酸丁酯及其盐酸盐的一般合成方法。     

Enantioselective Construction of Chiral 2,3‐cis‐Dimethyldihydrobenzofuran with an All‐Carbon Quaternary Center: An Efficient Approach to (+)/(−)‐PI‐220 and (+)‐3‐epi‐Furaquinocin C

A new strategy for the enantioselective construction of chiral 2,3‐cis‐dimethyldihydrobenzofurans has been developed by a ruthenium‐catalyzed asymmetric hydrogenation of racemic α‐aryloxybutanone via dynamic kinetic resolution (DKR), combined with a palladium‐catalyzed intramolecular reductive Heck cyclization. Starting from optically active 2,3‐cis‐dimethyldihydrobenzofuran, the natural products (+)/(−)‐PI‐220 and (+)‐3‐epi‐furaquinocin C were synthesized in high yields.

     

Organocatalytic Asymmetric Sulfa‐Michael Addition to α,β‐Unsaturated Ketones

The highly enantioselective organocatalytic sulfa‐Michael addition to α,β‐unsaturated ketones has been accomplished using benzyl and tert‐butyl mercaptans as the sulfur‐centered nucleophiles. Optically active products are obtained in high yields and good to excellent stereocontrol (up to 96 % ee) from a large variety of enones. Central to these studies has been the use of the catalytic primary amine salt A , derived from 9‐amino‐(9‐deoxy)‐epi‐hydroquinine and D ‐N‐Boc‐phenylglycine, in which both the cation and the anion are chiral, that exhibits high reactivity and selectivity for iminium ion catalysis with enones.     

Synthesis,post‐modification and characterization of linear polystyrene‐based supports for interaction with immobilized biocatalysts

A series of linear polystyrene‐based supports has been synthesized using conventional or controlled (reversible addition fragmentation chain transfer or nitroxide‐mediated) free radical polymerizations, in order to modulate their molecular weight characteristics. Polymer post‐modification via esterification of the primary hydroxyl alcohol functionalities, pendant from the polymer backbone, with a mandelic acid derivative introduces efficiently (>85% yield) racemic, enzyme‐cleavable moieties. A study of the interactions of these polymeric substrates with immobilized penicillin G acylase, in mixed organic/aqueous buffer solvent systems, results in a modest enantioselectivity (comparable with that of racemic mandelic acid methyl ester), but reveals that the biocatalytically active moieties on the linear polymers can be cleaved with first‐order rate kinetics, as observed with small molecules in solution; the reaction rates (which can differ by more than one order of magnitude) are influenced both by the synthetic polymer length and by the polymer structure resulting from the method of polymerization used. Copyright © 2012 Society of Chemical Industry     

Asymmetric Synthesis of 3‐Substituted Cyclohexylamine Derivatives from Prochiral Diketones via Three Biocatalytic Steps

Prochiral bicyclic diketones were transformed to a single diastereomer of 3‐substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a C C hydrolase (6‐oxocamphor hydrolase) in the first step and by an ω‐transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the C C hydrolytic step as well as the esterification could be run simultaneously in a one‐pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio‐amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω‐transaminase employed and the substrate the cis‐ as well as the trans‐diastereomers could be obtained in optically pure forms.     

In Vitro Double Oxidation of n‐Heptane with Direct Cofactor Regeneration

A novel concept for the direct oxidation of cycloalkanes to the corresponding cyclic ketones in a one‐pot synthesis in water with molecular oxygen as sole oxidizing agent was reported recently. Based on this concept we have developed a new strategy for the double oxidation of n‐heptane to enable a biocatalytic resolution for the direct synthesis of heptanone and (R)‐heptanols in a one‐pot reaction. The bicatalytic cascade employs an NADH driven P450 BM3 monooxygenase variant (WT^NADH, 19A12^NADH or CM1^NADH) and an (S)‐enantioselective alcohol dehydrogenase (RE‐ADH). In the initial step n‐heptane is hydroxylated under consumption of NADH to produce (R/S)‐heptanol. In the second oxidation step the (S)‐heptanol enantiomers are transformed to the corresponding ketones, reducing and thereby regenerating the cofactor. Characterization of initial hydroxylation step revealed high turnover frequencies (TOF) of up to 600 min⁻¹, as well as high coupling efficiencies using NADH as cofactor (up to 44%). In the cascade reaction a nearly 2‐fold improved product formation was achieved, compared to the single hydroxylation reaction. The total product concentration reached 1.1 mM, corresponding to a total turnover number (TTN) of 2500. Implementation of an additional cofactor regeneration system (D ‐glucose/glucose dehydrogenase) enabled a further enhancement in product formation with a total product concentration of 1.8 mM and a TTN of 3500.     

Organocatalytic Enantioselective Sulfenylation of β‐Keto Phosphonates: A Convenient Approach to Construct Hetero‐ Quaternary Stereocenters

The highly effective and enantioselective sulfenylation of β‐keto phosphonates catalyzed by α,α‐diaryl‐L ‐prolinols has been developed. The optically active α‐sulfenylated β‐keto phosphonates could be obtained under mild reaction conditions in good yields (up to 92%) and with excellent enantioselectivities (up to 92% ee).     

Synthesis and characterization of novel poly(amide imide)s based on bis(p‐amidobenzoic acid)‐n‐trimellitylimido‐L‐leucine

N‐Trimellitylimido‐L ‐leucine was reacted with thionyl chloride, and N‐trimellitylimido‐L ‐leucine diacid chloride was obtained in a quantitative yield. The reaction of this diacid chloride with p‐aminobenzoic acid was performed in dry tetrahydrofuran, and bis(p‐amidobenzoic acid)‐N‐trimellitylimido‐L ‐leucine (5) was obtained as a novel optically active aromatic imide–amide diacid monomer in a high yield. The direct polycondensation reaction of the monomer imide–amide diacid 5 with 4,4′‐diaminodiphenylsulfone, 4,4′‐diaminodiphenylether, 1,4‐phenylenediamine, 1,3‐phenylenediamine, 2,4‐diaminotoluene, and benzidine (4,4′‐diaminobiphenyl) was carried out in a medium consisting of triphenyl phosphite, N‐methyl‐2‐pyrolidone, pyridine, and calcium chloride. The resulting novel poly(amide imide)s (PAIs), with inherent viscosities of 0.22–0.52 dL g^?1, were obtained in high yields, were optically active, and had moderate thermal stability. All of the compounds were fully characterized with IR spectroscopy, elemental analyses, and specific rotation. Some structural characterization and physical properties of these new optically active PAIs are reported. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 35–43, 2002; DOI 10.1002/app.10181     

Single‐Biocatalyst Synthesis of Enantiopure d‐Arylalanines Exploiting an Engineered d‐Amino Acid Dehydrogenase

A practical and efficient biocatalytic synthesis of aromatic d ‐amino acids has been developed, based on the reductive amination of the corresponding α‐keto acids via a recombinant whole cell system composed of an engineered dehydrogenase and cofactor recycling apparatus. The reaction was shown to give excellent enantioselectivity (≥98%) and good yields at the preparative scale across a broad range of substrates. Additionally, the structure of the variant enzyme was solved to allow rationalisation of the observed reaction rates. The engineered whole cell catalyst was also used to mediate the production of d ‐phenylalanine derivatives from racemic mixtures and cheaper l ‐amino acids by combining it with an enantiocomplementary deaminase.

     

A highly stable whole‐cell biocatalyst for the enantioselective synthesis of optically active alpha‐hydroxy acids

BACKGROUND: Biocatalysts have gained increasing attention because of their inherent advantages over chemical catalysts. However, the poor operational stability has always prevented their broad application. In this study, (R)‐mandelic acid was chosen as a model compound of alpha‐hydroxy acids. The objective was to obtain a new biocatalyst with desired operational stability for the preparation of (R)‐mandelic acid as well as other optically pure alpha‐hydroxy acids of pharmaceutical importance. RESULTS: Using a two‐step screening strategy, Saccharomyces ellipsoideus GIM2.105 was selected as an effective biocatalyst with high enantioselectivity and remarkable operational stability. After 20 cycles of reuse, whole cells of S. ellipsoideus GIM2.105 maintained its activity, and no obvious decrease in conversion or enantiomeric excess (ee) was observed. Furthermore, effects of various reaction parameters, including pH, temperature, co‐substrate (type, concentration), substrate concentration and reaction time, on the bioreduction were studied. Under optimal conditions, (R)‐mandelic acid and four substituted aromatic (R)‐alpha‐hydroxy acids were prepared in high ee (95–>99%) and good conversion (>90%). CONCLUSION: The high enantioselectivity, remarkable operational stability and mild reaction conditions showed S. ellipsoideus GIM2.105 to be an economical biocatalyst with great industrial application potential for the production of optically active alpha‐hydroxy acids. Copyright © 2009 Society of Chemical Industry