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2,3-dihydroxy-2-methylbutyric acid, also known as 2,3-dimethylglyceric acid, constitutes the acyl and/or the alcoholic moiety of many bioactive natural esters. Herein, we describe a chemoenzymatic methodology which gives access to all the four possible stereoisomers of the 2,3-dimethylglyceric acid ethyl ester. The racemic ethyl α-acetolactate, produced by the N-heterocycle carbene (NHC)-catalyzed coupling of ethyl pyruvate and methylacetoin was employed as the starting material. The racemic mixture was resolved through (S)-selective reductions, promoted by the acetylacetoin reductase (AAR) affording the resulting ethyl (2R,3S)-2,3-dimethylglycerate; the isolated remaining (S)-ethyl α-acetolactate was successively treated with baker’s yeast to obtain the corresponding (2S,3S) stereoisomer. syn-2,3-Dimethylgliceric acid ethyl ester afforded by reducing the rac-α-acetolactate with NaBH4 in the presence of ZnCl2 was kinetically resolved through selective acetylation with lipase B from Candida antarctica (CAL-B) and vinyl acetate to access to (2S,3R) stereoisomer. Finally, the (2R,3R) stereoisomer, was prepared by C3 epimerization of the (2R,3S) stereoisomer recovered from the above kinetic resolution, achieved through the TEMPO-mediated oxidation, followed by the reduction of the produced ketone with NaBH4. The resulting 2,3-dimethylglycertate enriched in the (2R,3R) stereoisomer was submitted to stereospecicific acetylation with vinyl acetate and CAL-B in order to separate the major stereoisomer. The entire procedure enabled conversion of the racemic α-acetolactate into the four enantiopure stereoisomers of the ethyl 2,3-dihydroxy-2-methylbutyrate with the following overall yields: 42% for the (2R,3S), 40% for the (2S,3S), 42% for the (2S,3R) and 20% for the (2R,3R).

The thiamine diphosphate (ThDP) dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The recombinant enzyme shared close similarities with the acetylacetoin synthase (AAS) partially purified from Bacillus licheniformis suggesting that they could be the same enzyme. The product scope of the recombinant Ao:DCPIP OR was expanded to chiral tertiary α-hydroxy ketones through the rare aldehyde-ketone cross-carboligation reaction. Unprecedented is the use of methylacetoin as the acetyl anion donor in combination with a range of strongly to weakly activated ketones. In some cases, Ao:DCPIP OR produced the desired tertiary alcohols with stereochemistry opposite to that obtained with other ThDP-dependent enzymes. The combination of methylacetoin as acyl anion synthon and novel ThDP-dependent enzymes considerably expands the available range of C-C bond formations in asymmetric synthesis.

A general access to a novel class of sugar α-amino acids composed of iminofuranose and iminopyranose residues anomerically linked to the glycinyl group through an alkyl chain is described. A set of eight compounds was prepared by the same reaction sequence involving as an initial step the Grubbs Ru−carbene-catalyzed cross-metathesis (CM) of various N-Cbz-protected allyl C-iminoglycosides with N-Boc-vinyl- and N-Boc-allyloxazolidine. The isolated yields of the CM products (mixtures of E- and Z-alkenes) varied in the range 40−70%. Each mixture was elaborated by first reducing the carbon−carbon double bond using in situ generated diimide and then unveiling the N-Boc glycinyl group [CH(BocNH)CO2H] by oxidative cleavage of the oxazolidine ring by the Jones reagent. All amino acids were characterized as their methyl esters. The insertion of a model C-iminoglycosyl-2-aminopentanoic acid into a tripeptide via sequential carboxylic and amino group coupling with l-phenylalanine derivatives was carried out as a demo...

The introduction of a formyl group at the anomeric center of 2,3,5-tri-O-benzyl furanoses and substitution of the ring oxygen with a basic nitrogen atom (aminohomologation) was carried out via stereoselective addition of 2-lithiothiazole to N-benzyl, N-furanosylhydroxylamines (masked N-benzyl sugar nitrones), followed by reductive dehydroxylation of the resulting open-chain adducts, and then ring closure via intramolecular displacement of the free hydroxy group by the amino group and unmasking of the formyl group from the thiazole ring. The resulting formyl aza-C-glycosides were transformed into 2,5-dideoxy-2,5-imino-hexitols (pyrrolidine homoazasugars) by reduction of the formyl to the hydroxymethyl group and removal of the O- and N-benzyl groups by hydrogenolysis. This reaction sequence was applied to four furanoses (D-arabino, D-ribo, D-lyxo, L-xylo) to give the hydroxy- and amino-free homoazasugars, including the natural product 2,5-dideoxy-2,5-imino-D-mannitol, in 17% overall yields (six steps). The formyl aza-C-glycosides proved to be valuable intermediates for the synthesis of more complex derivatives. In fact, these sugar aldehydes were employed in Wittig-type coupling reactions with galactose and ribose phosphoranes to give bis-glycosylated alkenes, which upon reduction of the double bond were transformed into methylene isosteres of (1--6)- and (1--5)-linked disaccharides in which one of the two sugar moieties was an azasugar (aza-(1--x)-C-disaccharides).

ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzes the CC bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate. One of the two reactivities of CDH was selectively knocked down by mutation experiments. CDH-H28A is much less able to catalyze the CC bond formation, while the ability for CC bond cleavage is still intact. The double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94 % enantiomeric excess. In addition to pyruvate, methyl pyruvate and butane-2,3-dione are alternative donor substrates for CC bond formation. Thus, the very rare aldehyde-ketone cross-benzoin reaction has been solved by design of an enzyme variant.

Ofrom C(11) C(12) of secosteroids 4a and 4b affords the compounds 5a (5%) and 5b (20%), respectively.Onthe otherhand,in thebiotransformation of hyocholicacid with R.ruber the9,10-secosteroid4c is notdetected,but,rearrangingtoanintramolecularhemiacetalform,itevolvestothefinalfuranderivative6c(35%) by easy elimination of two molecules of H

A new entry to (1→6)- and (1→5)-aza- C -disaccharides featuring the pyrrolidine ring as the imino sugar component is described via Wittig condensation of polyhydroxylated 2-formylpyrrolidines with galactopyranose 6-phosphorane and ribofuranose 5-phosphorane, respectively.

The preparative-scale regio- and stereo-specific oxidation of hydroxy groups and reduction of keto functions at C(3) of several C21 bile acids, catalyzed by a new 3α-hydroxysteroid dehydrogenase (3α-HSDH), is reported. The crude enzyme, isolated from the cells of Pseudomonas paucimobilis, revealed the presence of a further enzymatic fraction containing a secondary alcohol dehydrogenase (SADH), that has been used to recycle the cofactor.

Preparation of various heteroaryl propanols 2a‐g and of the corresponding propanones 3a‐g as starting materials for microbial redox is described. The kinetic resolution of the racemic propanols 2a‐g is obtained via oxidation with Pseudomonas paucimobilisand Bacillus stearothermophilus[(R)-alcohols, ee 74‐100%]. Similar results are achieved with 3-(2-hydroxypropyl)trifluoromethylbenzene 7 (44%, ee 100% of the (R)-alcohol 6). The reduction of the propanones 3a‐d and 3g with baker’s yeast and other fungi gives the ( S)-alcohols (ee 100%). The pure (S)-alcohols are also obtained by reduction of 1-[3-(trifluoromethyl)phenyl]-2-propanone 7. 1-[(4,4-Dimethyl)2-( 2 )oxazolinyl]-2-propanone 3e and 1[2-( 2 )-thiazolinyl)-2-propanone 3f are not reduced. The heterocyclic rings of (S)-5-(2-hydroxypropyl)-3-methylisoxazole 2d and of (S)-2-(2-hydroxypropyl)-4-methylthiazole 2g are deblocked to the homochiral enamino ketone 8 (78%) and to the protected -hydroxy aldehyde 9 (73%), respectively. The (R)-3-(2-hydroxypropyl)trifluoromethylbenzene 6 is transfomed into the homochiral precursor of (S)-fenfluramine 10 (overall yield 65%). © 1998 Elsevier Science Ltd. All rights reserved.

Yarrowia lipolytica strains isolated from various habitats were tested in the reduction of prochiral carbonyl groups. The anti-Prelog reduction ( R -enantiomer) is observed with different yields and enantiomeric excesses depending on the structure of the ketones 1a-e .

Fortheoxidationofalcohols that require nicotinamide adenine nucleotide(NAD)oritsphosphateanalogue(NADP),orthereduc-tionofcarbonylcompoundsmediatedbythereducedformNADH(NADPH),regenerationofthecofactorisneces-sary. EnzymaticregenerationofNADH(orNADPH)forsynthesis usually employs the glucose/glucose dehy-drogenase

A convergent synthesis has been developed to afford C-linked glycosyl amino acids by cross-metathesis of vinyl, allyl, and butenyl C-glycosides with N-Boc-vinyloxazolidine using the Grubbs 1,3-dimesityl-4,5-dihydroimidazol-2-ylideneruthenium carbene. The method has been employed to introduce through an α-linkage, an α-aminopentanoic acid chain at the anomeric carbon of β-D-C-gentiobiose to give a totally artificial glycosyl α-amino acid of a disaccharide. This compound represents the isostere of the α-linked glycosylasparagine moiety of the natural glycopeptide nephritogenoside.

The synthesis of α-hydroxy-1,3-diketones 2 and 3 from the corresponding 1,2-diketones with Bacillus stearothermophilus ATCC2027 acetylacetoin synthase (AAS) was described. The enzyme catalyzed the condensation of the dialkyl- or alkyl-aryl-1,2-diketones 1 with the elimination of a carboxylic acid moiety. The reactions were carried out using either one diketone both acting as donor and acceptor (homo-coupling) or with two different reacting species (cross-coupling). The homo-coupling reactions of the asymmetric dialkyl-1,2-diketones 1c–d afforded a mixture of the regioisomers 2 (30–42%, ee 67–70%) and 3 (19–25%), while only the 1,3-diketones 2a (57%), 2b (60%), and 2e (45%, ee 76%) were obtained using 2,3-butanedione 1a, 3,4-hexanedione 1b, and 1-phenyl-1,2-propanedione 1e, respectively. The cross-coupling reactions of the diketones 1a and 1b, and 1b and 1e were carried out using various ratios of the donor and the acceptor. In both cases the only cross-coupling product was 3-ethyl-3-hydroxy-2,4-hexanedione 4 (62%, ee 91%).

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).

Fortheoxidationofalcohols that require nicotinamide adenine nucleotide(NAD)oritsphosphateanalogue(NADP),orthereduc-tionofcarbonylcompoundsmediatedbythereducedformNADH(NADPH),regenerationofthecofactorisneces-sary. EnzymaticregenerationofNADH(orNADPH)forsynthesis usually employs the glucose/glucose dehy-drogenase

The 1,3-dipolar cycloadditions of C-glycosyl nitrile oxides and acetylenes to an alkyne and an azide, respectively, bearing a masked glycinyl moiety furnished disubstituted isoxazoles and triazoles. Unveiling the glycinyl group in these cycloadducts afforded C-glycosyl alpha-amino acids in which the two bioactive entities were tethered through rigid five-membered heterocycles. Optimized entries to the same compounds involved the use of unmasked but protected alkyne- and azide-containing amino acids as the partners of 1,3-dipolar cycloadditions.

A convergent synthesis has been developed to afford C-linked glycosyl amino acids by cross-metathesis of vinyl, allyl, and butenyl C-glycosides with N-Boc-vinyloxazolidine using the Grubbs 1,3-dimesityl-4,5-dihydroimidazol-2-ylideneruthenium carbene. The method has been employed to introduce through an α-linkage, an α-aminopentanoic acid chain at the anomeric carbon of β-D-C-gentiobiose to give a totally artificial glycosyl α-amino acid of a disaccharide. This compound represents the isostere of the α-linked glycosylasparagine moiety of the natural glycopeptide nephritogenoside.

The cells of Bacillus stearothermophilus contain an NADH-dependent diacetyl (acetoin) reductase. The enzyme was easily purified to homogeneity, partially characterised, and found to be composed of two subunits with the same molecular weight. In the presence of NADH, it catalyses the stereospecific reduction of diacetyl first to (3S)-acetoin and then to (2S,3S)-butanediol; in the presence of NAD+, it catalyses the oxidation of (2S,3S)- and meso-butanediol, respectively, to (3S)-acetoin and to (3R)-acetoin, but is unable to oxidise these compounds to diacetyl. The enzyme is also able to catalyse redox reactions involving some endo-bicyclic octen- and heptenols and the related ketones, and its use is suggested also for the recycling of NAD+ and NADH in enzymatic redox reactions useful in organic syntheses.

Yarrowia lipolytica strains isolated from various habitats were tested in the reduction of prochiral carbonyl groups. The anti-Prelog reduction ( R -enantiomer) is observed with different yields and enantiomeric excesses depending on the structure of the ketones 1a-e .

An enzymatic strategy for the preparation of optically pure α-alkyl-α,β-dihydroxyketones is reported. Homo- and cross-coupling reactions of α-diketones catalyzed by acetylacetoin synthase (AAS) produce a set of α-alkyl-α-hydroxy-β-diketones (30–60%, ee 67–90%), which in turn are reduced regio-, diastereo-, and enantioselectively to the corresponding chiral α-alkyl-α,β-dihydroxyketones (60–70%, ee >95%) using acetylacetoin reductase (AAR) as catalyst. Both enzymes are obtained from Bacillus licheniformis and used in a crude form. The relative syn stereochemistry of the enantiopure α,β-dihydroxy products is assigned by NOE experiments, whereas their absolute configuration is determined by conversion of the selected 3,4-dihydroxy-3-methyl-pentan-2-one to the natural product (+)-citreodiol.

Two complementary routes, A and B, have been followed for the stepwise iterative assembly of beta-D-(1,6)-glucopyranose and galactopyranose residues through methylene bridges. In route A the building block was constituted by 2,3,4-tri-O-benzyl-6-O-tert-butyldiphenylsilyl (O-TBDPS) beta-linked galactosylmethylenephosphorane, while in route B the building block was a beta-linked formyl C-glycopyranoside with a similar orthogonal protection of hydroxy groups. In route A each cycle consisted of the reaction of the phosphorane building block with a sugar residue bearing a formyl group at the C-5 carbon atom (coupling) and transformation of the O-TBDPS-protected primary alcohol into the formyl group (arming). Accordingly, route A is defined as the aldehyde route. On the other hand, each cycle in route B involved the coupling of the sugar aldehyde building block with a substrate bearing a phosphorus ylide at C-6 and introduction of the phosphonium group in the arming step as a precursor of the ylide functionality. Accordingly, route B is defined as the ylide route. The efficiency of route A proved to be seriously hampered by the 1,2-elimination of BnOH under the basic reaction conditions of the Wittig olefination, giving rise to the formation of substantial amounts of enopyranose. On the other hand, the ylide route B proved to be more efficient since very good yields (70-93%) of the isolated Wittig products were obtained throughout four consecutive cycles. Individual olefins and polyolefins obtained by routes A and B using gluco and galacto substrates were reduced and debenzylated in one pot by H(2)/Pd(OH)(2) to give the corresponding beta-D-C-(1,6)-linked oligosaccharides up to the pentaose stage. The latter compounds were fully characterized by high-field NMR spectroscopy (500 MHz).