A four-step enzymatic cascade for the one-pot synthesis of non-natural carbohydrates from glycerol

A total of four enzymatic steps were combined, in a one-pot reaction, to synthesize carbohydrates starting from glycerol. First, phosphorylation of glycerol by reaction with pyrophosphate in the presence of phytase at pH 4.0 in 95% glycerol afforded racemic glycerol-3-phosphate in 100% yield. The L-enantiomer of the latter underwent selective aerobic oxidation to dihydroxyacetone phosphate (DHAP) at pH 7.5 in the presence of glycerolphosphate oxidase (GPO) and catalase. Subsequently, fructose-1,6-bisphosphate aldolase catalyzed the aldol reaction of DHAP with butanal. Finally, dephosphorylation of the aldol adduct was mediated by phytase at pH 4 affording 5-deoxy-5-ethyl-D-xylulose in 57% yield from L-glycerol-3-phosphate. The phytase on/off-switch by pH was the key to controlling phosphorylation and dephosphorylation.     

Mechanistic imperatives for aldose-ketose isomerization in water: specific, general base- and metal ion-catalyzed isomerization of glyceraldehyde with proton and hydride transfer.

The deuterium enrichment of dihydroxyacetone obtained from the aldose-ketose isomerization of D,L-glyceraldehyde in D(2)O at 25 degrees C was determined by (1)H NMR spectroscopy from the integrated areas of the signals for the alpha-CH(2) and alpha-CHD groups of the product. One mole equivalent of deuterium is incorporated into the product when the isomerization is carried out in 150 mM pyrophosphate buffer at pD 8.4, but only 0.6 mol equiv of deuterium is incorporated into the product of isomerization in the presence of 0.01 M deuterioxide ion, so that 40% of the latter isomerization reaction proceeds by the intramolecular transfer of hydride ion. Several pathways were identified for catalysis of the isomerization of glyceraldehyde to give dihydroxyacetone. The isomerization with hydride transfer is strongly catalyzed by added Zn(2+). Deprotonation of glyceraldehyde is rate-determining for isomerization with proton transfer, and this proton-transfer reaction is catalyzed by Br?nsted bases. Proton transfer also occurs by a termolecular pathway with catalysis by the combined action of Br?nsted bases and Zn(2+). These results show that there is no large advantage to the spontaneous isomerization of glyceraldehyde in alkaline solution with either proton or hydride transfer, and that effective catalytic pathways exist to stabilize the transition states for both of these reactions in water. The existence of separate enzymes that catalyze the isomerization of sugars with hydride transfer and the isomerization of sugar phosphates with proton transfer is proposed to be a consequence of the lack of any large advantage to reaction by either of these pathways for the corresponding nonenzymatic isomerization in water.     

One-pot synthesis of L-Fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase

Two methods have been developed for the highly efficient enzymatic synthesis of L-fructose: one is based on rhamnulose-1-phosphate aldolase and acid phosphatase using racemic glyceraldehyde and dihydroxyacetone phosphate as substrates; the other is to generate enantiomerically pure L-glyceraldehyde in situ from glycerol for the aldol reaction, using galactose oxidase catalyzed oxidation of glycerol in the presence of catalase. Using this four-enzyme system, enantiomerically pure L-fructose was obtained. Using the more expensive dihydroxyacetone phosphate, the yield was 55% after purification.     

Synthesis and structural characterization of a terminal hydroxide containing alumoxane via hydrolysis of aluminum hydrides

A novel terminal hydroxide containing dinuclear alumoxane LAl(OH)OAlL(OCH=N-tBu) (3; L = HC(CMeNAr)2, Ar = 2,6-iPr2C6H3) was prepared by treatment of aluminum dihydride LAlH2 (1) and tert-butyl isocyanate in the presence of trace amounts of water and alternatively from 1 and LAlH(OCH=N-tBu) (2) with water. Compound 2 was obtained from the reaction of 1 and tert-butyl isocyanate.     

Rhodium(I)-catalyzed nucleophilic ring-opening reactions of oxabicyclo adducts derived from the [4 + 2]-cycloaddition of 2-imido-substituted furans

A series of 2-imido-substituted furans containing tethered unsaturation were prepared by the addition of the lithium carbamate of furan-2-ylcarbamic acid tert-butyl ester to a solution of the mixed anhydride of an appropriately substituted 3-butenoic acid. The initially formed imido furans undergo a rapid intramolecular [4 + 2]-cycloaddition at room temperature to deliver the Diels-Alder cycloadducts in good to excellent yield. Isolation of the highly labile oxabicyclic adduct is believed to be a consequence of the lower reaction temperatures employed as well as the presence of the extra carbonyl group, which diminishes the basicity of the nitrogen atom, thereby retarding the ring cleavage/rearrangement reaction generally encountered with related systems. By using a Rh(I)-catalyzed ring opening of the oxabicyclic adduct with various nucleophilic reagents, it was possible to prepare highly functionalized hexahydro-1H-indol-2(3H)-one derivatives in good yield. The major stereoisomer obtained possesses a cis-relationship between the nucleophile and hydroxyl group in the ring-opened product. The stereochemistry was unequivocally established by X-ray crystallographic analysis. Coordination of Rh(I) to the alkenyl pi-bond followed by a nitrogen-assisted cleavage of the carbon-oxygen bond occurs to furnish a pi-allyl rhodium(III) species. Addition of the nucleophile then occurs from the least hindered terminus of the resulting pi-allyl rhodium(III) complex. Proton exchange followed by rhodium(I) decomplexation ultimately leads to the cis-diastereomer.     

Fructose-6-phosphate aldolases as versatile biocatalysts for nitrocyclitol syntheses

Efficient and stereoselective polyhydroxylated nitrocyclitol syntheses were performed via biocatalysed aldol reactions. The key step was based on a one-pot/one-enzyme cascade reaction process where two reactions occur: aldolase-catalysed aldolisation and spontaneous intramolecular nitroaldolisation. The synthetic methodology was investigated using fructose-6-phosphate aldolase A129S for the synthesis of known nitrocyclitols. Improvements were obtained which involved less steps and increased yields. Several new nitrocyclitols were also prepared using hydroxyacetone (HA) as the donor and FSA wt. From nitrocyclitol stereochemical analyses, the intramolecular nitro-Henry reaction stereoselectivity was dependent on the donor substrate used, HA or dihydroxyacetone (DHA). Whereas DHA provided two stereoisomers, four were obtained using HA.     

Novel Method for Selective Debenzylation Under Maintenance of Fluoro Atom Substituted on β-Amino Acids

tert-Butyl (R)-3-amino-3-(3-fluorophenyl)propanoate(5) was prepared with conventional debenzylation method. However, the tert-butyl (R)-3-[(S)-1-phenylethyl-amino]-3-(3-fluorophenyl) propanoate(6) and tert-butyl (R)-3-amino-3-phenylpropanoate(7) were generated as the byproducts under the general catalytic hydrogenation Pd(OH)2/C-H2 conditions. So a series of experiments was performed to optimize the reaction conditions so that product 5 could be obtained with high purity and yield. Finally an effective cata...     

Polymer-supported O-alkylisoureas: useful reagents for the O-alkylation of carboxylic acids

Polymer-supported O-alkylisoureas were prepared by reaction of an alcohol with a polymer-supported carbodiimide under copper(II) catalysis. These reagents were used to transform carboxylic acids into the corresponding methyl, benzyl, allyl, and p-nitrobenzyl esters in a highly chemoselective manner in high yields and in very high purity after simple resin filtration and solvent evaporation. The reactions could be carried out using both conventional or microwave heating, with reaction times as short as 3-5 min in the latter case, without compromising yield, purity, or chemoselectivity. Unfortunately, the corresponding solid-supported tert-butyl isoureas could not be prepared.     

Galacto,gluco, manno,and disaccharide-based C-glycosides of 2-amino-2-deoxy sugars

[reaction: see text] Starting from readily available precursors, selenoglycosides derived from GalNAc, GlcNAc, and ManNAc were prepared by either a one- or a two-step process. The anomeric selenides underwent facile C-Se homolysis to provide the corresponding anomeric radicals, which were trapped with alkenes to give C-glycosides. This provides a general entry to alpha-C-glycosides based on 2-amino-2-deoxy sugars that is also applicable to disaccharide variants.     

Heterocycles [h]fused onto 4-oxoquinoline-3-carboxylic acid, part IV. Convenient synthesis of substituted hexahydro [1,4]thiazepino[2,3-h]quinoline-9-carboxylic acid and its tetrahydroquino[7,8-b]benzothiazepine homolog

Substituted [1,4]thiazepino[2,3-h]quinolinecarboxylic acid 3 is prepared by PPA-catalyzed thermal lactamization of the respective 8-amino-7-[(2-carboxyethyl)thio]-1,4-dihydroquinoline-3-carboxylic acid 9. The latter synthon is obtained by reduction of the 8-nitro-1,4-dihydroquinoline precursor 8 which, in turn, is made accessible via interaction of 3-mercaptopropionic acid with 7-chloro-1-cyclopropyl-6-fluoro-8-nitro-1,4-dihydroquinoline-3-carboxylic acid 7 in the presence of triethylamine. A benzo-homolog of 3, namely tetrahydroquino[7,8-b]benzothiazepine-3-carboxylic acid 6, is analogously prepared via the reaction of 2-mercaptobenzoic acid with 7, followed by reduction of the resulting 7-[(2-carboxyphenyl)thio]-8-nitro product 10 into the corresponding 8-amino derivative 11, and subsequent lactamization. The structures assigned to 3, 6 and 8-11 are based on microanalytical and spectral (IR, MS, NMR) data.     

Aldol additions of dihydroxyacetone phosphate to N-Cbz-amino aldehydes catalyzed by L-fuculose-1-phosphate aldolase in emulsion systems: inversion of stereoselectivity as a function of the acceptor aldehyde

The potential of L-fuculose-1-phosphate aldolase (FucA) as a catalyst for the asymmetric aldol addition of dihydroxyacetone phosphate (DHAP) to N-protected amino aldehydes has been investigated. First, the reaction was studied in both emulsion systems and conventional dimethylformamide (DMF)/H2O (1:4 v/v) mixtures. At 100 mM DHAP, compared with the reactions in the DMF/H2O (1:4) mixture, the use of emulsion systems led to two- to three-fold improvements in the conversions of the FucA-catalyzed reactions. The N-protected aminopolyols thus obtained were converted to iminocyclitols by reductive amination with Pd/C. This reaction was highly diastereoselective with the exception of the reaction of the aldol adduct formed from (S)-N-Cbz-alaninal, which gave a 55:45 mixture of both epimers. From the stereochemical analysis of the resulting iminocyclitols, it was concluded that the stereoselectivity of the FucA-catalyzed reaction depended upon the structure of the N-Cbz-amino aldehyde acceptor. Whereas the enzymatic aldol reaction with both enantiomers of N-Cbz-alaninal exclusively gave the expected 3R,4R configuration, the stereochemistry at the C-4 position of the major aldol adducts produced in the reactions with N-Cbz-glycinal and N-Cbz-3-aminopropanal was inverted to the 3R,4S configuration. The study of the FucA-catalyzed addition of DHAP to phenylacetaldehyde and benzyloxyacetaldehyde revealed that the 4R product was kinetically favored, but rapidly disappeared in favor of the 4S diastereoisomer. Computational models were generated for the situations before and after C-C bond formation in the active site of FucA. Moreover, the lowest-energy conformations of each pair of the resulting epimeric adducts were determined. The data show that the products with a 3R,4S configuration were thermodynamically more stable and, therefore, the major products formed, in agreement with the experimental results.     

Continuous-flow reactor-based enzymatic synthesis of phosphorylated compounds on a large scale

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads-150 or Sepabeads EC-EP). After immobilization 70% of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D-glucose-6-phosphate, N-acetyl-D-glucosamine-6-phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol-1-phosphate, and inosine-5'-monophosphate from the corresponding primary alcohol on gram scale using either a fed-batch reactor or a continuous-flow packed-bed reactor.     

A novel stereoselective carbon-chain extension reaction at the C-6 position of 1,6-anhydropyranose

6-Bromo-1,6-anhydro-d-mannose triacetate reacted with a variety of carbon nucleophiles such as allylsilane, silylacetylenes, propargylsilane, and aromatic compounds in the presence of silver triflate to give the corresponding chain extended products at C-6 in high exo-selectivities. The product obtained from the reaction with propargylsilane was efficiently transformed into a naturally occurring heptopyranose derivative found in bacterial lipopolysaccharide.     

First transformations of pyrano[3,4-b]indol-3-ones to salvadoricine and 2,3-diacylindoles

The readily available methylated pyrano[3,4-b]indol-3-ones 1a and 1b were hydrolyzed to furnish the 2-acetylindol-3-alkanoic acids 2 and 4 . Compound 2 was easily transformed selectively to 2-acetyl-3-methylindole ( 3 , salvadoricine). Substrate 1b reacts with molecular oxygen from the air only in the presence of a catalyst to give 2,3-diacetylindole ( 5 ) while 1a reacts with nitrosobenzene via a proposed Diels-Alder step to yield 2-acetylindole-3-carbaldehyde ( 6 ). The latter product can also be obtained in low yield from the reaction of 1a with molecular oxygen from the air.     

Photochemistry of aryl tert-butyl ethers in methanol: the effect of substituents on an excited state cleavage reaction

The photolysis of a set of 10 substituted aryl tert-butyl ethers, 8a-j, in methanol gave, as the major product, the corresponding phenol along with tert-butyl-substituted phenols resulting from photo-Fries reaction. The corresponding 4-cyanophenyl 1-adamantyl ether, 9, also gave 1-methoxyadamantane 16 (16%), indicating that, at least for this ether, some of the products were ion-derived. Quenching studies with 2,3-dimethylbutadiene for the tert-butyl ethers indicated that these reactions were occurring from the singlet excited state. Rate constants for the reaction, obtained from quantum yields and singlet lifetimes, were found to correlate reasonably well with sigma(h)()(nu) values, rho = -0.77 (r = 0.975), a result that is unexpected for a reaction where the polarity of the bond breaking in the transition state is expected to be -O(delta-). C(delta+).     

Synthesis of Pyrimidines,Thienopyrimidines and Pyrazolopyrimidine

5‐Ethoxycarbonyl‐4‐methyl‐2‐phenylpyrimidin‐6(1H)‐thione ( 3 ), which was prepared from the reaction of ethyl β‐aminocrotonate 1 with benzoyl isothiocyanate ( 2 ) in refluxing acetone, was reacted with halo compounds to give S‐alkyl derivatives 4a‐h . Treatment of compounds 4a‐c with sod. ethoxide cyclized into thienopyrimidine 10a‐c . Hydrazinolysis of compound 3 gave hydroxypyrazolopyrimidine derivative 6 . Also the latter compound was obtained upon heating compound 4a with hydrazine hydrate under neat conditions, but when compound 4a refluxed with hydrazine hydrate in ethanol the corresponding carbohydrazide 5 was produced.     

Total synthesis of everninomicin 13,384-1--Part 4: explorations of methodology; stereocontrolled synthesis of 1,1'-disaccharides, 1,2-seleno migrations in carbohydrates, and solution- and solid-phase synthesis of 2-deoxy glycosides and orthoesters

Methods for the stereocontrolled construction of 1,1'-disaccharides, 2-deoxy glycosides, and orthoesters are reported. Specifically, a tin-acetal moiety was utilized to fix the anomeric stereochemistry of a carbohydrate acceptor leading to an efficient and stereoselective synthesis of 1,1'-disaccharides, while a newly discovered 1,2-phenylseleno migration reaction in carbohydrates opened entries to 2-deoxy glycosides and orthoesters. Thus, reaction of 2-hydroxy phenylselenoglycosides with DAST led to 2-phenylselenoglycosyl fluorides which reacted with carbohydrate acceptors to afford, stereoselectively, 2-phenylselenoglycosides. The latter compounds could be reductively deselenated to 2-deoxy glycosides or oxidatively converted to orthoesters via the corresponding ketene acetals.     

双核铜酶模型的合成及对安息香的催化氧化

为寻求更有效的仿酶模型,本文设计合成了含四个咪唑配基、两个咪唑基与铜配位的新型双核铜酶模型,并实现了在氧气存在下模型对安息香的催化氧化作用。以邻-氯苯酚为原料,经直接咪唑甲基化,醚化偶联,继与[Cu(CH~3CN)~4]CLO~4反应合成目标酶模型6-8。类似地,由对-氯苯酚合成酶模型9。以机械7为例,研究了在氧气下它们催化氧化安息香为二苯乙二酮的反应,高产率地合成目标产物,并求得催化氧化反应动力学参数。考查不同催化剂对反应的影响,发现它们的活性次序如下:7>4+[Cu(CH~3CN)~4]ClO~4>[Cu(CH~3(CN)~4]ClO~4>2+[Cu(CH~3CN)~4]ClO~4。     

Synthesis, and analgesic and antiparkinsonian activities of thiopyrimidine, pyrane, pyrazoline, and thiazolopyrimidine derivatives from 2-chloro-6-ethoxy-4-acetylpyridine

A series of substituted pyridine derivatives were prepared from 2-chloro-6-ethoxy-4-acetylpyridine, which was prepared from the corresponding citrazinic acid as starting material. Reaction of acetylpyridine with thiophene-2-carboxaldehyde afforded the 2-chloro-6-ethoxy-4-β-(2-thienyl)acryloylpyridine, which was reacted with malononitrile in refluxing ethanol in the presence of piperidine as a catalyst to afford the cyanoaminopyrane derivative. Acryloylpyridine was treated with urea or guanidine hydrochloride in refluxing ethanolic potassium hydroxide to give the corresponding pyrimidinone and aminopyrimidine derivatives. The latter was condensed with hydrazine hydrate or phenyl hydrazine to give pyrazoline and N-phenylpyrazoline derivatives. Finally, cycloaddition reaction of acryloylpyridine with thiourea yielded thioxopyrimidine, which was treated with 2-bromopropionic acid, 3-bromopropionic acid, or bromoacetic acid to yield methylthiazolo-, thiazino-, and thiazolopyrimidine derivatives. The arylmethylene derivative was prepared by reacting thiazolopyrimidine with benzaldehyde or by reacting thioxopyrimidine with benzaldehyde and bromoacetic acid in one step. The pharmacological screening showed that many of these compounds have good analgesic and antiparkinsonian activities comparable to Valdecoxib^® and Benzatropine^® as reference drugs.     

Location and analysis of 2-deoxy sugars on chromatograms and pherograms by fluorescence

A group of new reagents for the location of 2-deoxy sugars is introduced. A representative reagent is 4'-aminoacetophenone, which gives a green trimethine fluorogen with at least 0.5 microg of 2-deoxyribose, 2-deoxygalactose, or 2-deoxyglucose under neutral or slightly acidic conditions and the orange fluorescent anion under alkaline conditions at a lower limit of approximately 5 microg. Reagents such as 2-thiobarbituric acid, aniline-diphenylamine, and 2,4-pentanedione give vivid fluorescent colours with these sugars. Aniline-diphenylamine reagent was a useful fluorescent agent for locating the purine nucleosides and nucleotides and DNA. A new attachment for the spectropho-tofluorimeter has been evaluated. With this attachment fluorescence excitation and emission spectra are obtained directly from paper, glass fibre, or thin-layer chromatograms and pherograms in sizes up to 20 x 20 cm. Glass plates are handled as readily as paper chromatograms. The fluorescence spectra of the various fluorogens produced after separation were readily obtained directly from the chromatogram or pherogram. 2-Thiobarbituric acid and 4'-aminoacetophenone offered the greatest potential for future application to characterization and assay of 2-deoxy sugars. The aniline-diphenylamine procedure was the most useful for analysis of purine nucleosides and nucleotides and DNA. These various reagents show potential for application to air pollution studies in terms of location, characterization and assay of 2-deoxy sugars and their derivatives.