Tricyclic furanoid dichloroacetyl orthoesters of D-mannose from 1,2-O-trichloroethylidene-beta-D-mannofuranose

1,2-O-(R)-Trichloroethylidene-beta-D-mannofuranose (1) was obtained from the reaction of D-mannose with chloral. Reaction of 1 with potassium tert-butoxide (3 Mequiv) gave the thermodynamically stable 1,2,5-O-orthodichloroacetyl-beta-D-mannofuranose as the sole product whereas 1.5 Mequiv of reagent gave the kinetically controlled 1,2,3-O-orthodichloroacetyl-beta-D-mannofuranose (10) as the main product. Orthoester 10 gave the 5,6-isopropylidene derivative, which was also obtained from the reaction of 5,6-O-isopropylidene-1,2-O-(R)-trichloroethylidene-beta-D-mannofuranose with potassium tert-butoxide (1.5 Mequiv). These novel orthoesters are expected to prove useful as protecting groups and as building blocks in the formations of new mannofuranisidic units.     

A highly convergent and effective synthesis of the phytoalexin elicitor hexasaccharide.

The peracetylated hexasaccharide 1,2,4-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-6- O- (2,3,4-tri-O-acetyl-6-O-(2,4-di-O-acetyl-3,6-di-O-(2,3,4,6-tetra-O-acety l- beta-D-glucopyranosyl)-beta-D-glucopyranosyl)-beta-D-glucopyranosyl)-alp ha, beta-D-glucopyranose 21 was synthesized in a blockwise manner, employing trisaccharide trichloroacetimidate 2,4-di-O-acetyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)- alpha-D-glucopyranosyl trichloroacetimidate 17 as the glycosyl donor, and trisaccharide 4-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-6-O-(2,3,4 -tri -O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S)ethylidene-alpha-D-glucopyra nose 18 as the acceptor. The donor 17 and acceptor 18 were readily prepared from trisaccharides 3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-6-O-(2,3,4-tri-O-acet yl- 6-O-chloroacetyl-beta-D-glucopyranosyl)-1,2-O-(R,S)ethylidene-alpha-D- glucopyranose 10 and 3,6-di-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranose 11, respectively, which were obtained from rearrangement of orthoesters 3,4-di-O-acetyl-6-O-chloroacetyl-alpha-D-glucopyranose 1,2-(3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranosid-6-yl orthoacetate) 8 and 3,4,6-tri-O-acetyl-alpha-D-glucopyranose 1,2-(3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranosid-6-yl orthoacetate) 9, respectively. The orthoesters were prepared from selective coupling of the disaccharide 3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranose 4 with 'acetobromoglucose' (tetra-O-acetyl-alpha-D-glucopyranosyl bromide) and 6-O-chloroacetylated 'acetobromoglucose', respectively. To confirm the selectivity of the orthoester formation and rearrangement, the disaccharide 4-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S ) ethylidene-alpha-D-glucopyranose 7 was prepared from 4 by selective tritylation, acetylation and detritylation. The title compound, an elicitor-active D-glucohexaose 3-O-(beta-D-glucopyranosyl)-6-O-(6-O-(3,6-di-O-(beta-D-glucopyranosyl)-b eta -D-glucopyranosyl)-beta-D-glucopyranosyl)-alpha,beta-D-glucopyranose 1, was finally obtained by Zemplén deacetylation of 21 in quantitative yield.     

A new and efficient strategy for the synthesis of shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine and -uridine

Two shimofuridin analogs: 2'-O-(4-O-stearoyl-alpha-L-fucopyranosyl)thymidine (2) and -uridine (3) have been synthesized using D-arabinose, L-fucose, thymine, uracil, and stearoyl chloride as the starting materials. The synthetic procedures involve the facile preparation of 1-(3,5-di-O-benzyl-beta-D-ribofuranosyl)thymine (9) and -uracil (10) by coupling of 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) with silylated thymine and uracil, and then stereoselective formation of the 1,2-cis (alpha) interglycoside bonds through condensation of the nucleoside derivatives 9 and 10 with 2-(2,3-di-O-benzyl-4-O-stearoyl-beta-L-fucopyranosylsulfonyl) pyrimidine (18). The 1,2-anhydro-3,5-di-O-benzyl-alpha-D-ribofuranose (8) was prepared by an improved procedure from D-arabinose.     

Low-molecular-weight components of olive oil mill waste-waters

A new lignan 1-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(3-acetyl-4-hydroxy-5-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, the secoiridoid 2H-pyran-4-acetic acid,3-hydroxymethyl-2,3-dihydro-5-(methoxycarbonyl)-2-methyl-, methyl ester, the phenylglycoside 4-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-1,4-dihydroxy-2-methoxybenzene and the lactone 3-[1-(hydroxymethyl)-1-propenyl] delta-glutarolactone were isolated and identified on the basis of spectroscopic data including two-dimensional NMR, as components of olive oil mill waste-waters. The known aromatic compounds catechol, 4-hydroxybenzoic acid, protocatechuic acid, vanillic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, tyrosol, hydroxytyrosol, 2-(4-hydroxy-3-methoxy)phenylethanol, 2-(3,4-dihydroxy)phenyl-1,2-ethandiol, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, 1-O-[2-(3,4-dihydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, 1-O-[2-(4-hydroxy)phenylethyl]-(3,4-dihydroxy)phenyl-1,2-ethandiol, D(+)-erythro-1-(4-hydroxy-3-methoxy)-phenyl-1,2,3-propantriol, p-hydroxyphenethyl-beta-D-glucopyranoside,2(3,4-dihydroxyphenyl)ethanol 3beta-D-glucopyranoside, and 2(3,4-dihydroxyphenyl)ethanol 4beta-D-glucopyranoside were also confirmed as constituents of the waste-waters.     

Concentrated hydriodic acid in simultaneous deprotections of multifunctional inositols

l-1-Deoxy-1-fluoro-6-O-methyl-myo-inositol was epimerized by chloral/DCC in boiling 1,2-dichloroethane yielding D-1-O-cyclohexylcarbamoyl-2-deoxy-2-fluoro-3-O-methyl-5,6-O-[(R/S)-2,2,2-trichloroethylidene]-chiro-inositol. The latter and l-4-O-benzyl-3-O-cyclohexylcarbamoyl-5-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-muco-inositol, l-4-O-benzyl-3-O-cyclohexylcarbamoyl-1,2-O-ethylidene-5-O-methyl-muco-inositol, d-1-O-cyclohexylcarbamoyl-2-deoxy-5,6-O-ethylidene-2-fluoro-3-O-methyl-chiro-inositol, as well as D-5-O-benzyl-4-O-cyclohexylcarbamoyl-3-deoxy-3-(N,N'-dicyclohexylureido)-6-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-chiro-inositol were deprotected with boiling 57% aq hydrogen iodide. Ether, urethane and ethylidene acetal functions were simultaneously cleaved by the reagent, whereas the trichloroethylidene groups were still intact or were only removed in small quantities. Especially, the urea function of D-5-O-benzyl-4-O-cyclohexylcarbamoyl-3-deoxy-3-(N,N'-dicyclohexylureido)-6-O-methyl-1,2-O-(2,2,2-trichloroethylidene)-chiro-inositol was decomposed to a cyclohexylamino group. The hydrodechlorination of D-1-O-cyclohexylcarbamoyl-2-deoxy-2-fluoro-3-O-methyl-5,6-O-[(R/S)-2,2,2-trichloroethylidene]-chiro-inositol using Raney-Nickel yielded a mixture of the corresponding 5,6-O-ethylidene- and 5,6-O-chloroethylidene derivatives. The three synthetic steps-hydrodehalogenation, HI-deprotection and peracylation- were combined without purification of the intermediates.     

Syntheses of an alpha-D-Gal-(1-->6)-beta-D-Gal diglyceride, as lipase substrate

Two different routes were explored to afford 3-O-(6-O-alpha-D-galactopyranosyl-beta-D-galactopyranosyl)-1,2-di-O-dodecanoyl-sn-glycerol. In the first one, the key step was the glycosylation of the 3-O-(2,3,4-tri-O-benzyl-beta-D-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside as the donor. In the second one, the key step was the coupling of 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-D-galactopyranosyl trichloroacetimidate donor with 1,2-O-isopropylidene-sn-glycerol. Even though the number of steps was the same in both pathways, the first one afforded a better overall yield (12.4%) than the second one (6.5%). This eight-step synthesis allowed the preparation of the expected glycolipid, which was used as substrate for recombinant GPLRP2 galactolipase using the monomolecular film technique.     

Synthesis and evaluation of a series of 1-(3-alkyl-2,3-dideoxy-alpha,beta-D-erythro-pentofuranosyl)thymines

A series of 1-(3-alkyl-2,3-dideoxy-alpha,beta-D-erythro-pentofuranosyl)thymines (3'-alkyl-3'-deoxythymidines) has been prepared from 5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-D-glycero-pent-2- enono-1,4-lactone ((S)-5-[(tert-butyldiphenylsilyl)oxymethyl]-2(5H)- furanone) by Michael addition of the appropriate organocopper reagent, followed by reduction of the lactone, acetylation of the resulting hemiacetal, and trimethylsilyl triflate-catalyzed coupling with 2,4-di-O-(trimethylsilyl)thymine. The protected nucleosides were desilylated by using tetrabutylammonium fluoride to give anomeric mixtures of the free nucleosides. The unsubstituted 2',3'-dideoxynucleoside analog was similarly prepared from 5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-D-glycero-pentono- 1,4-lactone ((S)5-[(tert-butyldiphenylsilyl)-oxymethyl]-dihydro-2(3H)-fu r anone).     

Acylated protopanaxadiol-type ginsenosides from the root of Panax ginseng

Six new protopanaxadiol-type ginsenosides, named ginsenosides Ra(4) -Ra(9) (1-6, resp.), along with 14 known dammarane-type triterpene saponins, were isolated from the root of Panax ginseng, one of the most important Chinese medicinal herbs. The structures of the new compounds were determined by spectroscopic methods, including 1D- and 2D-NMR, HR-MS, and chemical transformation as (20S)- 3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (1), (20S)-3-O-[β-D-6-O-acetylglucopyranosyl-(1→2)-β-D-glucopyranosyl]-20-O-[β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (2), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (3), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (4), (20S)-3-O-{β-D-4-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (5), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (6). The sugar moiety at C(3) of the aglycone of each new ginsenoside is butenoylated or acetylated.     

Total synthesis of the modified ganglioside de-N-acetyl-GM3 and some analogs.

Methyl[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5- dideoxy-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosid]onat e was used for the glycosylation of benzyl O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)- and benzyl O-(2,3-di-O-benzyl-beta-D-galactopyranosyl)-(1----4)-3,6-di-O-benzyl- 2-O-pivaloyl-beta-D-glucopyranoside to give benzyl O-[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonate]-(2-- --3)-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(21) and benzyl O-[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5- dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonate]-(2----6) -O-(2,3-di- O-benzyl-beta-D-galactopyranosyl)-(1----4)-3,6-di-O-benzyl-2-O-pivaloyl- beta-D-glucopyranoside (18), respectively, accompanied by the beta-linked isomers 22 and 19, respectively. Compounds 18, 21, and 22 were converted into the corresponding glycotriosyl donors which, upon coupling with (2S,3R,4E)-3-O-benzoyl-2-N-tetracosanoylsphingenine, afforded completely protected ganglioside analogs 39, 40, and 41, respectively. Deprotection of 40, 41, and 39 completed the synthesis of the modified ganglioside de-N-acetyl-GM3, a stereoisomer, and a regioisomer. The N-deprotected forms of 40 and 39, on successive treatment with methyl isocyanate and O-deprotection, gave the N-(N-methylcarbamoyl) analogs of GM3 and its regioisomer.     

Total synthesis of 3-O-[2-acetamido-6-O-(N-acetyl-alpha-D-neuraminyl-2-deoxy- alpha-D-galactosyl]-L-serine and a stereoisomer

O-(5-Acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2- nonulopyranoxylonic acid)-(2----6)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1----3) -L-serine, a structural unit occurring in various submaxillary mucins, was synthesized for the first time by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D- galacto-2-nonulopyranosyl)onate]-(2----6)-3,4-di-O-acetyl-2- azido-2-deoxy-D- galactopyranosyl trichloroacetimidate (13) and N-(benzyloxycarbonyl)-L-serine benzyl ester as the key intermediates. The trichloroacetimidate 13 was prepared by starting from two monosaccharide synthons, namely, allyl 2-azido-2-deoxy-beta-D-galactopyranoside and methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-beta-D- galacto-2-nonulopyranosyl chloride)onate, which were coupled in the presence of silver triflate in tetrahydrofuran to give the desired alpha-(2----6)-linked disaccharide in moderate selectivity.     

Hantzsch 1,4-dihydropyridines containing a diazen-1-ium-1,2-diolate nitric oxide donor moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of racemic 3-isopropyl 5-[(2-piperazin-1-yl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (12a-c), 3-isopropyl 5-{2-[4-nitrosopiperazinyl]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (14a-c) and 3-isopropyl 5-{2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (22-30) were prepared using modified Hantzsch reactions. This group of compounds (12a-c, 14a-c, and 22-30) exhibited less potent calcium channel antagonist activity (IC(50)=0.11 to 3.35muM range) than the reference drug nifedipine (IC(50)=0.01 microM). The point of attachment of the isomeric C-4 substituent was a determinant of calcium channel antagonist activity providing the potency profile 2-pyridyl3-pyridyl4-pyridyl. The N-nitrosopiperazinyl compounds (14a-c) did not release nitric oxide. The prodrugs 22-30 that have a C-5 2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl ester substituent, upon incubation with guinea pig serum, undergo consecutive cleavage of the O(2)-acetoxymethyl moiety to give a nitric oxide donor diazenium-1-ium-1,2-diolate species that subsequently releases nitric oxide. The extent of nitric oxide released from the diazen-1-ium-1,2-diolate group is dependent upon the nature of the amino functionality attached directly to the diazen-1-ium N-1 position where the nitric oxide release profile is 1,4-piperazinyl>N-Et>N-(n-Bu)>N-Me upon exposure to guinea pig serum esterase(s). The results from this study suggest this class of hybrid calcium channel antagonist/nitric oxide donor prodrugs should release the vasodilator nitric oxide in vivo, preferentially in the vascular endothelium, to enhance the smooth muscle calcium channel antagonist effect to produce a combined synergistic antihypertensive effect.     

Synthesis of a fully protected derivative of O-(N-acetyl-alpha-D-neuraminyl)-(2----3)-O-beta-D-galactopyranosyl-(1- ---3)-O- [(N-acetyl-alpha-D-neuraminyl)-(2----6)]-O-(2-acetamido-2-deoxy-alpha- D-galactopyranosyl)-(1----3)-L-serine

N-(Benzyloxycarbonyl)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate]-(2----3)-O-(2,4,6-tri-O-acetyl-beta-D - galactopyranosyl)-(1----3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-O-(2-acetamido-4-O-acetyl-2- deoxy-alpha-D- galactopyranosyl)-(1----3)-L-serine benzyl ester was synthesized by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- di-deoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl)onate]- (2----3)-O-(2,4,6- tri-O-acetyl-beta-D-galactopyranosyl)-(1----3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-4-O-acetyl-2-azido-2-deoxy-a lpha- and -beta-D-galactopyranosyl trichloroacetimidate as a key glycotetraosyl donor which, upon reaction with N-(benzyloxycarbonyl)-L-serine benzyl ester, afforded a 44% yield of a mixture of the alpha- and beta-glycosides in the ratio of 2:5.     

Synthesis of cerebroside, lactosyl ceramide, and ganglioside GM3 analogs containing beta-thioglycosidically linked ceramide

Coupling of the sodium salt of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose, -beta-D-galactopyranose, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6-tri-O- acetyl- 1-thio-beta-D-glucopyranose, or O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2- nonulopyranosylonate)-(2----3)-O-(2,3-di-O-acetyl-6-O-bezoyl -beta-D- galactopyranosyl)-(1----4)-3-O-acetyl-2,6-di-O-benzoyl-1-thio-beta-D- glucopyranose, which were prepared from the corresponding 1-S-acetates, 1, 3, 6, and 9, with (2S,3R,4E)-2-azido-3-O-benzoyl-1-O-(p-tolylsulfonyl)-4-oc tadecene-1,3-diol (12) derived by tosylation of 11, gave the corresponding beta-thioglycosides 13, 17, 21, and 25, respectively in good yield. The beta-thioglycosides obtained were converted, via selective reduction of the azide group, condensation with octadecanoic acid, and removal of the protecting groups, into the title compounds.     

Transfer of apiose from UDP-apiose to 7-O-(beta-D-glucosyl)-apigenin and 7-O-(beta-D-glucosyl)-chrysoeriol with an enzyme preparation from parsley

An enzyme preparation from parsley (Petroselinum hortense Hoffm.) catalyses the formation of apiin (7-O-[beta-D-apio-furanosyl(1-->2)beta-D-glycosyl]-5,7,4'-trihydroxyflavone) from 7-O-(beta-D-glycosyl)-apigenin and UDP-apiose and of the corresponding chrysoeriol-7-apiosyl-glucoside from 7-O(beta-D-glucosyl)-chrysoeriol and UDP-apiose. Neither free apiose nor cyclic apiose-1,2-phosphate can function as a substrate for the transfer reaction.     

Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli

The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).     

A novel approach for the selective determination of tryptophan in blood serum in the presence of tyrosine based on the electrochemical reduction of oxidation product of tryptophan formed in situ on graphite electrode

In this study, a novel method was proposed for the selective determination of tryptophan (TRP) in blood serum in the presence of tyrosine. This method is based on the electrochemical reduction of 2-amino-3-(5-oxo-3,5-dihydro-2H-indol-3-yl)-propionic acid (5-O-3,5DH-TRP) formed by the oxidation of TRP on the electrochemically treated pencil graphite (ETPG) electrode surface at a suitable potential value. The parameters affecting the TRP determination were deeply investigated. The optimal pH value was determined as 3. The highest reduction current intensity was obtained at the accumulation potential and time values of +0.95 V and 120 s, respectively. The reduction peak current values of 5-O-3,5DH-TRP versus TRP concentration at the ETPG electrode showed linearity in the range from 0.5 μM to 50.0 μM (R(2)=0.9962) with a detection limit of 0.05 μM (S/N=3). The reduction peak intensity of 5-O-3,5DH-TRP on the ETPG electrode showed no significant change in the presence of different interfering substances. The analytical application of the proposed novel method was successfully tested by using human blood serum samples.     

（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联,实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta,获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时,添加终浓度1 mmol/L IPTG,30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达,它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中,添加5 mmol/L Zn2+时,获得产物(S)-苯乙二醇,产物光学纯度为91.3% e.e.,产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能,实现了(S)- 苯乙二醇的一步法转化,为简化手性醇制备途径提供了一条崭新的思路。     

Characterization and detection of lysine-arginine cross-links derived from dehydroascorbic acid

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. L-dehydroascorbic acid (DHA, 5), the oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. Identification is reported for the lysine-arginine cross-links N6-[2-[(4-amino-4-carboxybutyl)amino]-5-(2-hydroxyethyl)-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (9), N6-[2-[(4-amino-4-carboxybutyl)amino]-5-(1,2-dihydroxyethyl)-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (11), and N6-[2-[(4-amino-4-carboxybutyl)amino]-5-[(1S,2S)-1,2,3-trihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (13). The formation pathways could be established starting from dehydroascorbic acid (5), the degradation products 1,3,4-trihydroxybutan-2-one (7, L-erythrulose), 3,4-dihydroxy-2-oxobutanal (10, L-threosone), and L-threo-pentos-2-ulose (12, L-xylosone) were proven as precursors of the lysine-arginine cross-links 9, 11, and 13. Products 9 and 11 were synthesized starting from DHA 5, compound N6-[2-[(4-amino-4-carboxybutyl)amino]-5-[(1S,2R)-1,2,3-trihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (16) via the precursor D-erythro-pentos-2-ulose (15). The present study revealed that the modification of lysine and arginine side chains by DHA 5 is a complex process and could involve a number of reactive carbonyl species.     

云南金钱槭茎化学成分

对云南金钱槭枝条部分的化学成分进行了研究,从其乙醇提取物中共分离鉴定了16个化合物,通过波谱学方法鉴定为:erythro-4,7,9-三羟基-3,3'-二甲氧基-8-O-4'-新木脂素-9'-O-β-D-吡喃葡萄糖苷(1),Hyugano-side IIIa(2),Hyuganoside IIIb(3),erythro-Buddlenol B(4),erythro-7',8'-Didehydrobuddlenol B(5),(±)-丁香脂素(6),臭矢菜素A(7),柑橘苷A(8),(4R)-p-薄荷-1-烯-7,8-二醇7-O-β-D-吡喃葡萄糖苷(9),2-甲氧基-3-(3-吲哚基)丙酸(10),肌苷(11),Tachioside(12),Isotachioside(13),3-O-(β-D-吡喃葡萄糖基)-1-(3,5-二甲氧基-4-羟基苯基)-1-丙酮(14),反式异松柏苷(15),4-[(E)-3-乙氧基-1-丙烯基]-2-甲氧基苯酚(16)。其中化合物5为新的倍半木脂素,其余化合物均首次从该属植物中分离得到。     

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).