Synthesis of a cytosine nucleoside of 2-amino-2-deoxy-beta-D-xylofuranose.

1-(2-Amino-2-deoxy-beta-D-xylofuranosyl)cytosine (13) was synthesized by three routes: (a) coupling of 2-deoxy-3,5-di-O-p-nitrobenzoyl-2-(trifluoroacetamido)-D-xylofuranosyl chloride (5) with 2,4-dimethoxypyrimidine and subsequent treatment with methanolic ammonia, (b) coupling of 5 with 4-N-acetyl-2-O,4-N-bis(trimethylsilyl)cytosine followed by treatment with methanolic ammonia, and (c) thiation of 1-[3,5-di-O-acetyl-2-deoxy-2-(trifluoroacetamido)-beta-D-xylofuranosyl]uracil (6) by treatment with phosphorus pentasulfide in pyridine followed by amination of the resulting 4-thionucleoside 12 with metanolic ammonia. The best yield was obtained via route (a).     

Production of immunoadsorbents based on cellulose carbonates and their application to the isolation of specific immunoglobulin light chains

The purification of rabbit immunoglobulin molecules expressing kappa (κ) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (κ) IgG. Cellulose trans-2.3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preservng immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit a1a3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline celulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (κ) IgG, thus separating immunoglobulin, expressing kappa light chain, from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations.     

Synthesis of chitotetraose and chitohexaose based on dimethylmaleoyl protection

tert-Butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was readily transformed into the disaccharide glycosyl donor, 3,4,6-tri-O-acetyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-alpha/beta-D-glucopyranosyl trichloroacetimidate, and the disaccharide glycosyl acceptor, tert-butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside. A TMSOTf-catalysed coupling of the acceptor with the donor afforded the respective tetrasaccharide derivative, which can be transformed to chitotetraose. tert-Butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-4-O-phenoxyacetyl-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was converted into donor 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-4-O-phenoxyacetyl-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl trichloroacetimidate. Its coupling with benzyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside, followed by dephenoxyacetylation, gave benzyl 3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside, whose glycosylation furnished, after replacement of the DMM-group by the acetyl moiety and subsequent deprotection, chitohexaose.     

Coupling of Fermentation and Esterification: Microbial Esterification of Decanoic Acid with Ethanol Produced via Fermentation

Two different kinds of bioprocess, ethanol fermentation and subsequent microbial esterification, were coupled using Issatchenkia terricola IFO 0933 in an interface bioreactor. The strain produced ethyl decanoate (Et-DA) by esterification of exogenous decanoic acid (DA) with ethanol produced via fermentation. The efficiency of the new coupling system depended on the concentration of glucose in a carrier and DA in an organic phase (decane) in an agar plate interface bioreactor. Optimum glucose content and DA concentration were 4% and 29 mM, respectively.     

Coupling of fermentation and esterification: microbial esterification of decanoic acid with ethanol produced via fermentation

Two different kinds of bioprocess, ethanol fermentation and subsequent microbial esterification, were coupled using Issatchenkia terricola IFO 0933 in an interface bioreactor. The strain produced ethyl decanoate (Et-DA) by esterification of exogenous decanoic acid (DA) with ethanol produced via fermentation. The efficiency of the new coupling system depended on the concentration of glucose in a carrier and DA in an organic phase (decane) in an agar plate interface bioreactor. Optimum glucose content and DA concentration were 4% and 29 mM, respectively.     

Synthesis of a heptasaccharide fragment of the O-deacetylated GXM of C. neoformans serotype C

Beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)][beta-D-Xylp-(1-->4)]-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->4)]-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar C, was synthesized as its methyl glycoside. Thus, chloroacetylation of allyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-chloroacetyl-3-O-acetyl-6-O-benzoyl-alpha-D-mannopyranoside (4). Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6. Dechloroacetylation gave the disaccharide acceptor 7 and subsequent coupling with 5 produced the trisaccharide 8. Deacetylation of 8 gave the trisaccharide acceptor 9 and subsequent coupling with a disaccharide 10 produced the pentasaccharide 11. Reiteration of deallylation and trichloroacetimidate formation from 11 yielded the pentasaccharide donor 12. Coupling of a disaccharide acceptor 13 with 12 afforded the heptasaccharide 14. Subsequent deprotection gave the heptaoside 16, while selective 2-O-deacetylation of 14 gave the heptasaccharide acceptor 15. Condensation of 15 with glucopyranosyluronate imidate 17 did not yield the expected octaoside, instead, an orthoester product 18 was obtained. Rearrangement of 18 did not give the target octaoside; but produced 15. Meanwhile, there was no reaction between 15 and the glycosyl bromide donor 19.     

An efficient and concise synthesis of a beta-(1-->6)-linked D-galactofuranosyl hexasaccharide

A beta-(1-->6)-linked D-galactofuranosyl hexasaccharide was synthesized efficiently in a block construction manner by the well-known Schmidt glycosylation method using 6-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (1) and allyl 2,3,5-tri-O-benzoyl-beta-D-galactofuranoside (3) as the key synthons. Coupling of 3 with 1 gave beta-(1-->6)-linked disaccharide 4. Subsequent selective deacetylation of 4 afforded the disaccharide acceptor 5, while deallylation of 4 followed by trichloroacetimidate formation produced the disaccharide donor 6. Condensation of 5 with 6 gave the tetrasaccharide 7, and subsequent deacetylation afforded the tetrasaccharide acceptor 8. Finally, coupling of 8 with 6 followed by deacylation yielded the target beta-(1-->6)-linked galactofuranose hexasaccharide 10. All of the reactions in the synthesis were carried out smoothly and in high yield.     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

Facile synthesis of arabinomannose penta- and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, Rhodococcus equi

Pentasaccharide repeating unit 20 of the lipoarabinomannan from the equine pathogen, Rhodococcus equi, and its dimer 31, were synthesized. The pentasaccharide was obtained by assembling a benzoylated 2,6-branched mannosyl trisaccharide acceptor 13 with a free hydroxyl group at C-2' of the mannose residue attached to the core mannose residue by (1 --> 6)-linkage, followed by coupling with 2,3,5-tri-O-benzoyl-alpha-D-arabinofuranosyl-(1 --> 2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (18), and by deacylation. Meanwhile, the decamer 31 was obtained by firstly preparing a benzoylated mannose (1 --> 6)-linked tetrasaccharide backbone 26 with 2-, 2"-O-ClAc, and 2'-, 2'-O-Ac groups, respectively, then by dechloroacetylation and subsequent condensation with perbenzoylated trichloroacetimidate, and then by deacetylation and subsequent coupling with 18, and finally, by deacylation.     

Synthesis of a mannose heptasaccharide of the pathogenic yeast,Candida glabrata IFO 0622 strain

An effective synthesis of the mannose heptasaccharide existing in the pathogenic yeast, Candida glabrata IFO 0622 strain was achieved via TMSOTf-promoted condensation of a tetrasaccharide donor 13 with a trisaccharide acceptor 16, followed by deprotection. The tetrasaccharide 13 was constructed by coupling of 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7) with allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (10), followed by deallylation and trichloroacetimadation. The trisaccharide 16 was obtained by coupling of 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate with 10, and subsequent 6-O-deacetylation. The disaccharide 7 was prepared through coupling of perbenzoylated mannosyl trichloroacetimidate with 4,6-O-benzylidene-1,2-O-ethylidene-beta-D-mannopyranose, then simultaneous debenzylidenation and deethylidenation, and subsequent acetylation, selective 1-O-deacetylation, and trichloroacetimidation. The disaccharide 10 was obtained by self-condensation of 3,4,6-tri-O-benzoyl-1,2-O-allyloxyethylidene-beta-D-mannopyranose, followed by selective 2-O-deacetylation.     

Synthesis and antitumor activity of 2'-bromo- and 2'-chloro-3'-acetoxy-3'-deaminodaunorubicin analogs

Bromination of 3,4-di-O-acetyl-L-rhamnal (7) and subsequent glycosidic coupling under Koenigs-Knorr conditions with daunomycinone gave a mixture of three compounds having the beta-L-gluco (10), alpha-L-gluco (11), and alpha-L-manno (12) configurations. Analogous bromination of 3,4-di-O-acetyl-L-fucal (13) followed by coupling with daunomycinone gave a mixture of three glycosides having the beta-L-galacto (16), alpha-L-galacto (17), and alpha-L-talo (18) configurations. Chlorination of 7 and coupling with daunomycinone in the presence of silver triflate gave products having the alpha-L-gluco (21) and alpha-L-manno (22) configurations, whereas 13, under similar conditions, gave only one stereoisomeric product, that having the alpha-L-galacto (24) configuration. Compounds 12 and 22 showed high in vivo activity in the P-388 lymphocytic leukemia assay.     

Binding of glycosaminoglycans to cyano-activated agarose membranes: kinetic and diffusional effects on yield and homogeneity

Methods were developed for binding a glycosaminoglycan (GAG, a 50 kDa chondroitin sulfate) to thin agarose membranes using 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) as the activating agent. Process conditions were optimized to achieve high yields and spatially uniform concentrations of bound ligand. Yields were varied mainly by manipulating the duration and temperature of the aqueous washes prior to coupling, which affected the concentration of active sites available for subsequent GAG binding. The rate constants for degradation of the active cyanate esters in 0.1M bicarbonate solutions were 0.24+/-0.02 h(-1) at 4 degrees C and 0.08+/-0.03 h(-1) at 0 degrees C. Steric limitations in the 3% agarose gels severely restricted binding, with only about 0.1% of active sites being accessible to GAG molecules. The GAG binding occurred primarily in the outer 50-70 microm of the membranes, so that coupling was homogeneous only for thin gels. A model of GAG diffusion and reaction in the coupling step was developed to explain the observed effects of parameters such as the GAG concentration in solution and the membrane thickness. An analysis of the key time scales in the synthesis provides design principles that should be useful also for other cyanylating agents, other ligands, and for beads as well as membranes.     

Immobilization of glucoamylase on polyamide nets

The formation of reactive groups on polyamide nets (nylon 6) and the subsequent immobilization of glucoamylase were investigated. Different mesh sizes of the nets and two chemical methods of enzyme coupling - i( partial hydrolysis of the polyamide with subsequent glutaraldehyde binding and ii) O-alkylation of the carrier using a treatment with a benzene-methyl sulphate mixture – were used. The reactivity of immobilized glucoamylase (GA) was tested by hydrolysis reactions using 1% starch solutions. The highest reactivity (140 μg glc/)min × cm² was obtained for methylated nylon samples attached to a glass rod and by coupling glucoamylase on the nylon surface which had been treated with lysine and glutaraldehyde. This method resulted in a more reactive and more stable preparation of immobilized glucoamylase as compared to a simpler method of coupling glutaraldehyde to partially hydrolyzed nylon.     

An engineered monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel metabolic capability in Arabidopsis

Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.     

Structure of a Laccase-Mediated Product of Coupling of 2,4-Diamino-6-Nitrotoluene to Guaiacol, a Model for Coupling of 2,4,6-Trinitrotoluene Metabolites to a Humic Organic Soil Matrix

This work presents laccase-mediated model reactions for coupling of reduced 2,4,6-trinitrotoluene (TNT) metabolites to an organic soil matrix. The structure of an isolated coupling product of 2,4-diamino-6-nitrotoluene (2,4-DANT) to guaiacol as humic constituent was determined. Among several structures, the compound was identified conclusively to be the trinuclear coupling product 5-(2-amino-3-methyl-4-nitroanilino)-3,3(prm1)-dimethoxy-4,4(prm1)-diphenoqu inone. The compound has a weight of 409 g mol(sup-1) and may serve as a model reaction for the biogenic formation of bound residues in soil from TNT by coupling aminotoluenes (reduced TNT metabolites) to humic constituents. A linear correlation of the substrate consumption to the enzyme activity was detected. Based on this observation, the described reaction of 2,4-DANT coupling to guaiacol may be used for determination of laccase activity since the reaction was not inhibited by other compounds of culture supernatants. We propose a two-step mechanism for the coupling reaction because 2,4-DANT was not transformed by laccases in the absence of guaiacol and guaiacol oxidation was independent of the presence of 2,4-DANT. The first reaction step is a laccase-mediated dimerization of two guaiacol monomers with subsequent oxidation to a diphenoquinone. The second step is the nucleophilic addition of 2,4-DANT to the ortho position of the carbonyl group of the diphenoquinone structure.     

Highly Regioselective C-5 Iodination of Pyrimidine Nucleotides and Subsequent Chemoselective Sonogashira Coupling with Propargylamine

An efficient C-5 iodination of pyrimidine-5′-triphosphates and subsequent palladium-catalyzed Sonogashira coupling reaction with propargylamine is described. The iodination reaction is highly regioselective and the coupling reaction is highly chemoselective that furnishes exclusive 5-(3-aminopropargyl)-pyrimidine-5′-triphosphate in good yield with high purity (>99%).     

Surface modification of an acoustic biosensor allowing the detection of low concentrations of cancer markers

Analyte detection with biosensors is strongly influenced by the preparation of the biosensor surface including choice of sensing layers and coupling methods for corresponding capture molecules. We investigated the influence of different coupling procedures, especially considering coupling chemistry and incubation times for reagents, by means of surface acoustic wave (SAW) biosensors. The effect on the signal response was tested in two subsequent protein assays. Our optimized coupling procedure allowed the detection of the breast cancer markers HER-2 (human epidermal growth factor receptor-2) and TIMP-1 (tissue inhibitor of metalloproteinase-1) below the respective clinical cutoff values of only a few nanograms per milliliter.     

Synthesis of biosynthetic precursors of red fluorescent proteins' chromophores

A method for the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones, the corresponding chromophore of green fluorescent protein (GFP) with acylaminoalkyl substituents at position 2 of imidazole core have been developed. These structures represent biosynthetic precursors of the chromophores of red fluorescent proteins. The method is based on masking of the dehydrotyrosine fragment with beta-hydroxytyrosine moiety The key stages of the synthesis include peptide coupling of beta-hydroxytyrosine with the N-acetylamino acid of choice, unmasking of dehydrotyrosine by O-acylation with subsequent elimination, and cyclization of the obtained derivatives of 3-acylaminocinnamic acid in basic media.     

Solution-phase parallel synthesis and screening of anti-tumor activities from fenbufen and ethacrynic acid libraries

The derivatives with fenbufen and ethacrynic acid core compounds was synthesized through a facial preparation of 1-amino-4-azidobutane. The subsequent coupling with 102 members of carboxylic acids afforded amide products. The in situ screening using colorimetric assay with 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide showed that fenbufen but not ethacrynic acid butyl amide members displayed the cytotoxicities to tumor cells substantially, including two human cell lines (MCF7 and A549) and two murine cell lines (C26 and TRAMP-C1). Three fenbufen analogs were found to have a good anti-tumor activity comparable to cisplatin.     

Solid phase degradation of water insoluble peptides.

A method for attaching water insoluble peptides to amine resins for sequence determination is presented. Derivitization of peptides with 4-sulfophenylisothiocyanate (s-PITC) greatly increased coupling efficiency. Conditions for removal of N-terminal s-PITC were investigated to maximize subsequent degradation of the resin bound peptides. Degradation of a resin bound, 24 residue peptide with thioacetylthioglycolic acid is described.