A synthesis of psi-cytidine

2-O-Benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-, 4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-, and 2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-galactopyranosyl chloride were converted into the corresponding 2,2,2-trifluoroethanesulfonates, and these were treated with allyl 2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside, to give allyl 2-O-benzoyl-4-O-[2-O-benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-β-d-galactopyranosyl]-3,6-di-O-benzyl- α-d-galactopyranoside (26; 41% yield), allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl- α-d-galactopyranoside (27; 62% yield), and allyl 2-O-benzoyl-4-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-3,6-di-O-benzyl-α-d-galactopyranoside (28; 65% yield). All disaccharides were free from their α anomers. Disaccharides 26 and 27 were found to be base-sensitive, and were de-esterified by KCN in aqueous ethanol, and debenzylated with H₂-Pd. Attempts to produce (1→4)-β-d-galactopyranosides from the coupling of a number of fully esterified d-galactopyranosyl sulfonates to allyl 2,3,6-tri-O-benzoyl-α-d-galactopyranoside were unsuccessful.     

Chemical synthesis of diphosphoinositide

A chemical synthesis of DL-1-O-(1′-palmitoyl-2′-oleoyl-sn-glycero-3′-phosphoryl)-myo-inositol-4-phosphate (diphosphoinositide) is described. Selective phosphorylation of DL-2,3; 5,6-di-O-cyclohexylidene-myo-inositol with diphenylphosphochloridate led to the corresponding 1-diphenylphosphate which was transformed into silver DL-4-O-acetyl-2,3; 5,6-di-O-cyclohexylidene-myo-inositol-1-(benzyl)phosphate. Condensation of the latter with 1-palmitoyl-2-oleoyl-sn-glycero-3-iodohydrin gave a phosphotriester which after successive deacetylation, phosphorus oxychloride treatment and removal of the protective groups yielded diphosphoinositide. The intermediate DL-1-O-[1′-palmitoyl-2′-oleoyl-sn-glycero-3′-(benzyl)phosphoryl]-2,3; 5,6-di-O-cyclohexylidene-myo-inositol was used also for a new synthesis of phosphatidylinositol.     

Total synthesis of solamargine

Solamargine, (25R)-3β-{O-α-l-rhamnopyranosyl-(1→2)-[O-α-l-rhamnopyranosyl-(1→4)]-β-d-glucopyranosyloxy}-22α-N-spirosol-5-ene, has been synthesized in 13 steps in a 10.5% overall yield starting from the naturally abundant diosgenin. Condensation of a partially protected glucopyranosyl donor with an oxaza-spiro moiety, which was formed in one-pot azido reduction, significantly improved the synthesis of desired molecule. The target compound exhibited good cytotoxic activities against tumor cells HeLa, A549, MCF-7, K562, HCT116, U87, and HepG2 with IC₅₀ ranging from 2.1 to 8.0 μM.     

Microbial synthesis of riboflavin

This member of the vitamin-B complex is necessary in human diet to prevent soreness of mouth, lips and nose, and inflammation of the cornea. It is commercially produced from the fungi Ashbya gossypiiand Eremothecium ashbyii,and is used to enrich various foods and animal feedstuffs.     

Towards a modular synthesis of well-defined chitooligosaccharides: synthesis of the four chitodisaccharides

The total chemical synthesis of the four well-defined chitodisaccharides is described using N-trichloroacetyl (TCA) and N-benzyloxycarbonyl (Z) as C-2 protecting groups for acetamido and free amino groups, respectively. The synthesis is carried out according to a strategy that paves way to the elaboration of various homo- and hetero-chitooligosaccharides, with perfect control of the number and the position of GlcN and GlcNAc units along the oligomer chain.     

Biology of endocannabinoid synthesis system

Endocannabinoids (endogenous ligands of cannabinoid receptors) exert diverse physiological and pathophysiological functions in animal tissues. N-Arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) are two representative endocannabinoids. Both the compounds are arachidonic acid-containing lipid molecules generated from membrane glycerophospholipids, but their biosynthetic pathways are totally different. Anandamide is principally formed together with other N-acylethanolamines (NAEs) in a two-step pathway, which is composed of Ca²⁺-dependent N-acyltransferase and N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD). cDNA cloning of NAPE-PLD and subsequent analysis of its gene-disrupted mice led to the discovery of alternative pathways comprising multiple enzymes. As for the 2-AG biosynthesis, recent results, including cDNA cloning of diacylglycerol lipase and analyses of phospholipase Cβ-deficient mice, demonstrated that these two enzymes are responsible for the in vivo formation of 2-AG functioning as a retrograde messenger in synapses. In this review article, we will focus on recent progress in the studies on the enzymes responsible for the endocannabinoid biosyntheses.     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

The synthesis of 3,4-dideoxyhex-3-enitols

Syntheses of (E)-3,4-dideoxy-erythro-, (Z)-3,4-dideoxy-D-threo- and (E)-3,4-dideoxy-D-threo-hex-3-enitols are described. The action of potassium selenocyanate on 1,2:5,6-di-O-isopropylidene-D-mannitol 3,4-di-p-toluenesulfonate has been reexamined. Epoxidation of (E)-3,4-dideoxy-1,2:5,6-di-O-isopropylidene-D-threo-hex-3-enitol affords 3,4-anhydro-1,2:5,6-di-O-isopropylidene-D-mannitol and -D-iditol in the approximate proportions of 3:1. The configurations of the two epoxides were assigned on the basis of the reaction of the latter compound with sodium methoxide to give 1,2:5,6-di-O-isopropylidene-4-O-methyl-D-altritol.     

The synthesis of evernitrose and 3-epi-evernitrose

Evernitrose (2,3,6-trideoxy-3-C-methyl-4-O-methyl-3-nitro-L-arabino-hexopyranose) was synthesized from methyl 2,6-dideoxy-4-O-methyl-α-L-erythro-hexopyranosid-3-ulose (2) through introduction of an amino group attached to the tertiary branching carbon by the method of Bourgeois, and subsequent oxidation of the amino group by m-chloroperoxybenzoic acid to a nitro group. 3-Cyano-3-O-mesylation of 2 by Bourgeois's method gave exclusively the desired product having the L-ribo configuration; furthermore, the β anomer of 2 gave the L-ribo and L-arabino products in the ratio of 1:2. The latter compound was converted into 3-epi-evernitrose by a similar sequence of reactions.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Insulin regulation of RNA synthesis

During periods of nitrogen exportation from the cell, mitochondrial carbamoyl phosphate is synthesized, thus initiating the urea cycle. During times of nitrogen conservation by the liver cell, carbamoyl phosphate is synthesized in the cytosol of the cell, whereupon the de novo pyrimidine synthesis pathway is initiated. The de novo pathway provides pyrimidines for increased ribonucleic acid synthesis. Formerly, it was believed that these two pathways functioned irrespective of one another. However, recent experimental evidence indicates that, when excess ammonia is present, mitochondrial carbamoyl phosphate passes from the mitochondria into the cell cytosol, where it is metabolized by the de novo pyrimidine synthesis pathway. When ornithine and excess ammonia are both present, mitochondrial carbamoyl phosphate no longer passes from the mitochondria into the cytosol to be metabolized by the de nova pathway. Thus the metabolic fate of mitochondrial carbamoyl phosphate, and that of excess nitrogen, is determined by the presence or absence of ornithine. In turn, this key molecule is the substrate for the cytoplasmic enzyme ornithine decarboxylase. When ornithine decarboxylase is stimulated by insulin, ornithine is metabolized to putrescine. The activated ornithine decarboxylase combines with ribonucleic acid polymerase, activating the later enzyme. When ornithine is acted upon by ornithine decarboxylase, it is no longer available for the perpetuation of the urea cycle and mitochondrial carbamoyl phosphate levels rise until the carbamoyl phosphate passes into the cytosol to be metabolized by the de novo pathway. Increased amounts of pyrimidines are available for the activated ribonucleic acid polymerase. Therefore insulin, through its stimulation of ornithine decarboxylase, achieves cellular nitrogen retention by regulating nitrogen incorporation into newly synthesized ribonucleic acid.     

Novel biocatalytic process of N-substituted acrylamide synthesis

The enzymatic synthesis of N-substituted acrylamides (N-isopropyl acrylamide and N, N-dimethylaminopropyl acrylamide) was demonstrated for the first time. The Rhodococcus erythropolis 37 strain, exhibiting acylamidase activity, was used as a source of enzyme, and water-dissolved acrylamide and isopropylamine/dimethylaminopropylamine served as substrates. The optimum conditions for the synthesis of acrylamide N-substitutes were determined using N-isopropyl acrylamide. The yield of the product was maximum at pH 9.5–10.5, substrate (acrylamide/isopropylamine) ratio within the range from 1.3: 1 to 2: 1, and absolute substrate concentrations of 8.0 (acrylamide) and 4.0% (isopropylamine). These conditions allowed for the synthesis of 22 g/L of N-isopropyl acrylamide.     

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C₃₆, C₃₄ and C₃₂, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.     

The synthesis of chlorodeoxyhexofuranoid derivatives

The reaction of 1,2-O-isopropylidene-α- d-glucofuranose with sulfuryl chloride at 0° and at 50° afforded 6-chloro-6-deoxy-1,2-O-isopropylidene-α- d-glucofuranose 3,5-bis(chlorosulfate) ( 3) and 5,6-dichloro-5,6-dideoxy-1,2-O-isopropylidene-β- l-idofuranose 3-chlorosulfate ( 7, not characterised), respectively. Dechlorosulfation of 3 afforded the hydroxy derivative, whereas treatment of 3 with pyridine gave the 3,5-(cyclic sulfate). Dechlorosulfation of 7 afforded 5,6-dichloro-5,6-dideoxy-1,2-O-isopropylidene-β- l-idofuranose which, on acid hydrolysis, was converted into 3,6-anhydro-5-chloro-5-deoxy- l-idofuranose. 5-Chloro-5-deoxy-α- l-idofuranosidurono-6,3-lactone and 5-chloro-5-deoxy-β- l-idofuranurono-6,3-lactone derivatives were also prepared.     

In vitro synthesis of phase-specific flagellin of Salmonella

Chromatography of Salmonella flagellin at pH 8 on DEAE-cellulose separated at least four serologically distinct kinds of flagellin, a, enx, i and 1,2, eluting in that order with increasing concentration of sodium chloride. By this chromatographic technique, the preincubated cell-free extract of Escherichia coli given saltprecipitable RNA of Salmonella was shown to synthesize flagellin characteristic of the flagellar antigen type of the cells from which the RNA was derived. Two of the in vitro synthesized flagellins specifically reacted with their corresponding antiserum.When RNA was extracted from the cells of the diphasic strain propagated from a single colony, expressing either phase 1 or phase 2, the in vitro synthesized flagellin was predominantly the same as that produced by the original colony. Translation of messenger RNA specific for phase 1 flagellin was not inhibited by the presence of messenger RNA specific for phase 2. RNA extracted from the cells of a diphasic strain without any selection directed synthesis of both phase 1 and phase 2 flagellins in the ratio expected if the culture was at equilibrium with respect to phase variation. Experimental evidence is presented to support the hypothesis that phase variation is due to the alternative synthesis of phase-specific messenger RNA.     

Patterns of urease synthesis in developing soybeans

An examination of in vivo polysome-bound activity indicates that soybean (Glycine max, cv. Prize) seed urease is synthesized on large polysomes (n ≥ 15). In vitro urease synthesis is directed by a large RNA (3,000-3,300 nucleotides). Urease synthesis occurs throughout the normal protein biosynthetic phase of the developing seed. Surprisingly, the activity/antigen ratios of urease increase throughout development. Urease appears to be in a more highly polymerized state in mature beans versus beans in early development.     

UMP synthesis in the kinetoplastida

All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular form of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmi, whereas orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.     

Control of bacteriophage T4 DNA polymerase synthesis

Analysis of sodium dodecyl sulphate/acrylamide gels of ¹⁴C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43⁺ infections or increased synthesis in gene 43^? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal.     

Novel synthesis of 3-iodo-ortho-carborane

A new and convenient route to 3-iodo-ortho-carborane was developed starting from thallium ortho-dicarbollide. This stable dicarbollide derivative can be isolated and purified thus avoiding undesirable by-product formation observed in a more conventional synthetic approach. Thallium dicarbollide readily reacts with boron triiodide in hexane to give the title compound in 70-80% yield and on a scale ranging from several milligrams to tens of grams of 3-iodo-ortho-carborane.