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.     

New oligodeoxyribonucleotide derivatives bearing internucleotide <Emphasis Type="Italic">N</Emphasis>-tosyl phosphoramidate groups: Synthesis and complementary binding to DNA and RNA

N-Sulfonyl phosphoramidate derivatives of oligodeoxyribonucleotides containing N-tosyl phosphoramidate groups are first reported. The synthesis is based on Staudinger reaction between tosyl azide and 3′,5′-dinucleoside β-cyanoethyl phosphite comprising the immobilized oligonucleotide, which is obtained by the phosphoramidite coupling during the solid-phase oligonucleotide synthesis. The N-tosyl phosphoramidate group was stable under conditions of the oligonucleotide synthesis, in particular, upon acidic detritylation followed by the removal of protective groups and cleavage from the polymer support by the treatment with concentrated aqueous ammonia at 55°C. The stability of DNA and RNA duplexes of the model oligonucleotides containing N-tosyl phosphoramidate groups was only slightly lower than that of native DNA:DNA and DNA:RNA duplexes, respectively.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligonucleotide phosphotriester method using O-nucleophilic intramolecular catalysis at the stage of the internucleotide bond formation. The former synthons may be used for the automatic synthesis of 2′-modified oligonucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high yields.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

Inhibition of dextran synthesis by glucoamylase and endodextranase

In a model experiment, glucoamylase was shown to inhibit α-D-glucan synthesis as catalyzed by potato phosphorylase. Both glucoamylase and endodextranase inhibited dextran synthesis with dextransucrases of Leuconostoc mesenteroides. The inhibition could be ascribed to competition between glucoamylase and dextransucrase for the glucosyl groups at the non-reducing end of dextran. The inhibition caused by endodextranase may result from rapid and random hydrolysis of acceptor dextrans. Moreover, significantly low units of glucoamylase, as compared with endodextranase, effectively inhibited dextran synthesis. These results thus present evidence that bio-synthesis of dextran occurs by the addition of glucosyl groups at the non-reducing end of the growing dextran. The measurement of initial velocity suggested that the ping-pong Bi-Bi mechanism proposed for the levansucrase of Bacillus subtilis is also applicable to dextransucrase.     

Facile and efficient approach for the synthesis of N2-dimethylaminomethylene-2′-O-methylguanosine

A convenient method for the synthesis of N²-dimethylaminomethylene-2′-O-methylguanosine (1), which is a useful intermediate for oligonucleotide construction, was developed. We chose the di-tert-butylsilyl group and the triisopropylbenzenesulfonyl group as sugar and base protecting groups, respectively. These protecting groups were stable during the 2′-O-methylation step with MeI and NaH. Our six-step synthesis of 1 is easy to perform using commercially available reagents, and requires only three chromatographic purifications. Compound 1 was obtained in 56% yield from guanosine.     

Synthesis of galactosylphytosphingosine and galactosylceramides containing phytosphingosine

Phytosphingosine prepared from the yeast Hansenula ciferrii was used for synthesis of galactosphingolipids. Glycosidation of N-dichloroacetyl and 3,4-di-O-benzoyl protected phytosphingosine was performed with acetobromogalactose in the presence of mercury cyanide. By subsequent alkaline hydrolysis of the protective groups 1-O (β-D-galactopyranosyl)-phytosphingosine was obtained. The psychosine-like intermediate was N-acylated with p-nitrophenylesters of octadecanoic and 2D-hydroxyoctadecanoic acid to give cerebrosides. The overall yield for the synthesis was 30%. Melting points and optical rotation were recorded and the structures of the cerebrosides confirmed by infrared and mass spectra. The thin-layer chromatographic behaviour of corresponding synthetic sphingosine and phytosphingosine compounds was compared.     

A universal approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N-and O-glycosides differing in the conformation mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Towards new antibiotics targeting bacterial transglycosylase: Synthesis of a Lipid II analog as stable transition-state mimic inhibitor

Described here is the asymmetric synthesis of iminosugar 2b, a Lipid II analog, designed to mimic the transition state of transglycosylation catalyzed by the bacterial transglycosylase. The high density of functional groups, together with a rich stereochemistry, represents an extraordinary challenge for chemical synthesis. The key 2,6-anti- stereochemistry of the iminosugar ring was established through an iridium-catalyzed asymmetric allylic amination. The developed synthetic route is suitable for the synthesis of focused libraries to enable the structure–activity relationship study and late-stage modification of iminosugar scaffold with variable lipid, peptide and sugar substituents. Compound 2b showed 70% inhibition of transglycosylase from Acinetobacter baumannii, providing a basis for further improvement.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.     

Synthesis of phospholipids. III. Synthesis of 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene)

The chemical synthesis of two new glycerophosphatide analogues containing steroid groups, i.e., 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene) is described.     

Synthesis of substituted 2,7-dioxabicyclo[4.1.0]heptanes: 1,2-anhydro-3,4,6-tri-O-benzyl- and 1,2-anhydro-3,4,6-tri-O-(p-bromobenzyl)-α-d-galactopyranose

The title compounds were synthesised from d-galactose via 9 steps. For obtaining stable, 1,2-blocked d-galactopyranose ethers, the 1,2-hydroxyl groups were protected with an ethylidene group instead of a 1-ethoxyethylidene group. The key intermediates for the synthesis were 2-O-acetyl-3,4,6-tri-O-benzyl- and 2-O-acetyl-3,4,6-tri-O-(p-bromobenzyl)-β-d-galactopyranosyl fluoride that were prepared from the corresponding, substituted α-d-galactopyranosyl chlorides with silver fluoride. Ring closure of the β-d-galactopyranosyl fluorides was quantitative with potassium tert-butoxide in oxolane under reflux, and crystalline target compounds were obtained. Most of the reactions involved in the synthesis were carried out readily in high yield.     

Zinc-binding proteins (ligands) in brains of severely zinc-deficient rats

Previous studies from this laboratory reported the presence of a metallothionein-like protein in brain with an apparent estimated molecular weight of 13,000–15,000 daltons. The synthesis of this protein, which incorporates large quantity of cysteine, is stimulated following administration of zinc and copper and is blocked by actinomycin D. In this study, we report that the synthesis of this metallothionein-like protein is considerably lower in brains of severely zinc-deficient rats in comparison with pair-fed orad libitum fed groups. Furthermore, incubation of partially purified metallothionein-like protein with⁶⁵Zn and chromatography on DEAE A-25 Sephadex produced similar elution patterns in the three experimental groups. However, the extent of binding of⁶⁵Zn to the metallothionein-like protein from the zinc-deficient rats was significantly (p<0.05) lower than the control groups. On the other hand, the total concentration of zinc in brains of zinc deficient rats did not vary from control groups. Since the synthesis of this metallothionein-like protein is reduced by zinc deficiency and is stimulated following administration of zinc, we postulate that the free pool of zinc may regulate the synthesis of its binding protein in the brain.     

Synthetic routes to higher-carbon sugars. 1-C-Formylation of a 2-deoxyaldose via the anion of its diethyl dithioacetal

Diphenylmethylation of carbohydrate hydroxyl groups may be effected by the thermal reaction with diazo(diphenyl)methane in the absence of catalysts. Migration of the labile ester groups of methyl 2,3,4-tri-O-acetyl-α-d-glucopyranoside and 3-O-benzoyl-1,2-O-isopropylidene-α-d-glucofuranose does not occur during diphenylmethylation by this procedure. The diphenylmethyl group may be readily removed by catalytic hydrogenolysis, and is sufficiently acid-stable to enable the selective hydrolysis of acetal groups. Its use as an O-4 protecting-group and as a non-participating O-2 protecting-group in α-glycoside synthesis has been demonstrated in syntheses of methyl 2,3,6-tri-O-methyl-α-d-glucopyranoside and kojibiose octa-acetate, respectively.     

Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii

The synthesis of the major chloroplast membrane polypeptides has been studied during synchronous growth of Chlamydomonas reinhardtii. Under these conditions, chlorophyll is synthesized during the latter part of the light period and cell division takes place during the dark period. The profile of the chloroplast membrane polypeptides of C. reinhardtii has been well characterized and shown to contain two major classes by size (Hoober, J. 1970. J. Biol. Chem. 245:4327). Polypeptides of group I have a mol wt range of 50,000–55,000 daltons. The second region consists of at least three polypeptide groups, IIa, IIb, and IIc, having mol wt of 40,000, 31,000, and 27,000 daltons, respectively. The synthesis of these polypeptides has been measured using a double-labeling technique and a computer-aided statistical analysis. The rate of labeling of group I polypeptides is highest during the early light period and decreases after 6 h of growth. Group IIa is labeled from the beginning of the light period, but little synthesis of IIb occurs before 3 h, and significant amounts of label are not found in IIc before 5 h of growth. After approximately 8 h of light, groups IIb and IIc are synthesized at rates significantly greater than those of the other membrane polypeptides. The synthesis of the major polypeptide groups ceases in the dark. We conclude that the biosynthesis of the chloroplast membranes is a sequential or stepwise process.     

Chemo-enzymatic synthesis of a glycosylated peptide containing a complex <Emphasis Type="Italic">N</Emphasis>-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.     

The methylation of transfer ribonucleic acid during regeneration of the liver

Transfer ribonucleic acid¹ is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-¹⁴C and ³H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.     

Synthesis and pharmacological evaluation of novel N-aryl-3,4-dihydro-1′H-spiro[chromene-2,4′-piperidine]-1′-carboxamides as TRPM8 antagonists

A novel series of N-aryl-3,4-dihydro-1′H-spiro[chromene-2,4′-piperidine]-1′-carboxamides was identified as transient receptor potential melastatin 8 (TRPM8) channel blockers through analogue-based rational design, synthesis and screening. Details of the synthesis, effect of aryl groups and their substituents on in-vitro potency were studied. The effects of selected functional groups on the 4-position of the chromene ring were also studied, which showed interesting results. The 4-hydroxy derivatives showed excellent potency and selectivity. Optical resolution and screening of alcohols revealed that (R)-(–)-isomers were in general more potent than the corresponding (S)-(+)-isomers. The isomer (R)-(–)-10e (IC₅₀: 8.9 nM) showed a good pharmacokinetic profile upon oral dosing at 10 mg/kg in Sprague–Dawley (SD) rats. The compound (R)-(–)-10e also showed excellent efficacy in relevant rodent models of neuropathic pain.     

The preparation and study of some novel glycosides of D-galactose

The tert-butyl- and the 2,2,2-trichloroethyl α- and β-D-galactopyranosides were prepared and both α-D-glycosides were selectively benzoylated to give the 2,3,6-tri-O-benzoyl-D-galactopyranosides. Cleaving of the glycosidic groups is described. The glycosides are useful intermediates for oligosaccharide synthesis where generation of a reducing sugar terminus under mild conditions is necessary.     

Exeristes,Itoplectis, Aphaereta,Brachymeria, and Hyposoter species: In vitro glyceride synthesis and regulation of fatty acid composition

Microsomes from two species of parasitic Hymenoptera, Exeristes roborator and Itoplectis conquisitor, exhibited little or no de novo glyceride synthesis but actively acylated endogenous mono- and diacylglycerides. It is suggested that this lack of de novo synthesis is related to the fact that the fatty acid composition of these parasitoid species closely resembles that of the hosts on which they are reared. Microsomes from three other species of parasitic Hymenoptera, Aphaereta pallipes, Brachymeria lasus, and Hyposoter exigua, whose fatty acid compositions are little influenced by the host species, exhibited active de novo glyceride synthesis as well as acylation of endogenous mono- and diacylglycerides. Radiotracer studies indicated that E. roborator microsomes and cytosol did not contain noncompetitive or uncompetitive inhibitors of glycerophosphate acyltransferase. E. roborator microsomes acylated exogenous phosphatidic acid but not dihydroxyacetone phosphate or glycerol. The maximum rate of glycerophosphate acylation was less than 0.1 nmole/min/mg microsomal protein after 15 min incubation. The incorporation was subject to rapid lipolysis on further incubation. The addition of bovine serum albumin (BSA) reduced the ability of E. roborator microsomes to acylate mono- and diacylglycerides with endogenous acyl groups. In the absence of BSA, palmitoyl-CoA was a more effective substrate than stearoyl-CoA for both mono- and diacylglyceride acyltransferases.