Allyl deprotection of galacturonic acid derivatives: mechanistic aspects of mercuric-catalyzed prop-1-enyl acetal cleavage

Different deallylation methods were assayed for selective deprotection of allyl galactopyranosiduronic acid derivatives. A two-step procedure using DABCO and (Ph(3)P)(3)RhCl followed by mercuric-assisted cleavage gave quantitative yields. Reaction in the presence of [(18)O]water allowed us to obtain evidence about the mechanism of prop-1-enyl cleavage.     

Simultaneous regioselective protection of phenyl 1-thioglucosides at the C-3 and C-6 or at the C-2 and C-6 hydroxy groups

Simultaneous regioselective 3,6- or 2,6-selective protection of 1-thio-beta- or alpha-D-glucopyranosides is described. The C-3 and C-6 hydroxy groups of the beta-thioglucoside were selectively protected with triisopropylsilyl or tert-butyldiphenylsilyl trifluoromethanesulfonate. The C-2 and C-6 hydroxy groups of the alpha-thioglucoside were selectively protected with tert-butyldiphenylsilyl trifluoromethanesulfonate.     

1,5-Anhydro- -fructose: regioselective acylation with fatty acids

Regioselective acylation of 1,5-anhydro- -fructose was performed with dodecanoic acid to give 1,5-anhydro-6-O-dodecanoyl- -fructose, chemically in 50% yield and enzymatically in quantitative yield. Quantitative conversions were also obtained using hexadecanoic and octadecanoic acids as acyl donors.     

Regioselective formation of 6-O-acylsucroses and 6,3'-di-O-acylsucroses via the stannylene acetal method

Regioselective formation of 6-O-acylsucroses and 6,3′-di-O-acylsucroses in one pot with good yields was achieved for the first time by a typical acylation method of sucrose via its dibutylstannylene acetal. Pure monoesters at OH-6 and diesters at OH-6,3′ obtained by these procedures were readily isolated by simple column chromatography, thus overcoming the main difficulties associated with regioselectivity, efficiency, and isolation techniques for the practical preparation. Explanations for the regioselectivities observed during this stannylene acetal-mediated reaction were also proposed based on the structures of the stannylene acetal in solution and the intramolecular migration of stannylenes.     

Mechanism of the reductive cleavage reaction of permethylated methyl -glycopyranosides

The mechanism of the reductive cleavage reaction of permethylated methyl -glycopyranosides was investigated by measuring the rate of reaction. Glycosides employed were of α-Glc, β-Glc, α-Man, α-Gal, and β-Gal. Seven silanes were used to explore the reactivities of the reducing agents as well as to examine the stereoelectronic effects of the agents. Trimethylsilyl trifluoromethanesulfonate was employed as catalyst. In general, the rates of β anomers were about twice as fast as those of the α anomers. The rates of anomerization were about five to ten times lower than those of reduction. A cyclic oxonium ion has been proposed as a sole intermediate for the reductive cleavage of the α-glycoside linkage, but the attack of the reducing agent on both cyclic and acyclic forms as well as on the substrate–Lewis acid complex seems to be involved for the β anomer.     

Effect of temperature modulations on TEMPO-mediated regioselective oxidation of unprotected carbohydrates and nucleosides

Regioselective oxidation of unprotected and partially protected oligosaccharides is a much sought-after goal. Herein, we report a notable improvement in the efficiency of TEMPO-catalyzed oxidation by modulating the temperature of the reaction. Mono-, di-, and tri-saccharides are oxidized regioselectively in yields of 75 to 92%. The present method is simple to implement and is also applicable for selective oxidations of other mono- and poly-hydroxy compounds including unprotected and partially protected nucleosides.     

Structure at restriction endonuclease MboI cleavage sites protected by actinomycin D or distamycin A

Restriction endonuclease MboI cleavage of DNA was inhibited by actinomycin D and distamycin A. The two inhibitors protected different subsets of the 8 cleavage sites in polyoma DNA. The cleavage reactions were analyzed both in the presence of minimal inhibitory concentrations of the compounds and at higher concentrations, allowing cleavage at only 1 site/DNA molecule. The experiments showed that cleavage sites most efficiently protected by actinomycin D had putative inhibitor binding sites at a distance of 1-2 base pairs from the MboI recognition sequence. Distamycin A, in contrast, apparently has to bind immediately adjacent to the MboI recognition sequence to protect from cleavage.     

Synthesis, characterization, and C-H/C-C cleavage reactions of two rhodium-trispyrazolylborate dihydrides

Treatment of Tp′Rh(PMe₃)Cl₂ and Tp′Rh(CNCH₂CMe₃)Cl₂ with Cp₂ZrH₂ produces Tp′Rh(PMe₃)H₂ and Tp′Rh(CNCH₂CMe₃)H₂, respectively, in excellent yield. Photolysis of benzene solutions of each dihydride complex generates hydrogen and the fragment [Tp′Rh(L)] which inserts into the solvent C-H bond. The phosphine dihydride has also been shown to be a catalyst for the hydrogenation of biphenylene, showing a capability to cleave C-C bonds. Reductive elimination of benzene from Tp′Rh(PMe₃)PhH is nearly 250 times slower than from Cp*Rh(PMe₃)PhH.     

Copper·Lys-Gly-His-Lys mediated cleavage of tRNA: Studies of reaction mechanism and cleavage specificity

The reactivity of [Cu²⁺·Lys-Gly-His-Lys-NH₂]²⁺ and [Cu²⁺·Lys-Gly-His-Lys]⁺ toward tRNA^Phe has been evaluated. The amidated and carboxylate forms of the copper peptides display complex binding behavior with strong and weak sites evident (, for the amide form; and , for the carboxylate form), while Cu²⁺(aq) yielded and . The time-dependence of the reaction of [Cu²⁺·Lys-Gly-His-Lys]⁺ and [Cu²⁺·Lys-Gly-His-Lys-NH₂]²⁺ with tRNA^Phe yielded k_obs ∼ 0.075 h⁻¹ for both complexes. HPLC analysis of the reaction products demonstrated guanine as the sole base product. Mass spectrometric data shows a limited number of cleavage fragments with product peak masses consistent with chemistry occurring at a discrete site defined by the structurally contiguous D and TΨC loops, and in a domain where high affinity magnesium centers have previously been observed to promote hydrolysis of the tRNA^Phe backbone. This cleavage pattern is more selective than that previously observed by Long and coworkers for nickel complexes of a series of C-terminally amidated peptides (Gly-Gly-His, Lys-Gly-His, and Arg-Gly-His), and may reflect variations in structural recognition and a distinct reaction path by the nickel derivatives. The data emphasizes the optimal positioning of the metal-associated reactive oxygen species, relative to scissile bonds, as a major criterion for development of efficient catalytic nucleases or therapeutics.     

New regioselective derivatives of sucrose with amino acid and acrylic groups

We report here a range of new sucrose derivatives obtained from '3-ketosucrose' in aqueous medium with few reaction steps. As an intermediate, 3-amino-3-deoxy-alpha-D-allopyranosyl beta-D-fructofuranoside (1) was obtained via the classical route of reductive amination with much improved yield and high stereoselectivity. Building blocks for polymerization were synthesized by introduction of acrylic-type side chains, for example, with methacrylic anhydride. Corresponding polymers were synthesized. Aminoacyl and peptide conjugates were obtained through conventional peptide synthesis with activated and protected amino acids. Deprotection yielded new glycoderivatives having an unconventional substitution pattern, namely 3-(aminoacylamino) allosaccharides. Both mono- and di-peptide conjugates of allosucrose have been synthesized.     

Ultrasonic cleavage of DNA: Quantitative analysis of sequence specificity

Looking for new means of assessing local conformational and dynamic heterogeneities in DNA structure, we have estimated the rates of phosphodiester bond cleavage in DNA fragments of known sequence caused by ultrasonic treatment. Among the 16 dinucleotide steps possible, those with 5′-ward cytosine [5′-d(CpN)-3′] are distinguished by significantly higher cleavage rates: CG > CA = CT > CC. The possible causes of this intriguing phenomenon are considered.     

Phylogenetic analysis and evolutionary studies of plant carotenoid cleavage dioxygenase gene

The oxidative breakdown of carotenoid evidences the formation of apocarotenoids through carotenoid cleavage dioxygenases (CCDs). Numerous CCDs and apocarotenoids have been identified and characterized in plants. Using available sequence data, a study was performed to investigate the phylogenetic relationship among CCD genes and to statistically estimate the sequence conservation and functional divergence. In total, 77 genes were identified from 39 species belonging to 21 families. Our result of phylogenetic analysis indicated the existence of well-conserved subfamilies. Moreover, comparative genomic analysis showed that the gene structures of the CCDs were highly conserved across some different lineage species. Through functional divergence analysis, a substantial divergence was found between CCD subfamilies. In addition, examination of the site-specific profile revealed the critical amino acid residues accounting for functional divergence. This study mainly focused on the evolution of CCD genes and their functional divergence which may deliver an initial step for further experimental verifications.     

Syntheses of acetylated steroid glycosides and selective cleavage of O-acetyl groups in sugar moiety

Acetylated 3β-O-β-glycosyl steroid derivatives were synthesized by the reaction of a new polyhydroxysteroid 3β,5α,6β-trihydroxypregn-16-en-20-one (2) with the peracetylated 1-bromo derivatives of d-glucose and d-galactose, respectively. Subsequent protection by excess acetic anhydride in pyridine selectively gave the 6β-O-acetylated steroid glycosides. Deprotection of the acetylated steroid glycosides separately with moderate catalysis dibutyltin oxide in methanol selectively removed all acetyl groups of sugar moiety, whereas the acetyl group of the steroid part was retained. The structures of the steroid glycosides were confirmed by mass spectrometry, NMR and IR. The complete protocol was shown to be non-destructive at all stages to the sugar moieties and the steroid nucleus. These regioselective reactions open a route to the synthesis of a series of closely related isomers of 2 and other widespread polyhydroxysteroids and steroid glycosides in marine organisms and some terrestrial species.     

The relation of time to direction and equality of cleavage in Ilyanassa embryos

Summary

Cleavage inhibition experiments using cytochalasin B and hydrostatic pressure demonstrate the existence of a “clock mechanism” specifying cleavage time and form in Ilyanassa obsoleta embryos. Cytokinesis but not karyokinesis is inhibited during these treatments. When second cleavage is inhibited, the following cleavage occurs approximately on schedule with controls. Two micromeres are produced in this cleavage even though treated embryos consist of only two cells. When third cleavage is inhibited, the following micromere cleavage occurs in a counter clockwise direction, typical for the controls. Treatment with nocodazole, an antitubulin drug, inhibits both cytokinesis and karyokinesis but does not affect the cleavage clock mechanism. Treatment with 2,4-dinitrophenol stops both cleavage and the clock mechanism. These results indicate that the cleavage clock in Ilyanassa requires energy but does not depend on centrosomal behavior or on the form of previous cleavages. With regard to the production of micromeres the clock may involve an interaction between the aster-spindle complex and special regions of the animal pole cortex.     

Fertilization and the first cleavage mitosis in insects

Fertilization in animals is now considered to be of the "sea urchin type"; that is, haploid male and female pronuclei completely fuse shortly after sperm entry into the egg, followed by the formation of a mitotic spindle to allow cleavage mitoses to proceed. However, two other patterns of fertilization and early embryonic mitosis in some animal species are known: an Ascaris type and a gonomeric type. The gonomeric type of fertilization in insects and other arthropods is not well known and is quite different from the sea urchin and Ascaris types. In the present article, the author examines the peculiar gonomeric fertilization, using mainly the silkworm as an example.     

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oocyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indicated that the rate of GVBD was not influenced by the treatment, but polar body extrusion, parthenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti β-tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drug, the already formed spindle structure was not affected, but the oocytes were arrested at metaphases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis.     

Immobilisation of bovine enterokinase and application of the immobilised enzyme in fusion protein cleavage

Two immobilisation methods for enterokinase were developed, which yielded high remaining activities for the cleavage of the fusion protein MUC1-IgG Fc. Different carrier materials were compared regarding remaining enzyme activity and storage stability. Immobilisation procedures involving support material activation using glutardialdehyde were found to result in low remaining activities. Applying less aggressive activation procedures, remaining activities of approximately 60% were received when immobilising enterokinase on either Estapor paramagnetic microspheres or hexamethylamino Sepabeads. In case of hexamethylamino Sepabeads we were able to increase the half-life time 4.3-fold at 23 degrees C and 3.8-fold at 4 degrees C compared to the free enzyme at the same temperatures. By immobilising the biocatalyst the downstream process is simplified allowing the easy removal of the enzyme from the reaction mixture. The immobilised enterokinase cleaves the fusion protein MUC1-IgG Fc in at least two repeated batches, proving the efficiency of the immobilisation method and the reusability of the biocatalyst.     

Phosphodiester hydrolysis and specific DNA binding and cleavage promoted by guanidinium-functionalized zinc complexes

Two new Zn(II) complexes containing guanidinium groups, [Zn(L¹)Cl₂](ClO₄)₂ · H₂O · CH₃OH (1) and [Zn(L²)Cl₂](ClO₄)₂ · 0.5H₂O (2), were synthesized and characterized (L¹ = 5,5′-di[1-(guanidyl)methyl]-2,2′-bipyridyl bication and L² = 6,6′-di[1-(guanidyl)methyl]-2,2′-bipyridyl bication). Both complexes are able to catalyze bis(p-nitrophenyl) phosphate (BNPP) hydrolysis efficiently. Obtained kinetic data reveal that both 1 and 2 show nearly 300- and 600-fold rate enhancement of BNPP hydrolysis, respectively, compared to their simple analogue without the guanidinium groups [Zn(bpy)Cl₂] (bpy = 2,2′-bipyridy) (3). Enhanced acceleration for cleavage of BNPP could be attributed to cooperative interaction between the Zn(II) ion and the guanidinium groups by electrostatic interaction and H-bonding. Studies on inhibition of sequence-specific endonucleases (DraI and SmaI) by complexes show that 1 and 2 are able to recognize nucleotide sequence, -TTT^AAA-, and highly effectively cleave the plasmid DNA in the presence of hydrogen peroxide, while 3 has no specific binding to the DNA target sequences and only shows low DNA cleavage activity.