The reactions of the hydroxymethyl radical with 1,3-dimethyluracil and 1,3-dimethylthymine

Hydroxymethyl radicals .CH2OH, generated by the radiolysis of methanol (0.5 mol dm-3) in N2O-saturated aqueous solutions, were reacted with 1,3-dimethyluracil or 1,3-dimethylthymine (10(-3) mol dm-3). The products were identified and their G values determined. It has been concluded that in 1,3-dimethyluracil .CH2OH attack occurs only at C(6) while in 1,3-dimethylthymine there is partitioning between addition (two-thirds) and H-abstraction from the C(5)-methyl group (one-third). A rate constant for CH2OH addition to 1,3-dimethyluracil of about 10(4) dm3 mol-1 s-1 is estimated. Complexities that may arise in the radiolysis of pyrimidines such as 1,3-dimethylthymine, apparently as a consequence of the formation of 5-alkylidenepyrimidines, are discussed. A value of 0.15 has been estimated for the disproportionation/combination ratio for the hydroxymethyl radical self-termination reaction.     

Identification and characterization of three novel beta 1,3-N-acetylglucosaminyltransferases structurally related to the beta 1,3-galactosyltransferase family

We have isolated three types of cDNAs encoding novel beta1,3-N-acetylglucosaminyltransferases (designated beta3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the beta1,3-galactosyltransferase family and a beta1,3-N-acetylglucosaminyltransferase (designated beta3Gn-T1), but show no structural similarity to another type of beta1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed beta1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not beta1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with beta3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these beta3Gn-Ts were different among 35 human tissues. beta3Gn-T2 was ubiquitously expressed, whereas expression of beta3Gn-T3 and -T4 was relatively restricted. beta3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. beta3Gn-T4 was mainly expressed in brain. These results have revealed that several beta1,3-N-acetylglucosaminyltransferases form a family with structural similarity to the beta1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each beta1,3-N-acetylglucosaminyltransferase may play different roles.     

Synthesis of 1,3-Dithiane-2-thiones

A number of new 1, 3-dithiane-2-thione derivatives were synthesized from bismesylates of substituted 1, 3-dihydroxypropanes by the reaction with sodium trithiocarbonate provided from Na₂S and CS₂. The cyclic structures were elucidated on the basis of the IR, UV, NMR, and mass spectra, together with elemental analyses and chemical reactions.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Selective inhibition of Trypanosoma brucei GAPDH by 1,3-bisphospho-D-glyceric acid (1,3-diPG) analogues

Various phosphono-phosphates and diphosphonates were synthesized as 1,3-diphosphoglycerate (1,3-diPG) analogues by using a beta-ketophosphonate, an alpha-fluoro,beta-ketophosphonate or a beta-ketophosphoramidate to mimic the unstable carboxyphosphate part of the natural substrate. The inhibitory effect of these analogues on glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Trypanosoma brucei (Tb) and rabbit muscle were measured with respect to both substrates, glyceraldehyde-3-phosphate (GAP) and 1,3-diPG. Interestingly, all 1,5-diphosphono,2-oxopentanes without substitution at the C-3 position selectively inhibit the Tb GAPDH with respect to 1,3-diPG and are without effect on Rm GAPDH. All 1-phospho,3-oxo,4-phosphonobutanes show themselves to be non-selective inhibitors either with regard to substrates or organisms, but they will be of a great interest as 1,3-diPG stable models for structural studies of co-crystals with GAPDHs.     

An Enzyme-Linked Lectin Assay for α1,3-Galactosyltransferase

UDP-Gal:Galβ1-4GlcNAc α1,3-galactosyltransferase (α3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galα1-3Galβ1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of α3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galβ1-4GlcNAc (acceptor) are incubated with α3GalT in the presence of “cold” UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of α3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of α3GalT, no enzymatic conversion of Le^x into GalαLe^x was observed using this assay. Human αGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.     

Preparation of pseudo-arsonolipids: 2-acyloxypropane-1,3-bis(arsonic acids). The crystal structure of 2-hydroxypropane-1,3-bis(arsonic acid)

Epihalohydrins react with alkaline arsenite to give in very good yields 2-hydroxypropane-1,3-bis(arsonic acid) (7), a key compound for the synthesis of pseudo-arsonolipids and more complex arsinolipids. Through a series of reduction to -As(SPh)(2), acylation, and oxidation to -AsO(3)H(2), pseudo-arsonolipids, i.e. 2-acyloxypropane-1,3-bis(arsonic acids), were obtained. These pseudo-lipids are very sensitive to bases, being de-acylated. The bis(arsonic acid) (7) crystallizes in the orthorombic space group P2(1)2(1)2(1) with unit cell constants a=6.911(3), b=17.496(8), c=7.002(3) A. Both arsenic atoms are essentially tetrahedral being bound to three oxygens and one carbon. All hydrogen atoms have been located. There is no intramolecular but only intermolecular hydrogen bonding involving all the As=O, As-OH, and C-OH groups. The C-OH group acts as a hydrogen donor to an acidic As-OH, and this As-OH, in turn, acts as a hydrogen donor to an As=O group.     

Reactions between ruthenium cluster carbonyls and 1,4-diphenylbuta-1,3-diyne

The complexes RuC(CCPh)=CPhC(CCPh)=CPh(CO)₃(NMe₃) (3), Ru₂μ-C(CCPh)=CPhC(CCPh)=CPh(CO)₆ (1), Ru₂μ-[C(CCPh)=CPh]₂CO(CO)₆ (2), Ru₃(μ₃-PhC₂CCPh)(μ-CO)(CO)₉ (4) and Ru₄(μ₄-PhC₂CCPh)(CO)₁₂ (5) have been isolated from reactions between PhC₂C₂Ph and Ru₃(CO)₁₂ or RU₃(CO)₁₀(NCMe)₂. The molecular structures of complexes 1, 2, 3 and 5 have been determined from single-crystal X-ray studies. All complexes have precedents in similar products obtained from reactions involving mono-ynes; in the present cases, each alkyne fragment retains a phenylethynyl (PhCC---) group as a non-coordinated substituent.     

The SO4(.-)-induced chain reaction of 1,3-dimethyluracil with peroxodisulphate

The sulphate radical SO4(.-) reacts with 1,3-dimethyluracil (1,3-DMU) (k = 5 X 10(9) dm3 mol-1 s-1) thereby forming with greater than or equal to 90 per cent yield the 1,3-DMU C(5)-OH adduct radical 4 as evidenced by its absorption spectrum and its reactivity toward tetranitromethane. Pulse-conductometric experiments have shown that a 1,3-DMU-SO4(.-) aduct 3 as well as the 1,3-DMU radical cation 1, if formed, must be very short-lived (t1/2 less than or equal to 1 microsecond). The 1,3-DMU C(5)-OH adduct 4 reacts slowly with peroxodisulphate (k = 2.1 X 10(5) dm3 mol-1 s-1). It is suggested that the observed new species is the 1,3-DMU-5-OH-6-SO4(.-) radical 7. At low dose rates a chain reaction is observed. The product of this chain reaction is the cis-5,6-dihydro-5,6-dihydroxy-1,3-dimethyluracil 2. At a dose rate of 2.8 X 10(-3) Gys-1 a G value of approximately 200 was observed ([1,3-DMU] = 5 X 10(-3) mol dm-3; [S2O8(2-)] = 10(-2) mol dm-3; [t-butanol] = 10(-2) mol dm-3). The peculiarities of this chain reaction (strong effect of [1,3-DMU], smaller effect of [S2O(2-)8]) is explained by 7 being an important chain carrier. It is proposed that 7 reacts with 1,3-DMU by electron transfer, albeit more slowly (k approximately 1.2 X 10(4) dm3 mol-1 s-1) than does SO4(.-). The resulting sulphate 6 is considered to hydrolyse into 2 and sulphuric acid which is formed in amounts equivalent to those of 2. Computer simulations provide support for the proposed mechanism. The results of some SCF calculations on the electron distribution in the radical cations derived from uracil and 1-methyluracil are also presented.     

Antidiabetic activity of N-(6-substituted-1,3-benzothiazol-2-yl)benzenesulfonamides

N-(6-Substituted-1,3-benzothiazol-2-yl)benzenesulfonamide derivatives 1–8 were synthesized and evaluated for their in vivo antidiabetic activity in a non-insulin-dependent diabetes mellitus rat model. Several compounds synthesized showed significant lowering of plasma glucose level in this model. As a possible mode of action, the compounds were in vitro evaluated as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. The most active compounds (3 and 4) were docked into the crystal structure of 11β-HSD1. Docking results indicate potential hydrogen bond interactions with catalytic amino acid residues.     

Cloning and expression analysis of two beta-1,3-glucanase genes from strawberry

We isolated from strawberry (Fragariae x ananassa Duch) a genomic clone of a beta-1,3-glucanase gene, designated as FaBG2-2. In addition, a related cDNA clone, designated as FaBG2-3, was also isolated. FaBG2-2 and FaBG2-3 are similar in their coding regions, except that FaBG2-2 does not appear to contain a signal peptide coding sequence. The 5' and 3' flanking regions of FaBG2-2 and FaBG2-3 are differentt. Using real-time PCR, the expression patterns of FaBG2-3 and a previously isolated beta-1,3-glucanase gene, FaBG2-1, in strawberry plants infected with Colletotrichum fragariae or Colletotrichum acutatum were analyzed at different time points post-infection. The results showed that expressions of both genes in the leaves of infected plants were induced by the two fungi, but the level of induction was several fold greater with C. fragariae. Comparison of the expression levels of the two genes revealed that the level of FaBG2-3 expression was several hundred to over a thousand fold higher than that of FaBG2-1. Furthermore, the expression levels of the two genes in the leaf, fruit, crown and root of uninfected strawberry plants were analyzed.     

Fuc-TIX: a versatile alpha1,3-fucosyltransferase with a distinct acceptor- and site-specificity profile

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcbeta1-3'LN (underscored site reacted) and GlcNAcbeta1-3'LNbeta1-3'LN (both LNs reacted), but very poorly with the reducing end LN of LNbeta1-3'LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNbeta1-3'LN and LNbeta1-3'LNbeta1-3'LNbeta1-3'LN, confirming our previous data on LNbeta1-3'LNbeta1-OR. In contrast, the sialylated glycan Neu5Acalpha2-3'LNbeta1-3'LNbeta1-3'LNbeta1-3'LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile alpha1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of alpha2,3-sialylated i-type polylactosamines.     

Propterol B,a further 1,3-diarylpropan-2-ol from Pterocarpus marsupium

The structure of propterol B, a heartwood extract of Pterocarpus marsupium, has been established as the hitherto unreported 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)propan-2-ol. The trimethyl ether of propterol B on oxidation with Jones reagent gave 1-(2,4-dimethoxyphenyl)-3-(4-methoxyphenyl)propan-2-one.     

The structure of 1,3-diphenyl-1,3-propandionatobis(triphenylantimony)diphenylrhodium(III)dibenzene

1,3-Diphenyl-1,3-propandionatobis(triphenylantimony)diphenylrhodium(III)dibenzene, [Rh(DPD)(SbPh₃)₂Ph₂]·2(C₆H₆) has been isolated as the product of the reaction between the Rh(I) complex 1,3-diphenyl-1,3-propandionatodicarbonylrhodium(I), [Rh(DPD)(CO)₂], and triphenylantimony in acetone and in n-hexane medium. The crystal and molecular structure was determined from single crystal X-ray diffractometer data. The unit cell is triclinic with a = 19.083, b = 13.167, c = 13.536 Å, α = 81.81°, β = 111.59°, γ = 100.49°, Z = 2 and space group P_$\text{1}$. The structure was refined to a R-value of 0.079 for 6637 contributing reflections. The coordination polyhedron can be described as a slightly distorted octahedron in which the Rh-atom is coordinated by two phenyl groups, two oxygen atoms of a chelate ring, which are in cis position to one another, and two antimony-atoms of the two SbPh₃ ligands, which are in trans positions.     

Evidence that heme d1 is a 1,3-porphyrindione

Heme d1 is the noncovalently associated heme prosthetic group of the bacterial nitrite reductase known as cytochrome cd1. Additional evidence has been obtained in support of a dioxoisobacteriochlorin, or 1,3-porphyrindione, skeleton for this heme. The new data include the natural abundance 13C NMR spectrum of the free base methyl ester derivative of d1, mass spectrometric determinations of the molecular mass of the free base methyl ester and the Cu and the Zn chelates, visible and 1H NMR spectral comparisons between d1 and synthetic porphyrindione model compounds, and the isolation and characterization of several byproducts formed during the purification of the free base methyl ester of d1. The accumulated evidence strongly supports the following structure for the skeleton of d1: 1-oxo-2-methyl-2'-acetyl-3-oxo-4-methyl-4'-acetyl-5-methyl-6-acrylyl+ ++-7- propionyl-8-methylporphyrin.     

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.     

Endo-beta-1,3-glucanase using affinity inhibition

Synthetic 2',3'-epoxypropyl-1-thio-beta-D-glucopyranoside selectively modifies a catalytically essential nucleophylic group in the active site of beta-1,3-glucanase LIV from the marine mollusc Spisula sachalinensis, the inactivation being as high as 95%. The properties of native and epoxypropylthioglucopyranoside-inhibited glucanase LIV were compared, using UV-spectroscopy, SDS polyacrylamide gel electrophoresis and isoelectrofocusing. It was found that the addition of laminarine and laminarioligosaccharides to a solution of the inhibited enzyme induces UV-differential spectra typical for the tryptophanyl residue involved in the formation of the enzyme-inhibitor-substrate complex. The glucone-1,5-lactone does not produce such spectrum. It was shown that epoxypropylthioglucopyranoside protects the accessible tryptophanyl residues in the enzyme active center against the oxidation by N-bromosuccinimide.     

Synthesis and antiviral activity of 1-(1,3-disubstitutedimidazolidyn-2-ylidene)-3-ethoxycarbonylmethylurea derivatives

Novel 1-(1,3-disubstituted-imidazolidyn-2-ylidene)-3-ethoxycarbonylmethylurea derivatives (3a–3j) were obtained from appropriate 1-aryl-3-arylsulfonyl-1H-imidazolidine-2-imines (1a–1j) and ethyl isocyanatoacetate (2), which were subjected to condensation. Seven compounds were tested for their antiviral activity against HSV-1 and CVB3 viruses. Among the tested compounds, 3c was found to be active against HSV-1, proving that 4-methoxy substituent as R and 4-methyl substituent as R₁ are most beneficial for activity against this virus. Furthermore, 3e and 3g were active against CVB3, which demonstrated that both 4-methyl and 4-chloro substitution is tolerated as R₁, whereas 4-chloro and 2-methoxy substituents are best as R. It was also shown that the active compounds are characterized by relatively big surface area, small ovality, and greatest HOMO and LUMO energies in comparison to the rest of the compounds.     

1,3-Disubstituted benzazepines as neuropeptide Y Y1 receptor antagonists.

A novel class of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Through a blind screening we found the compound 1-N-(3-(N'-(tert-butoxycarbonyl)amino)benzyl)-7-methoxy-(3-(3)-methyl ureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (9: IC50 = 1.6 microM). Chemical modifications of 9 gave a potent NPY Y1 antagonist 3-(N-(4-hydroxyphenyl)-N'-methylguanidino)-1-N-(3-(N'-(tert-butoxy carbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (14c: IC5(0=43 nM), which had no affinity for NPY Y2 and Y5 receptors.