Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Convenient synthesis of 2'-deoxy-2-fluoroadenosine from 2-fluoroadenine

A convenient synthesis of 2'-deoxy-2-fluoroadenosine from commercially available 2-fluoroadenine is described. The coupling reaction of silylated 2-fluoroadenine with phenyl 3,5-bis[O-(t-butyldimethylsilyl)]-2-deoxy-1-thio-D-erythro-pentofuranoside gave the corresponding 2-fluoro-2'-deoxyadenosine derivative (alpha/beta = 1:1) in good yield. The alpha- and beta-anomers were separated by chromatography, and then desilylated to give compounds 1a and 1b.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.     

The inhibitory activities of 2-acetamido-2,3-dideoxy-D-hex-2-enonolactones on 2-acetamido-2-deoxy-beta-D-glucosidase.

Treatment of 2-acetamido-2-deoxy-D-mannono-1,4-lactone with dicyclohexylamine in ethanolic solution afforded an unsaturated 1,4-lactone, 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enono-1,4-lactone (1), in good yield. 2-Acetamido-2,3-dideoxy-D-threo-hex-2-enono-1,4-lactone (2) was similarly prepared from 2-acetamido-2-deoxy-D-galactono-1,4-lactone. An unsaturated 1,5-lactone, 2-acetamido-2,3-dideoxy-D-threo-hex-2-enono-1,5-lactone (4), was obtained through the oxidation of 2-acetamido-2-doexy-4,6-0-isopropylidene-D-galactopyranose with silver carbonate on Celite, followed by mild hydrolysis. The inhibitory activity of four isomeric 2-acetamido-2,3-dideoxy-D-hex-2-enonolactones [1, 2, 4, and 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactone (3)] was assayed against 2-acetamido-2-deoxy-beta-D-glucosidase from bull epididymis. Only the erythro lactones 1 and 3 are weak competitive inhibitors, whereas the threo lactones 2 and 4 are practically inactive. The 1,4-lactone 1 inhibited 2-acetamido-2-deoxy-beta-D-glucosidase more strongly than the 1,5-lactone 3. The lactones 1-4 were found to be quite stable in aqueous solution or under inhibitory-assay conditions. In addition, two 2-acetamido-2-deoxy-D-glycals, 2-acetamido-1,5-anhydrohex-1-enitol (7) were tested; both are 10 times as active as 1.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Intra-H-2 recombination in the mouse. III. I-Region characterization of haplotypes H-2at2, H-2at3 and H-2a2

Serological characterization of three K-S interval recombinant strains, TBR2 (H-2at2), TBR3 (H-2at3) and AIR 1 (H-2a2) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between the I-A and I-E subregions. In both cases, the H-2K and I-A subregions were derived fron the H-2t1 of chromosome, while the I-E, S and H-2D regions were derived from the H-2b chromosome (KsAkEbSbDb). The H-2a2 chromosome resulted from a crossover event between the H-2a1 and H-2i9 chromosomes. Ia and Ss typing of AIR 1 suggested that the K to I-E regions originated from H-2a1 and the S and D regions originated from H-2i9 (KkAkEkSbDd).     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Triplex forming ability of oligonucleotides containing 2'-O-methyl-2-thiouridine or 2-thiothymidine

The triplex forming ability of oligonucleotides containing 2'-O-methyl-2-thiouridine (s2Um) and 2-thiothymidine (s2T) was studied. The UV melting experiments revealed that triplex forming oligonucleotides (TFOs) containing both s2Um or s2T stabilized significantly parallel triplexes. The main reason for stabilization of triplexes was due to the stacking effect of the 2-thiocarbonyl group. Moreover, it turned out that these modified TFOs had a high selectivity in recognition of a matched Hoogsteen base from a mismatched one.     

Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).

The lipopolysaccharide (LPS) of Klebsiella serotype O2 is antigenically heterogeneous; some strains express multiple antigenic factors. To study this heterogeneity, we determined the structure of the O-antigen polysaccharides in isolates belonging to serotypes O2(2a), O2(2a,2b), and O2(2a,2c), by using composition analysis, methylation analysis, and both 1H and 13C nuclear magnetic resonance spectroscopy. The repeating unit structure of the 2a polysaccharide was identified as the disaccharide [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] and was identical to D-galactan I, one of two O polysaccharides present in the LPS of Klebsiella pneumoniae serotype O1 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). LPS from serotype O2(2a,2b) also contained D-galactan I as the only O polysaccharide, suggesting that the 2b antigen is not an O antigen. The LPS of serotype O2(2a,2c) contained a mixture of two structurally distinct O polysaccharides and provides a second example of this phenomenon in Klebsiella spp. One polymer was identical to D-galactan I, and the other polysaccharide, the 2c antigen, was a polymer with a disaccharide repeating unit structure, [----3)-beta-D-GlcpNAc-(1----5)-beta-D-Galf-(1----]. The 2c structure does not resemble previously reported O polysaccharides from Klebsiella spp. Periodate oxidation confirmed that D-galactan I and the 2c polysaccharide are distinct glycans, rather than representing domains within a single polysaccharide chain. Monoclonal antibodies against the 2c antigen indicated that only LPS molecules with the longest O-polysaccharide chains contained the 2c epitope.     

Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyriboside methylphosphonates.

A synthetic method was developed for the synthesis of oligodeoxyribonucleotides and oligodeoxyribonucleoside methylphosphonates comprised exclusively of the fluorescent 2-pyrimidinone base for the first time. The method utilized the solid-phase 2-cyanoethylphosphoramidite and methylphosphonamidite chemistry for internucleotide couplings and a baselabile oxalyl linkage to anchor the oligomers onto the CPG support. Cleavage of the oligomers from the support was effected by a short treatment of the support with 5% ammonium hydroxide in methanol at room temperature, without any degradation of the base-sensitive 2-pyrimidinone residues or the base-sensitive methylphosphonate backbone.     

Structural characterization of the head-to-head isomers of the [Pd2(Ph2Ppy)2Cl2] and [PtPd(Ph2Ppy)2I2] complexes (Ph2Ppy = 2-(diphenylphosphino)pyridine)

The molecular structures of the thermodynamically unstable head-to-head isomers, HH-[Pd₂(Ph₂Ppy)₂Cl₂] and HH-[PtPd(Ph₂Ppy)₂I₂], have been determined by single crystal X-ray diffraction. The two complexes have proved to be isostructural. The severe distortions of the bond angles from the ideal square planar geometry around the metal centers ligating the trans phosphorus donor atoms are indicative of a more pronounced internal strain in the HH isomers as compared to the HT counterparts. The enhanced internal strain is thought to be the major driving force responsible for the spontaneous conversion of the head-to-head isomers to their head-to-tail congeners. ¹³C NMR spectra in solution phase as well as solid-state ³¹P MAS NMR spectra have proved to be informative regarding the orientation of the asymmetric Ph₂Ppy ligands.     

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.     

The inhibitory activity of 2-acetamido-2-deoxy-D-gluconolactones and their isopropylidene derivatives on 2-acetamido-2-deoxy-beta-D-glucosidase

2-Acetamido-2-deoxy-D-glucono-1,4-lactone (1) and 2-acetamido-2-deoxy-D-gluconic acid (3) have been examined for inhibitory activity against 2-acetamido-2-deoxy-β-D-glucosidase from bull epididymis. Crystalline 1 and 3 were compared with the known, crystalline 2-acetamido-2-deoxy-D-glucono-1,5-lactone (2), and a correlation of the activities of these compounds with various factors is presented. The inhibition constant of the 1,5-lactone 2 is lower (0.45μM) than that (4.43μM) of the 1,4-lactone 1. The effect of time is the opposite; whereas the activity of solutions of 2 decreases with time, solutions of 1 show an increase in inhibitory power, but both reach an equilibrium after 5 h. The free acid 3 exhibits no inhibitory activity. 2-Acetamido-2-deoxy-5,6-O-isopropylidene-D-glucono- 1,4-lactone (4) and 2-acetamido-2-deoxy-4,6-O-isopropylidene-D-glucono-1,5-lactone (5), which are appropriately protected to prevent conversion into the other lactone isomer, were also tested; 4 has 1/1000th the activity of 5.     

2-(Aminomethyl)-2-decarboxyprostaglandin F2 alpha type analogs

The 2-(aminomethyl)-2-decarboxy analogs of prostaglandin F2 alpha (PGF2 alpha), (15S)-15-methyl-PGF2 alpha, 16-phenoxy-omega-tetranor-PGF2 alpha and 16,16-dimethyl-PGF2 alpha were synthesized. The amino analogs closely resemble the parent PGF2 alpha compounds as antifertility agents in the hamster.     

Antimicrobial actions of the human epididymis 2 (HE2) protein isoforms,HE2alpha,HE2beta1 and HE2beta2

Background  

The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis.     

Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.     

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions

In order to elucidate the relationship between hypertension and hypertrophy in the production of heat shock proteins, we studied the induction of the HSP72 synthesis by the heart and gracilis muscles of normo (WKY) and hypertensive (SHR) rats subjected to hyperthermia (42°C±0.5 for 15 min). Two age groups were investigated in each strain: young (2 months, with developing cardiac hypertrophy) and old (18 months, with fully developed chronic cardiac hypertrophy). The gracilis muscle never developed hypertrophy, independently of hypertension or aging. 72 kDa inducible protein was determined by Western blot analysis using a specific monoclonal antibody. We also used a commercial standard, loaded on each blot, to quantitate densitometrically the signal.The heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8±24.7% vs 88.3 ±8.5%, p<0.001). This response is not maintained during aging as we did not find any significant difference between normo-and hypertensive old rats after exposure to hyperthermia (43.6±5.3% vs 65.3±10.4%).Unlike the heart, the gracilis muscle shows a basal spontaneous HSP72 synthesis in both the SHR (71.4±10.8%) and WKY (40.6±11.7%) animals. There was a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to their control (186.2±18.7% vs 115.8±9.9%, p<0.02) which was maintained also during aging (171.9±17.3% vs 95.2±10.5%, p<0.01).In conclusion, these data show that hypertension results in an increased synthesis of HSP72 both in cardiac and gracilis muscle in response to heat shock. This abnormal response is attenuated by aging in the heart but not in the gracilis muscle. Thus, the abnormality seems to be independent from hypertrophy and linked to genetic determination of the disease.     

Ethylene oligomerization using nickel dithiolene complexes Ni(S2C2R2)2 (R=Ph, CF3) and the crystal structure of Ni[S2C2(CF3)2]2

Nickel bis(dithiolene) complexes have been known for over four decades, yet little is known regarding the chemistry of this important subclass of inorganic coordination complexes in olefin oligomerization or polymerization. We report here that Ni(S(2)C(2)R(2))(2) (R=Ph, CF(3)) are converted to active catalysts for ethylene oligomerization when activated with methylaluminoxane (MAO). The catalyst activity is comparable to some nickel coordination complexes with N-donor ligands under similar conditions. The products are mainly butenes and hexenes, with small amounts of higher oligomers. The product distribution pattern is consistent with a nickel hydride species being the active center, where fast beta-hydride elimination limits the products to mostly butenes and hexenes. The exact nature of the active center and the reaction mechanism remain to be investigated. In addition, we determined the crystal structure for Ni[S(2)C(2)(CF(3))(2)](2). The molecule crystallizes in the P2(1)/n space group and adopts a planar geometry with expected bond lengths and angles. Comparing this structure with that for the donor-acceptor complex with perylene reveals elongation of both the Ni-S and the S-C bonds in the latter, suggesting reduction of Ni[S(2)C(2)(CF(3))(2)](2) may have occurred in the latter.