5'-Fluoro-5'-deoxyaristeromycin

5'-Fluoro-5'-deoxyaristeromycin (2) has been prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylidenedioxy)-4-fluoromethylcyclopentan-1-ol with N6-bis-boc protected adenine. This procedure is adaptable to preparing a number of 5'-fluoro-5'-deoxycarbocyclic nucleoside analogs with diversity in the heterocyclic base. Antiviral analysis found promising activity for 2 toward measles but no other viruses. No cytotoxicity was observed for 2.     

Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.     

Independence of brachymesophalangia-5 from brachymesophalangia-5 with cone mid-5

Analysis of hand radiographs of juvenile siblings of juvenile propositi indicates that brachymesophalangia-5 alone (without cones) is separately inherited without apparent sex bias while brachymesophalangia-5 with the cone-epiphysis of mid-5 and the cone-epiphysis of mid-5 alone are both apparently inherited as a complex and with a marked excess of females over males.     

Synthesis and biological activity of the new 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid

A series of 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid 6, were synthesized and evaluated for their antitumor activity.     

The presence of guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine in brine shrimp embryos

Acid-soluble extracts of dormant embryos of the brine shrimp, Artemia salina, contain small amounts of two previously undescribed dinucleotides which we have identified to be guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine. These compounds each comprise about 0.03% of the dry weight of the encysted embryos and are related chemically to guanosine 5'-triphospho-5'-guanosine and guanosine 5'-tetraphospho-5'-guanosine which have been shown previously to be major constituents of the nucleotide pool of Artemia cysts. These new dinucleotides were purified from perchloric acid extracts of dormant cysts by ion exchange column chromatography and identified by means of chemical, spectrophotometric, and enzymatic analyses compared to commercially available compounds. The possible role of these new compounds in nucleotide and nucleic acid metabolism in Artemia embryos is discussed.     

5'-deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: studies with 5'-nucleotidase and alkaline phosphatase

The interaction of 5'-deoxy-5'-thioadenosine 5'-monophosphate (A(S)MP) and 5'-deoxy-5'-thioinosine 5'-monophosphate (I(S)MP) with snake venom, 5'nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5'-monophosphate and inosine 5'-monophosphate in which sulfur replaces oxygen as the bridge between the 5'-carbon of the ribose and the phosphorous. The P-S bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The Km for A(S)MP was 270 microM and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 microM and 530 nmol/min/mg protein, respectively. In contrast, 5'-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5'-nucleotidase hydrolysis of AMP and IMP, respectively, with Ki values of 975 and 13 microM. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the Ki for I(S)MP by 100-fold, to a value of 0.15 microM.     

Amino substituted derivatives of 5'-amino-5'-deoxy-5'-noraristeromycin

The potent antiviral potential of 5'-amino-5'-deoxy-5'-noraristeromycin (2) is limited by associated toxicity. To seek derivatives of 2 that circumvent this undesirable property, three amino substituted derivatives (acetyl, 3; formyl, 4; and methyl, 5) of 2 have been prepared in 4-7 steps from the same intermediate, (1S,4R)-4-(6-chloropurin-9-yl)cyclopent-2-en-1-ol (6). Key steps involved an improved Pd(0)-catalyzed allylic azidation and a novel Pd(0)-catalyzed allylic amidation. The three target compounds were evaluated against a large number of viruses and found to be inactive except for a very weak effect of 5 on human cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. There was also no noteworthy cytotoxicity associated with the new derivatives. Thus, these results indicate variation of the cyclopentyl amine of 2 does not offer a means to improve upon its antiviral potential.     

Preparation, characterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphate, and 5-deazariboflavin 5'-diphosphate, 5'leads to5'-adenosine ester.

In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given.     

5''-Halogeno-2'',3''-cyclic sulphite isomers in the preparation of 5''-halogeno nucleosides. Synthesis of 5''-deoxyuridine and 5''-deoxy-5-fluorouridine.

When uridine (Ia) is reacted with thionyl chloride in hexamethylphosphoric triamide a mixture of isomeric 5'-chloro-2',3'-sulphites is formed, which can be separated to individual epimers IIa and IIIa, in 45% and 15% yields, respectively. Analogously, crystalline epimers IIb (37%) and IIIb (17%) can be obtained from 5-fluorouridine (Ib). Both isomers IIa, IIIa (or IIb, IIIb) afford a single 5'-chloro derivative IVa (or IVb, respectively) if treated with 0.1N sodium methoxide. From the mixture of sulphites IIa and IIIa (or IIb and IIIb) crystalline 5'-chlorouridine IVa is formed in 84.5% yield, calculated per starting uridine Ia (or crystalline 5'-chloro-5-fluorouridine IVb, 85.5% per starting 5-fluorouridine Ib, respectively). On reduction of 5'-chlorouridine IVa with tributyltin hydride 5'-deoxyuridine (Va) is formed in 79% yield. During the reduction of 5'-chloro-5-fluoro derivative IVb to 5'-deoxy-5-fluorouridine (Vb, 57%) a partial reductive elimination of 5-fluorine takes place under formation of 5'-deoxyuridine (Va, 9%).     

An improved synthesis of 5'-fluoro-5'-deoxyadenosines

Synthesis of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and structurally similar compounds is generally a poor yielding process. This is attributed to the instability of the selected synthetic intermediates. Herein, we report a general synthesis of 5'-fluoro-5'-deoxy-N6-substituted adenosines including a high yielding access to 5'-FDA.     

Solid-phase synthesis of 5'-deoxy-5'-amino-clitocine analogues

Several 6-substituted-amino-5'-deoxy-5'-amino-clitocine analogues were synthesized in a parallel fashion in solid phase. The desired scaffold was generated by coupling 2,3-O-bis-(t-butyldimethylsilyl)-5-N-(monomethoxytrityl-polystyrene-resin)-1,5-diamino-5-deoxy-beta-D-ribofuranose and 4, 6-dichloro-5-nitropyrimidine. The scaffold was then reacted with a variety of amines to generate a small library of 14 analogues of 5'-deoxy-5'-amino-clitocine following a protocol developed earlier.     

A ligand for CD5 is CD5

Recognition by scavenger receptor cysteine-rich domains on membrane proteins regulates innate and adaptive immune responses. Two receptors expressed primarily on T cells, CD5 and CD6, are linked genetically and are structurally similar, both containing three scavenger receptor cysteine-rich domains in their extracellular regions. A specific cell surface interaction for CD5 has been difficult to define at the molecular level because of the susceptibility of CD5 protein to denaturation. By using soluble CD5 purified at neutral pH to preserve biological activity, we show that CD5 mediates species-specific homophilic interactions. CD5 domain 1 only is involved in the interaction. CD5 mAbs that have functional effects in humans, rats, and mice block homophilic binding. Ag-specific responses by mouse T cells in vitro were increased when engagement of human CD5 domain 1 was inhibited by mutation or by IgG or Fab fragment from a CD5 mAb. This showed that homophilic binding results in productive engagement. Enhancement of polyclonal immune responses of rat lymph node cells by a Fab fragment from a CD5 mAb shown to block homophilic interactions provided evidence that the extracellular region of CD5 regulates inhibition in normal cells. These biochemical and in vitro functional assays provide evidence that the extracellular region of CD5 regulates immunity through species-specific homophilic interactions.     

Evidence that intracellular synthesis of 5-fluorouridine-5'-phosphate from 5-fluorouracil and 5-fluorouridine is compartmentalized

L1210 cells were exposed to equitoxic concentrations of [14C]5-fluorouracil and [3H]5-fluorouridine for 4 hours. The RNA from these cells was separated into cytosolic and nuclear fractions, and then further fractionated by chromatography on poly-U Sepharose, Sephadex G-200 and DEAE-cellulose. The ratio of tritium to carbon-14 incorporated into various species of RNA differed by as much as 6-fold, indicating that the respective 5-fluorouridine-5'-monophosphates synthesized from the two precursors are localized in separate pools that do not mix rapidly.     

Stat5及JAK-Stat5通路

信号转导与转录因子（Stats）家族是一个由7个成员组成的转录调控家族,其中Stat5由于在多种细胞因子刺激时均可起到重要作用而引起了广泛的关注。Stat5具有两种不同类型,即Stat5a和Stat5b,它们均可被多种细胞因子所激活,启动JAK-Stat5信号通路,从而调节相应基因的表达。在两种Stat5的激活中,特殊位点的丝氨酸和酪氨酸的磷酸化对Stat5的激活起重要的作用。对信号通路严格的控制对于生物体来说具有重要的意义,在JAK-Stat5信号通路中,以SOCS3为代表的SOCS家族对JAK-Stat5的负反馈调节具有重要作用。     

Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell

Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.     

Complement component 5a (C5a)

The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.     

Tissue-Specific Differences in DNA Modifications (5-Hydroxymethylcytosine, 5-Formylcytosine, 5-Carboxylcytosine and 5-Hydroxymethyluracil) and Their Interrelationships

Background

Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.

Results

Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine, as well as 5-hydroxymethyl-2′-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2′-deoxycytidine in both porcine (R² = 0.88) and rat tissues (R² = 0.83); no such relationship was observed for 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2′-deoxycytidine and its product 5-formyl-2′-deoxycytidine, as well as between 5-formyl-2′-deoxycytidine and 5-carboxyl-2′-deoxycytidine (R² = 0.60 and R² = 0.71, respectively).

Conclusions

Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2′-deoxycytidine and/or 5-carboxyl-2′-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2′-deoxycytidine which defines the active demethylation process.     

5'-Amino-5'-deoxyaristeromycin and its antiviral properties

An efficient, three-step synthesis of 5'-amino-5'-deoxyaristeromycin (5), from a protected form of aristeromycin (6), is described. Compound 5 was evaluated against a large number of viruses. It showed weak activity towards vaccinia, herpes simplex virus 2, and cytomegalovirus. No other activity was observed. Compound 5 displayed some cytotoxicity towards the host cell lines human foreskin fibroblast, Daudi, and human T-lymphocyte (CEM).