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.     

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.     

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。     

Analogues of ribose 5-phosphate and 5-phosphoribosyl pyrophosphate. The preparation and properties of ribose 5-phosphorothioate and 5-phosphoribosyl 1-methylenediphosphonate

1. 5-Phosphoribosyl 1-methylenediphosphonate was isolated after reaction of ribose 5-phosphate and O-adenylyl methylenediphosphonate with 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. 2. The analogue reacted with adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase [K(m) (analogue)/K(m) (5-phosphoribosyl pyrophosphate) 0.17, 0.19 and 6.3 respectively; V(max.) (analogue)/V(max.) (5-phosphoribosyl pyrophosphate) 0.011, 0.26 and 1.1 respectively]. 3. The analogue was not a substrate for 5-phosphoribosyl pyrophosphate amidotransferase or orotate phosphoribosyltransferase. 4. Ribose 5-phosphorothioate was synthesized by allowing ribose to react with thiophosphoryl chloride in triethyl phosphate. The analogue was a substrate for 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. When this reaction was coupled to either adenine phosphoribosyltransferase or hypoxanthine phosphoribosyltransferase, adenosine 5'-phosphorothioate or inosine 5'-phosphorothioate was formed respectively.     

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.     

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.     

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.     

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.     

Synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives of ribavirin

The synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives (13 and 15, respectively) of the antiviral agent ribavirin are described. Direct glycosylation of 2',3'-O-isopropylideneribavirin with either tetra-O-acetyl-alpha-D-glucopyranosyl bromide (4) or tetra-O-acetyl-alpha-D-galactopyranosyl bromide (8) under Koenigs-Knorr conditions (i.e., silver carbonate, silver perchlorate, and Drierite in dichloromethane) followed by O-deacetylation of the reaction product gave the corresponding ortho esters. However, treatment of 2',3'-di-O-acetyl-5'-O-tritylribavirin (11) with 4 under the Bredereck modification of the Koenigs-Knorr reaction (i.e., silver perchlorate and Drierite in nitromethane) and subsequent deacetylation furnished the desired 1-(5-O-beta-D-glucopyranosyl-beta-D-ribofuranosyl)-1,2,4-triazole-3-carb oxamide (13). Similarly, reaction of 11 with 8 in the presence of AgClO4, and deprotection of the condensation product, gave 5'-O-beta-D-galactopyranosylribavirin (15). The beta-anomeric configuration of the D-glucosyl and D-galactosyl groups of 13 and 15 was assigned by 1H-n.m.r. studies.     

Interaction of thymidylate synthase with the 5'-thiophosphates, 5'-dithiophosphates, 5'-H-phosphonates and 5'-S-thiosulfates of 2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine.

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5'-thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS2, dTMPS2 and FdUMPS2), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-S-thiosulfates (dUSSO3, dTSSO3 and FdUSSO3), have been synthesized and their interactions studied with highly purified mammalian thymidylate synthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS2 classic competitive inhibitors, only slightly weaker than dTMP. Their 5-fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, the corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak activities, only FdUMP-H and FdUSSO3 exhibiting significant interactions with the enzyme, as weak competitive slow-binding inhibitors versus dUMR The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pKa values of the phosphate and thiophosphate groups, the profile of FdUMPS being shifted (approximately 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitude lower than for the parent 5'-phosphates) with the enzyme is further evidence that the enzyme's active center prefers the dianionic phosphate group for optimum binding.     

Preparation of Oligonucleotides Containing 5-Bromouracil and 5-Methylcytidine.

Abstract

A previously described side reaction on 5-bromouracil during standard oligonucleotide deprotection conditions has been studied in detail. The side product, 5-amino-2′-deoxyuridine, is isolated and characterized. The use of several 5-methylcytidine protected derivatives for the preparation of oligonucleotides containing 5-bromouracil and 5-methylcytidine free of 5-amino-2′-deoxyuridine is discussed.     

麦芽根中5'-磷酸二酯酶和5'-磷酸单酯酶的提取

建立一种高效快速的从麦芽根中提取5'-磷酸二酯酶和磷酸单酯酶的方法.通过将麦芽根粉碎后,取一定粒度的麦芽根加一定量的去离子水浸泡,过滤后得粗酶液.用紫外分光光度法测定5'-磷酸二酯酶和5'-磷酸单酯酶的酶活,研究粉碎粒度、料液比、抽提温度、抽提时间等因素对麦芽根中5'-磷酸二酯酶和5'-磷酸单酯酶提取的影响.将原料粉碎至100目,以去离子水为提取剂,于4℃提取20h,料液质量比为1:10,5'-磷酸二酯酶的酶活力可达160U/ml.该法简单、快速、准确、适应性强,为5'-磷酸二酯酶的提取与检测提供了有效的工具.