A transition state analogue for an RNA-editing reaction

Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR.RNA complexes.     

Substrate analogues for an RNA-editing adenosine deaminase: mechanistic investigation and inhibitor design

ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. ADARs capable of editing biologically relevant RNA substrates have been identified. In addition, the consequence of the RNA-editing reaction on the function of the gene product is known in several cases. However, our understanding of the chemical mechanism of the ADAR-catalyzed adenosine deamination in RNA is lagging. By studying analogues of a naturally occurring substrate for ADAR2, we infer features of the enzyme's active site and reaction mechanism. 8-Aza substitution at adenosine in various RNA substrates accelerates the rate of deamination at these sites by ADAR2 (2.8-17-fold). The magnitude of this "aza effect" depends on the RNA structural context of the reacting nucleotide. N(6)-Methyladenosine in RNA is a slow substrate for ADAR2 (rate is 2% that of adenosine), with no product observed with N(6)-ethyladenosine, suggesting a limited size of the leaving group pocket. 2,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which catalyzes a similar reaction on nucleosides. This and other results indicate that ADAR2 uses a base recognition strategy different from that of ADA. Consistent with the large 8-aza effect observed for the ADAR2 reaction, we find that 8-azanebularine, as the free nucleoside, inhibits the ADAR2 reaction (IC(50) = 15 +/- 3 mM) with no inhibition observed with nebularine or coformycin.     

Efficient C2'alpha-hydroxylation of deoxyribose in protein-induced Z-form DNA

DNA local conformations are thought to play an important biological role in processes such as gene expression by altering DNA-protein interactions. Although left-handed Z-form DNA is one of the best-characterized and significant local structures of DNA, having been extensively investigated for more than two decades, the biological relevance of Z-form DNA remains unclear. This is presumably due to the lack of a versatile detection method in a living cell. Previously, we demonstrated that the incorporation of a methyl group at the guanine C8 position (m(8)G) dramatically stabilizes the Z-form of short oligonucleotides in a variety of sequences. To develop a photochemical method to detect Z-form DNA, we examined the photoreaction of 5-iodouracil-containing Z-form d(CGCG(I)UGCG)(ODN 1)/d(Cm(8)GCAm(8)GCG)(ODN 2) in 2 M NaCl and found stereospecific C2'alpha-hydroxylation occurred at G(4) to provide d(CGCrGUGCG), 5. Recently, Rich and co-workers [Schwartz et al. Science 1999, 284, 1841. Schwartz et al. Nat. Struct. Biol. 2001, 8, 761] found that an ubiquitous RNA editing enzyme, adenosine deaminase 1 (ADAR1), and tumor-associated protein DML-1 specifically bind to Z-form DNA. In the present study, we investigate the photoreactivity of octanucleotide ODN 1-2 in Z-form induced by Zalpha, which is the NH(2)-terminal domain of ADAR1 responsible for tight binding of ADAR1. Detailed product analysis revealed that the C2'alpha-hydroxylated products 5 and 6 produced significantly higher yields in Z-form ODN 1-2 induced by Zalpha compared with that in 2 M NaCl. Upon treatment with ribonuclease T1, 5 and 6 were quantitatively hydrolyzed at the 3'-phosphodiester bond of the rG residue to provide d(UGCG) as a common hydrolyzed fragment on the 3' side. Quantitative analysis demonstrated that the amount of photochemically formed 5 and 6 from ODN 1-2 directly correlated with the proportion of Z-form induced by Zalpha or NaCl. These results suggest that this photochemical and enzymatic procedure can be used as a specific probe for the existence of local Z-form structure in cellular DNA.     

The binding selectivity of ADAR2's dsRBMs contributes to RNA-editing selectivity

ADAR2 is an RNA editing enzyme that deaminates adenosines in certain duplex structures. Here, we describe the role of its RNA binding domain, consisting of two copies of a common dsRNA binding motif (dsRBM), in editing site selectivity. ADAR2's dsRBMs bind selectively on a duplex RNA that mimics the Q/R editing site in the glutamate receptor B-subunit pre-mRNA. This selectivity is different from that of PKR's dsRBM I, indicating that dsRBMs from different proteins possess intrinsic binding selectivity. Using directed hydroxyl radical cleavage data, molecular models were developed that predict important recognition surfaces on the RNA for identified dsRBM binding sites. Blocking these surfaces by benzyl modification of guanosine 2-amino groups impeded RNA-editing, demonstrating a correlation between deamination efficiency by ADAR2 and selective binding by its dsRBMs. In addition, the editing activity of a mutant of ADAR2 lacking dsRBM I on N(2)-benzylguanosine-modified RNA suggests the location of the dsRBM I binding site that leads to editing at the GluR-B Q/R site.     

苯乙酸对肝癌细胞系SMMC-7721的增殖抑制作用及与RNA编辑酶ADAR1表达的相关性

为探讨苯乙酸(PA)对肝癌细胞系SMMC-7721的增殖抑制作用及其与RNA编辑酶ADAR1表达的相关性, 应用细胞计数及MTT法检测了不同浓度(0.5, 1.0, 2.0和4.0 mmol/L)PA对肝癌细胞系SMMC-7721的增殖抑制作用, 通过流式细胞术(FCM)分析了各细胞周期的细胞百分比, 应用半定量逆转录-聚合酶链式反应(RT-PCR)及免疫印迹杂交分析使用不同浓度(0.5, 1.0, 2.0 mmol/L)PA作用后肝癌细胞系SMMC-7721中RNA编辑酶ADAR1 mRNA及蛋白表达的变化. 结果表明, 肝癌细胞系SMMC-7721经不同浓度PA作用后, 增殖抑制率随作用时间延长及PA浓度增加而明显提高(P<0.05), 但2.0和4.0 mmol/L PA作用72 h后组间差异比较无统计学意义(P>0.05). 肝癌细胞系SMMC-7721中RNA编辑酶ADAR1 mRNA及蛋白表达随PA浓度增加而明显降低(P<0.05). 通过沉默SMMC-7721细胞中ADAR1的表达发现, ADAR1表达下调可有效抑制肝癌细胞增殖. 结果表明, PA可阻抑肝癌细胞系SMMC-7721细胞增殖, 且存在时间及剂量的依赖性, 作用机制与PA下调ADAR1表达相关.     

内包金属富勒烯衍生物La@C82-(C4N2H8)mHn的合成与表征

在常温、Ar气保护下研究了金属富勒烯与哌嗪(Piperazine)的反应, 并用硅胶柱分离了3种金属富勒烯衍生物. 用激光解析飞行时间质谱、紫外-可见-近红外光谱和傅里叶变换红外光谱等手段分析确定其结构分别为La@C82-C4N2H8, La@C82-C4N2H8-H8和La@C82-(C4N2H8)2-H6. 对比C60与哌嗪的反应结果发现, 与空富勒烯相比, 金属富勒烯反应活性更高, 产物加成数目更多.     

Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.     

Synthesis of 2',4'-propylene-bridged (carba-ENA) thymidine and its analogues: the engineering of electrostatic and steric effects at the bottom of the minor groove for nuclease and thermodynamic stabilities and elicitation of RNase H

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH(2)/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C═N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON:RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance but 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH(2) substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase H1 with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.     

Long‐range heteronuclear coupling constants in 2,6‐disubstituted purine derivatives

Four‐ and five‐bond heteronuclear J‐couplings between the hydrogen H‐8 and carbons C‐6 and C‐2 in a series of 7‐ and 9‐benzyl substituted purine derivaties with variuous substituents in positions 2 and 6 were studied by coupled ¹³ C NMR and H,C‐HMBC experiments and by DFT calculations. We have found that for some of the derivatives, the five‐bond coupling H8‐C2 is higher than the four‐bond H8‐C6 coupling, which is also evidenced by a stronger crosspeak in the HMBC. This finding contradicts the generally accepted opinion that only strong three‐bond crosspeaks and one weak four‐bond H8‐C6 crosspeak can be observed in the HMBC spectra of purine derivatives. The misinterpretation of HMBC spectra may lead to an incorrect determination of the purine derivatives' structure. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of novel ligands for the Z-DNA binding protein by structure-based virtual screening

We describe the first discovery of small molecules that bind to the Z-DNA binding domain of human ADAR1 (Adenosine Deaminase Acting on RNA 1) by structure-based virtual screening of chemical database. These molecules bind to Z-DNA binding domain to inhibit the interaction with the Z-DNA. Many viruses have Z-DNA binding proteins, which are structurally similar to Z-DNA binding domain of human ADAR1, and the ability of Z-DNA binding protein to bind the Z-DNA is essential for their pathogenicity. Therefore, the molecules identified in this study may serve as novel leads for the design of agents that inhibit biological functions of those pathogenic viruses.     

Discotic liquid crystals of transition metal complexes: 22. synthesis and mesomorphism of octa-alkoxy-substituted tetraphenylporphyrin derivatives and their copper(II) complexes

Ten novel long-chain-substituted porphyrin derivatives, tetrakis (3,4-dialkoxyphenyl)porphyrins [abbreviated as (CnO8)TPPM: M=H2, n=4, 6, 8, 10, 12, 16, 18; M=Cu, n=8, 12 and (2EtC6O)8TPPH2], were synthesized and their mesomorphism was investigated. Among them four derivatives, (CnO)8TPPH2(n=12, 16, 18) and (C12O)8TPPCu, exhibited a monotropic discotic lamellar (DL) mesophase.     

Synthesis and antiviral activity of highly water-soluble polycarboxylic derivatives of [70]fullerene

We report here a facile preparation of highly water-soluble derivatives C(70)[p-C(6)H(4)(CH(2))(n)COOH](8) (n = 2, 3) starting from readily available chlorinated [70]fullerene precursors C(70)Cl(8) and C(70)Cl(10). The synthesized fullerene derivatives showed pronounced antiviral activity in vitro, particularly against human immunodeficiency virus (HIV) and influenza A virus (subtypes H1N1 and H3N2).     

Synthesis and reactions of fluorous carbobenzyloxy (FCbz) derivatives of alpha-amino acids

Fluorous carbobenzyloxy ((F)Cbz) reagents RfCH(2)CH(2)C(6)H(4)CH(2)OC(O)OSu (where Su is succinimidoyl and Rf is C(6)F(13) and C(8)F(17)) have been used to make (F)Cbz derivatives of 18 of the 20 natural amino acids. The potential utility of this new family of reagents in both standard fluorous synthesis with spe separation and fluorous quasiracemic synthesis is illustrated with representative reactions of the (F)Cbz-Phe derivatives.     

Hydrogenation of the ansa-Chain of Rifamycins. X-ray crystal structure of (16S)-16,17,18,19-tetrahydrorifamycin S

The catalytic hydrogenation of rifamycin S ( 2 ) over Pd/C, followed by oxidation with K₃[Fe(CN)₆], generates a pair of 16,17,18,19-tetrahydrorifamycins S ( 3/4 ), epimeric at C (16). The use of PtO₂ as catalyst leads to the hydrogenation also of the C(28)?C(29) bond giving, after oxidation by K₃[Fe(CN)₆], a mixture of the epimers (16R)- and (16S)-16,17,18,19,28,29-hexahydrorifamycins S ( 5/6 ). Furthermore, we synthesized the (16R)- and (16S)-3-bromo derivatives 7/8 and (16R)- and (16S)-3-(piperidin-1-yl) derivatives 9/10 . The determination of the X-ray crystal structure of the most abundant epimer 4 of the tetrahydrorifamycins allowed the assignment of the absolute configuration at C(16) of all derivative. A Structure-activity relationship study showed that in general the (16R)-epimers are more potent inhibitors of bacterial RNA polymerase than the (16S)-epimers.     

含氟链不对称苯并菲盘状液晶的合成及疏氟效应对介晶性的影响

盘状液晶分子中引入氟代烃链并利用疏氟效应（fluorophobic effect）能有效稳定分子的柱状堆积;低对称性的盘状分子有较低的熔点和宽的介晶相温度范围．基于此,本文设计并合成了一系列半氟酯链的不对称苯并菲化合物C18H6（OCnH2n＋1）4（OCH3）（O2CC2H4C6F13）（1）,及相对应的不含氟化合物C18H6（OCnH2n＋1）4（OCH3）（O2CC8H17）（2）,n=4—8．化合物结构通过核磁和质谱表征．介晶性通过差示扫描量热法和偏光显微镜进行了研究．结果显示：化合物均为柱状互变热致液晶．含氟链化合物1a—1e与相对应的化合物2a-2e比较,有更高的熔点和清亮点．合成的多数化合物为室温液晶．     

The Reaction of the Bis-spirocyclic Phosphazene [N 3 P 3 (O 2 C 12 H 8 ) 2 Cl 2 ] (O 2 C 12 H 8 = 2,2′-Dioxybiphenyl) with Thiophenols,in the Presence of Alkali Carbonates

The reactions of the spirocyclic phosphazene [N 3 P 3 (O 2 C 12 H 8 ) 2 Cl 2 ] (O 2 C 12 H 8 = 2,2'-dioxybiphenyl) with the thiophenols HS--C 6 H 4 --R and M 2 CO 3 (M = K or Cs) in refluxing acetone gave respectively the spirocyclic substituted derivatives [N 3 P 3 (O 2 C 12 H 8 ) 2 (SC 6 H 4 --R) 2 ] R = H ( 2a ), Br ( 2b ), OMe ( 2c ), NO 2 ( 2d ). The reaction is a two-step process the second of which is much faster than the first and the monosubstituted intermediate [N 3 P 3 (O 2 C 12 H 8 ) 2 (SC 6 H 4 --R)Cl] cannot be detected. By contrast, in the analogous reactions with the phenols HO--C 6 H 4 --R and M 2 CO 3 (M = K or Cs) in acetone or THF, to give the known derivatives [N 3 P 3 (O 2 C 12 H 8 ) 2 (OC 6 H 4 --R) 2 ], the first step is faster although both are very dependent on R, M and the solvent. Thus, in the case of the phenol HO--C 6 H 4 --OMe the reaction conditions could be adjusted to give the useful synthetic intermediate monosubstituted derivative [N 3 P 3 (O 2 C 12 H 8 ) 2 (OC 6 H 4 --OMe)Cl] ( 3 ). The reaction of [N 3 P 3 (O 2 C 12 H 8 ) 2 Cl 2 ] with the bifunctional reagent mercaptophenol HS--C 6 H 4 --OH was not specific and led to mixtures of cyclic and oligomeric products.     

Inhibitory Effects of Phenylacetic Acid on Proliferation of Human Pancreatic Carcinoma BXPC-3 Cells by the Regulation of Adenosine Deaminases Acting on RNA

The effect and mechanism of phenylacetic acid on the proliferation of pancreatic carcinoma cells were investigated in cultured pancreatic carcinoma BXPC-3 cells by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry assay.The results show that the treatment of pancreatic carcinoma cells with phenylacetic acid significantly inhibited the cell proliferation in time-dependent and dose-dependent manners.The proliferation of BXPC-3 cells was inhibited at the stage of S phase,the cells at the end stage of S phase were accumulated abundantly,and thus DNA synthesis could not be accomplished entirely.In addition,the expression of adenosine deaminases acting on RNA(ADARs) mRNA in BXPC-3 cells and pancreatic carcinoma specimen were detected by RT-PCR.Having been treated with phenylacetic acid,ADAR2 mRNA in BXPC-3 cells was significantly decreased,the differences were of statistical significance(P0.01).Taken together,these results suggest that phenylacetic acid may likely regulate the proliferation of pancreatic carcinoma cells through the regulation of ADAR2 mRNA expression.