Some Aspects on Acyclonucleoside Synthesis

Abstract

An acyclonucleoside synthesis was investigated on the regioselective introduction of an acyclochain. We found that iodotrimethylsilane catalyzed the reaction of acyclochain introduction as well as its migration from S² to N¹ of 2-thiothymine and from N⁷ to N⁹ position of guanine. By taking the findings into account, several acyclo-nucleosides were synthesized in a simple one-pot procedure.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.     

A convenient route to synthesize N2-(isobutyryl)-9-(carboxymethyl)guanine for aeg-PNA backbone

Abstract

Synthesis of exclusive N²-(isobutyryl)-9-(carboxymethyl)guanine, an important moiety for peptide nucleic acid synthesis has been reported through a high-yielding reaction scheme starting from 6-chloro-2-amino purine. Crystal structures of two intermediates confirmed the formation of N9-regioisomer. This new synthetic route can potentially replace the conventional tedious method with moderate overall yield.     

Reactivity of an extremenly sterically crowded monofunctional Pt complex, [Pt(1-MeC-N3)3(H2O)]2+ (1-MeC=1-methylcytosine), toward model nucleobases and selectivity toward guanine in single- and double-stranded deoxyoligonucleotides

 Reactions of [Pt(1-MeC-N3)₃Cl]NO₃ (1-MeC-N3=1-methylcytosine, bound to Pt via N3) and the respective aqua species [Pt(1-MeC-N3)₃(H₂O)]²⁺ with the model nucleobases 9-ethylguanine (9-EtGH), 9-methyladenine (9-MeA), single-stranded 5′d(T₃GT₃), and double-stranded [5′d(GAGA₂GCT₂CTC)]₂ have been studied in solution by means of ¹H NMR spectroscopy, HPLC, and electrospray ionization mass spectrometry. Reactions are generally slow, in particular with the chloro species, and guanine is the only reactive base in the oligonucleotides. However, unlike (dien)Pt^II, which binds randomly to the guanines in the ds dodecamer, (1-MeC-N3)₃Pt^II binds selectively to the terminal guanine only, probably because base fraying takes place at the duplex ends. The X-ray crystal structures of [Pt(1-MeC-N3)₃(9-EtG-N7)]ClO₄·8H₂O (1b) and of [Pt(1-MeC-N3)₃(9-MeA-N7)](ClO₄)₂·0.5H₂O as well as NMR spectroscopic studies of [Pt(1-MeC-N3)₃(9-EtGH-N7)] (NO₃)₂·H₂O (1a) are reported. The tetrakis(nucleobase) complexes adopt a head-tail-head orientation of the three 1-MeC bases and an orientation of the fourth base (purine) that permits a maximum of intracomplex H bonds between exocyclic groups. As far as the guanine adduct (1a, 1b) is concerned, relative orientations of the four bases are identical in the model and in the oligonucleotide adduct. Received: 19 June 1998 / Accepted: 1 October 1998     

Stereoselective Approach to the Z-Isomers of Methylenecyclopropane Analogues of Nucleosides: A New Synthesis of Antiviral Synguanol

Stereoselective synthesis of antiviral synguanol (1) is described. Reaction of 6-benzyloxy-2-(dimethylaminomethyleneamino)purine (10) with ethyl (cis,trans)-2-chloro-2-(chloromethyl) cyclopropane-1-carboxylate (2c) under the conditions of alkylation-elimination gave (Z)-6- benzyloxy-2-formylamino-9-[(2-carbethoxycyclopropylidene)methyl]purine (11) but no E,N⁹-isomer. Minor amounts of (Z)-6-benzyloxy-2-formylamino-7-[(2-carbethoxy-cyclopropylidene)methyl]purine (13) were also obtained. Hydrolysis of compounds 11 and 13 in 80% acetic acid afforded (Z)-9-[2-(carbethoxycyclopropylidene)methyl]guanine (14) and (Z)-7-[2-(carbethoxy- cyclopropylidene)methyl]guanine (15). Reduction of 14 furnished synguanol (1). Reaction of N⁴-acetylcytosine (7) with ester 2c led to (Z,E)-1-(2-carbethoxycyclopropropylidenemethyl)cytosine (8, Z/E ratio 6.1:1). Basicity of purine base, lower reactivity of alkylation intermediates as well as interaction of the purine N³ or cytosine O² atoms with the carbonyl group of ester moiety seem to be essential for the observed high stereoselectivity of the alkylation-elimination. The Z-selectivity is interpreted in terms of E1cB mechanism leading to a transitory “cyclic” cyclopropenes which undergo a cyclopropene-methylenecyclopropane rearrangement.     

Synthesis and Triplex Forming Properties of an Acyclic N7-Glycosylated Guanine Nucleoside

Abstract

A chiral acyclic nucleoside, one in which the ribose carbohydrate has been replaced with a glycerol-based linker, is prepared by glycosylating guanine at the N⁷-nitrogen. The stereochemically pure derivative is converted to a DMT-protected phosphoramidite for incorporation into DNA sequences. Sequence containing the acyclic N⁷-dG nucleoside are capable of forming DNA triplexes in which it is likely that the N¹-H and N²-amino groups of the N⁷-dG are involved in recognition of the guanine base in G-C base pairs.     

High performance liquid chromatography studies on the interactions of cis-Pt(NH3)2−(H2O)22+ with guanine and methylated g

Time dependence studies, using high performance liquid chromatography (HPLC), on the reaction between cis-diamminediaquoplatinum and guanine, N1-methylguanine, N7-methylguanine, N9-methylguanine, and N1 ,N7-dimethylguanine are reported. Each reaction gave rise to eight or more compounds; the major components have been prepared and characterization by ¹H and ¹⁹⁵Pt nuclear magnetic resonance has been attempted. Species of the form ((NH₃)₂Pt(NO₃)-(G-H)-(NO₃)-Pt(NH₃)₂)⁺, (NH₃)₂,Pt(G-H)(NO₃) monomer and (NH₃)₂Pt(G-H)(NO₃) dimer, where G-H indicates the guanine monoanion, are postulated.     

An Efficient Synthesis Of Acyclic N7- and N9-Adenine Nucleosides Via Alkylation With Secondary Carbon Electrophiles to Introduce Versatile Functional Groups At the C-1 Position of Acyclic Moiety

The introduction of versatile functional groups, allyl and ester, at the C-1 position of the acyclic chain in acyclic adenine nucleosides was achieved for the first time directly by alkylation of adenine and N⁶-protected adenine. Thus, the C-1′-substituted N⁹-adenine acyclic nucleoside, adenine-9-yl-pent-4-enoic acid ethyl ester (11), was prepared by direct alkylation of adenine with 2-bromopent-4-enoic acid ethyl ester (6), while the corresponding N⁷-regioisomer, 2-[6, (dimethylaminomethyleneamino)-purin-7-yl]-pent-4-enoic acid ethyl ester (10), was obtained in one step by the coupling of N,N-dimethyl-N′- (9H-purin-6-yl)-formamidine (9) with 2-bromopent-4-enoic acid ethyl ester (6). The functional groups, ester and allyl, were converted to the desired hydroxymethyl and hydroxyethyl groups, and subsequently to phosphonomethyl derivatives and corresponding pyrophosphorylphosphonates.     

Synthesis of Some Purine Carbocyclic Isosteres of 2′,3′-Dideoxy-3′-C-Hydroxymethyl Nucleosides as Potential Inhibitors of HIV

Abstract

The synthesis of some enantiomerically pure carbocyclic 2′,3′-dideoxy-3′-C-hydroxymethyl derivatives of adenine, inosine and guanine is described. The Mitsunobu reaction was used in the coupling procedure giving exclusively N⁹-coupling. The nucleosides were tested for inhibition of HIV multiplication in vitro and were found to be inactive in the assay.     

Synthesis of Base Substituted 2-Hydroxy-3-(purin-9-yl)-propanoic Acids and 4-(Purin-9-yl)-3-butenoic Acids

Abstract

Alkylation of 6-chloropurine and 2-amino-6-chloropurine with bromoacetaldehyde diethyl acetal afforded 6-chloro-9-(2,2-diethoxyethyl)purine (3a) and its 2-amino congener (3b). Treatment of compounds 3 with primary and secondary amines gave the N⁶-substituted adenines (5a–5c) and 2,6-diaminopurines (5d–5f). Hydrolysis of 3 resulted in hypoxanthine (6a) and guanine (6b) derivatives, while their reaction with thiourea led to 6-sulfanylpurine (7a) and 2-amino-6-sulfanylpurine (7b) compounds. Treatment with diluted acid followed by potassium cyanide treatment and acid hydrolysis afforded 6-substituted 3-(purin-9-yl)- and 3-(2-aminopurin-9-yl)-2-hydroxypropanoic acids (8–10). Reaction of compounds 3 with malonic acid in aqueous solution gave exclusively the product of isomerisation, 6-substituted 4-(purin-9-yl)-3-butenoic acids (15).     

Efficient Synthesis of 8-Thiosubstituted Guanine Derivatives as Potential Tools for Biochemical and Biological Studies

Abstract

A method for the selective introduction of the N²-(dimethylamino)methylene group into 8-thio-9-(2-hydroxyethoxymethyl)guanine (1) has been developed. The effect of the N²-amidine protection on the S-alkylation of 1 was studied.     

Effects of N7-methylation, N7-platination, and C8-hydroxylation of guanine on H-bond formation with cytosine: platinum coordination strengthens the Watson-Crick pair

d ₆, applying concentration-dependent ¹H NMR spectroscopy: 9-ethylguanine, 7,9-dimethylguanine (7,9-DimeGH⁺), and 7,8-dihydro-8-oxo-9-methylguanine (8-O-9-MeGH), as well as three 9-ethylguanine complexes carrying different Pt(II) moieties at the N7 position. The association constants K for the Watson-Crick pairing schemes are by a factor 2–3 higher in the cases of platinated guanine complexes compared to the Watson-Crick pair between 9-ethylguanine and 1-methylcytosine (K=6.9±1.3 M⁻¹). Similar enhanced stabilities are observed for the pairs formed between 1-MeC and 7,9-DimeGH⁺ or 8-O-9-MeGH. The increase in N1H acidity of the guanine derivative upon modification at the N7 or C8 positions can be correlated with the association constants K; the result is a bell-shaped curve meaning that acidification initially stabilizes hydrogen bond formation up to a certain maximum; further acidification then leads to a destabilization. For two of the examples studied in solution, hydrogen bonding according to Watson-Crick between N7-platinated 9-ethylguanine and 1-methylcytosine has also been established by X-ray crystallography. Received: 29 December 1999 / Accepted: 15 February 2000     

Novel oligodeoxynucleotide agonists of TLR9 containing N3-Me-dC or N1-Me-dG modifications

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N³-Me-cytosine or N¹-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs). IMOs containing N-Me-cytosine or N-Me-guanine in C- or G-position, respectively, of the CpG dinucleotide showed activation of HEK293 cells expressing TLR9, but not TLR3, 7 or 8. IMOs containing N-Me-cytosine or N-Me-guanine modification showed activity in mouse spleen cell cultures, in vivo in mice, and in human cell cultures. In addition, IMOs containing N-Me-substitutions reversed antigen-induced Th2 immune responses towards a Th1-type in OVA-sensitized mouse spleen cell cultures. These studies suggest that TLR9 tolerates a methyl group at N1-position of G and a methyl group at N3-position of C may interfere with TLR9 activation to some extent. These are the first studies elucidating the role of N3-position of cytosine and N1-position of guanine in a CpG motif for TLR9 activation and immune stimulation.     

Facile Method for the Preparation of 7-Methyl-8-oxoguanosines as an Immunomodulator¶

Abstract

Reaction of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.

     

EFFICIENT METHODS FOR THE SYNTHESIS OF [2-15N]GUANOSINE AND 2′-DEOXY[2-15N]GUANOSINE DERIVATIVES

The nucleophilic addition–elimination reaction of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(4-nitrophenyl)ethyl]inosine (8) with [¹⁵N]benzylamine in the presence of triethylamine afforded the N ²-benzyl[2-¹⁵N]guanosine derivative (13) in a high yield, which was further converted into the N ²-benzoyl[2-¹⁵N] guanosine derivative by treatment with ruthenium trichloride and tetrabutyl-ammonium periodate. A similar sequence of reactions of 2′,3′,5′-tri-O-acetyl-2-fluoro-O ⁶-[2-(methylthio)ethyl]inosine (9) and the 6-chloro-2-fluoro-9-(β-D-ribofuranosyl)-9H-purine derivative (11), which were respectively prepared from guanosine, with potassium [¹⁵N]phthalimide afforded the N ²-phthaloyl [2-¹⁵N]guanosine derivative (15; 62%) and 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-[¹⁵N]phthalimido-9H-purine (17; 64%), respectively. Compounds 15 and 17 were then efficiently converted into 2′,3′,5′-tri-O-acetyl[2-¹⁵N]guanosine. The corresponding 2′-deoxy derivatives (16 and 18) were also synthesized through similar procedures.     

Mutagenicity of Intermediates Produced in the Early Stage of the Browning Reaction of Triose Reductone with Nucleic Acid Related Compounds on Bacterial Tests

Two types of reductive intermediates, linear and tricyclic forms, isolated from browning mixtures of triose reductone (TR) with guanine and its derivatives showed evident mutagenicity on Salmonella typhimurium TA 100 without S-9 mixture. The linear intermediates, N²-(3-oxo-2-hydroxypropenyl) compounds of guanine, guanosine, 2′(3′)-guanylic acid and 5′-guanylic acid were more effective than the tricyclic one, l, N²-(2-hydroxypropenylidene)guanine, though they were far less active than 4-nitroquinoline-N-oxide. No acceleration in mutagenicity was observed with Cu^{2 +} and other metal ions. The reaction mixtures of TR and nucleic acid bases were also mutagenic on TA 100. Intermediates of TR with guanine and its derivatives did not have a lethal effect in Recassays with Bacillus subtilis.     

Study of the interaction between a histidine-deoxyguanosine hybrid and cisplatin

 A histidine-2′-deoxyguanosine hybrid, Phac-Hse(p^5′dG)-His-OH (I), was synthesized, and its reaction with cisplatin was monitored by reversed-phase HPLC and ¹H NMR. Two new compounds, II and III, were observed to be simultaneously formed, II in larger proportion than III. These products were isolated after HPLC purification and extensively characterized. Both II and III contained platinum, had the same mass and showed a bathochromic displacement of their absorption maxima with respect to that of I. Both remained undegraded upon enzymatic digestion and yielded I when treated with NaCN. From these data and the information provided by ¹H NMR analysis, we inferred that II and III were constitutional isomers, in particular chelates in which platinum was coordinated to the N7 of guanine and either the N^π or the N^τ imidazole nitrogens, respectively. No preference of the metal for either of these N-donors was observed. Received: 3 May 1999 / Accepted: 24 August 1999     

Immunogenicity and Protective Efficacy of a Vero Cell Culture-Derived Whole-Virus H7N9 Vaccine in Mice and Guinea Pigs

BackgroundA novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models.MethodsAntibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine.ResultsThe whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic.ConclusionsThe induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.     

Excited-state relaxation paths of oxo/hydroxy and N9H/N7H tautomers of guanine: a CC2 theoretical study

We performed a theoretical investigation, at the CC2/aug-cc-pVDZ level, of the ring-deformation mechanisms of four guanine tautomers (oxo, hydroxy, N9H, and N7H). The study showed that the optimized conical intersections S₀/S₁ are accessible through the ¹ππ* excited states of tautomers. The optimized conical intersections S₀/S₁, which show deformation at the pyrimidine ring, have high energies. This means that the relaxations of the ¹ππ* excited states via internal conversion are disfavored. For two tautomers we found crossing points ¹ππ*/¹πσ* of the excited-state reaction paths, revealing the possibility of a population of the ¹πσ* excited state by the ¹ππ* excited state.

近20年中国部分地区鸡源H9N2亚型禽流感病毒HA基因遗传演化及其变异频率北大核心CSCD

【目的】通过比较不同时期的H9N2亚型禽流感流行毒株HA基因的分子特征和变异频率,揭示免疫压力下病毒的遗传演化趋势。【方法】选取源于课题组的40株鸡源H9N2毒株,以及从Gen Bank下载的136株中国鸡源H9N2流行毒株和7株经典毒株的序列,利用Lasergen 7.1和MEGA 5.1等软件,对其HA基因进行系统演化、分子特征和变异频率分析。【结果】系统发育分析表明,近20年的鸡源H9N2流行株分属于BJ94、Y280和S2等谱系,优势流行株的分布与年代密切相关。氨基酸序列比较显示,H9N2病毒不同谱系之间具有各自的特征,且存在着明显的氨基酸变异积累。以Ck/BJ/1/1994 HA基因为参照,1994–2014年间,H9N2流行株核苷酸和氨基酸的年均进化率分别为5.73×10^(–3)和4.25×10^(–3)。其中,2011–2014年的核苷酸(氨基酸)年均进化率为6.35×10^(–3)(5.32×10^(–3)),明显高于2006–2010年5.22×10^(–3)(3.70×10^(–3)),更显著高于疫苗推广初期1999–2005年的0.74×10^(–3)(0.50×10^(–3))。【结论】H9N2疫苗株和流行毒株的不匹配是病毒变异频率加快的重要原因。