Synthesis and anti-HCV Evaluation of 4′(α)-ethyl and 2′(β)-methyl-carbodine Analogues

Novel 4′(α)-ethyl-2′(β)-methyl carbocyclic nucleoside analogues have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The construction of cyclopentene intermediate 12β was successfully made via sequential Johnson-Claisen orthoester rearrangement and ring-closing metathesis (RCM) starting from Weinreb amide 5. Selective dihydroxylation and desilylation gave the target carbodine analogues. The synthesized nucleoside analogues mentioned above 18 and 19 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides neither showed any significant antiviral activity nor toxicity up to 50 μM.     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

Molecular Structure of the Core-Modified siRNA Duplexes Containing Diastereomeric Pair of [C6′(R)-OH]- versus [C6′(S)-OH]-carba-LNAs Suggests a Model for RNAi Action

Molecular structures of native and a pair of modified small interfering RNA–RNA duplexes containing carbocyclic [6 ′-(R)-OH/7 ′-(S)-methyl]- and [6 ′-(S)-OH/7 ′-(S)-methyl]-carba-LNA-thymine nucleotides, which are two diastereomeric analogs of the native T nucleotide, incorporated at position 13 in the antisense (AS) strand of siRNA, have been simulated using molecular mechanics/dynamics techniques. The main aim of the project has been to find a plausible structural explanation of why modification of siRNA at T¹³ position by the [6 ′(R)-O-(p-Toluoyl)-7 ′(S)-methyl]-carba-LNA-Thymine [IC₅₀ of 3.32 ± 0.17 nM] is ca 24 times more active as an RNA silencing agent against the target HIV-1 TAR RNA than the [6 ′(S)-O-(p-Toluoyl)-7 ′(S)-methyl]-counterpart [IC₅₀ of 79.8 ± 17 nM] [1]. The simulations reveal that introduction of both C6 ′(R)-OH and C6 ′(S)-OH stereoisomers does not lead even to local perturbation of the siRNA–RNA duplex structures compared to the native, and the only significant difference between 6 ′(S)- and 6 ′(R)-diastereomers found is the exposure of the 6 ′-OH group of the 6 ′(R)-diastereoisomer toward the edge of the duplex while the 6 ′-hydroxyl group of the 6 ′(S)-diastereoisomer is somewhat buried in the minor groove of the duplex. This rules out a hypothesis about any possible local distortion by the nature of chemical modification of the siRNA-target the RNA duplex, which might have influenced the formation of the effective RNA silencing complex (RISC) and puts some weight on the hypothesis about the 6 ′-hydroxy group being directly involved with most probably Ago protein, since it is known from exhaustive X-ray studies [2, 3] that the core residues are indeed involved with hydrogen bonding with the internucleotidyl phosphates. Further systematic investigation is in progress to map the position-dependent functional and nonfunctional interactions of the modified [6 ′(R or S)-O-(p-Toluoyl)-7 ′(S)-methyl]-carba-LNA-T with the Ago2 protein of the RISC.     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Poly(U)-directed peptide-bond formation from the 2′(3′)-glycyl esters of adenosine derivatives

Summary The self-condensation of 2(3)-O-glycyl esters of adenosine, adenosine-5-(O-methylphosphate) and P₁, P₂-diadenosine-5-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5°C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10°C (5°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and below 23°C (15°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5°C of 2(3)-O-(glycyl)-adenosine, 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate), 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate, and 5-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2(3)-O-(glycyl)-adenosine yields 5% of 5-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).Abbreviations DKP diketopiperazine - (gly)₂ glycylglycine - (gly)₃ glycylglycylglycine - AppA-gly 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - Ado-2(3)-gly 2(3)-O-(glycyl)-adenosine - Ado-5-gly 5-O-(glycyl)-adenosine - Boc-gly N-tert-butyloxycarbonylglycine - AppA P₁, P₂-diadenosine-5-pyrophosphate - MepA adenosine-5-(O-methylphosphate) - AppA-Boc-gly 2(3)-O-(Boc-glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - Ado-5-Boc-gly 5-O-(Boc-glycyl)-adenosine - Ado-2(3)-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine     

Receptor-independent effects of 2′(3′)-O-(4-benzoylbenzoyl)ATP triethylammonium salt on cytosolic pH

The effect of the relatively potent P2X7 receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (BzATP-TEA) on cytosolic pH (pH_i) was studied using MC3T3-E1 osteoblast-like cells, which endogenously express P2X7 receptors. pH_i was measured fluorimetrically using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. BzATP-TEA (0.3–1.5 mM) elicited fast-onset alkalinization responses. In contrast, adenosine 5′-triphosphate disodium salt (5 mM) failed to reproduce the BzATP-TEA-induced responses, indicating a P2 receptor-independent mechanism. We speculated that triethylamine, which is present in solutions of BzATP-TEA, permeates the plasma membrane, and is protonated intracellularly, leading to an increase in pH_i. Consistent with this hypothesis, triethylammonium (TEA) chloride mimicked the effects of BzATP-TEA on pH_i. Moreover, measurements using a Cytosensor microphysiometer revealed that TEA chloride transiently suppressed proton efflux from cells, whereas washout of TEA transiently enhanced proton efflux. BzATP-TEA also elicited a sustained increase in proton efflux that was blocked specifically by the P2X7 antagonist A-438079. Taken together, we conclude that BzATP-TEA-induced alkalinization is unrelated to P2X7 activation, but is due to the presence of TEA. This effect may confound assessment of the outcomes of P2X7 activation by BzATP-TEA in other systems. Thus, control experiments using TEA chloride are recommended to distinguish between receptor-mediated and nonspecific effects of this widely used agonist. We performed such a control and confirmed that BzATP-TEA, but not TEA chloride, caused the elevation of cytosolic free Ca²⁺ in MC3T3-E1 cells, ruling out the possibility that receptor-independent effects on pH_i underlie BzATP-TEA-induced Ca²⁺ signaling.     

Conformationally Locked Nucleosides. Synthesis of Oligodeoxynucleotides Containing 3′-Amino-3′-deoxy-3′-N,5′(R)-C-ethylenethymidine

Abstract

3′-Amino-3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-N,5′(R)-C-ethylenethymidine (6) was synthesized starting from 3′-azido-3′-deoxythymidine. Condensation of 6 with 5′-O-(H-phosphonyl)thymidine and 5′-O-(p-nitrophenoxycarbonyl)thymidine derivatives gave dinucleotide and dinucleoside derivatives, respectively, which were incorporated into oligodeoxynucleotides (ODNs). Tm data of the modified ODNs are also presented.     

The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

Stereospecific Synthesis and Anti-HIV Activity of (Z)2′- and (E)3′-Deoxy-2′(3′)-C-(chloromethylene) Pyrimidine Nucleosides

Abstract

Reaction of 1-[2,5(and 3,5)-di-O-trityl-β-D-erythro-pentofuran-3 (and 2)-ulosyl]uracil derivatives 5 and 6 with (chloromethyl)triphenylphosphorane resulted in the stereoselective formation of (E)-3′- and (Z)-2′-chloromethylene derivatives 7 (69%) and 8 (53%), respectively, deprotection of which gave 9 and 10. Transformation of the uracil nucleoside 7 into cytosine one followed by deprotection yielded 12. The latter was converted into the arabinoside 14. The fully deprotected chloromethylene nucleosides were tested for their activity against HIV-1 and HIV-2.     

Ability of Adenosine-2′(3′)-deoxy-3′(2′)-triphosphates and Related Analogues to Replace ATP as Phosphate Donor for all Human and Drosphila melanogaster Deoxyribonucleoside Kinases

Abstract

Six non-conventional adenosine-2′- and 3′-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3′-deoxyadenosine-2′-triphosphate was an effective donor (5–60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5′-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

The elongation factor Tu·GTPase reaction: Effect of 2′(3′)-O-aminoacyl oligoribonucleotides

The activity of synthetic (2′(3′)-O-aminoacyl trinucleotides, C-C-A-Phe, C-C-U-Phe, C-U-A-Phe, U-C-A-Phe and C-A-A-Phe, in promoting the EF-Tu·70 S ribosome-catalyzed GTP hydrolysis was investigated. It was found that the activity decreases in the order C-C-A-Phe > C-U-A-Phe > U-C-A-Phe > C-A-A-Phe ⪢ C-C-U-Phe. Thus, the substitution in ‘natural’ C-C-A sequence with other nucleobases weakens binding of 2′(3′)-O-aminoacyl trinucleotides to EF-Tu, with the substitution at the 3′-position having the most profound effect. Since the 2′(3′)-O-aminoacyl oligonucleotides mimic the effect of the aa-tRNA 3′-terminus on EF-Tu·GTPase, it follows that EF-Tu probably directly recognizes structure of nucleobases in the aa-tRNA 3′-terminus, with the 3′-terminal adenine playing the most important role.     

Accuracy of Optimized Chemical-exchange Parameters Derived by Fitting CPMG R 2 Dispersion Profiles when R 2 0a ≠ R 2 0b

The transverse relaxation rate, R₂, measured as a function of the effective field (R₂ dispersion) using a Carr-Purcell-Meiboom-Gill (CPMG) pulse train, is well suited to detect conformational exchange in proteins. The dispersion data are commonly fitted by a two-site (sites a and b) exchange model with four parameters: the relative population, p_a, the difference in chemical shifts of the two sites, δω, the correlation time for exchange, τ_ex, and the intrinsic relaxation rate (i.e., transverse relaxation rate in the absence of chemical exchange), R₂⁰. Although the intrinsic relaxation rates of the two sites, R₂^0a and R₂^0b, can differ, they are normally assumed to be the same (i.e., R₂^0a = R₂^0b = R₂⁰) when fitting dispersion data. The purpose of this investigation is to determine the magnitudes of the errors in the optimized exchange parameters that are introduced by the assumption that R₂^0a = R₂^0b. In order to accomplish this goal, we first generated synthetic constant-time CPMG R₂ dispersion data assuming two-site exchange with R₂^0a ≠ R₂^0b, and then fitted the synthetic data assuming two-site exchange with R₂⁰ = R₂^0a = R₂^0b. Although all the synthetic data generated assuming R₂^0a ≠ R₂^0b were well fitted (assuming R₂^0a = R₂^0b), the optimized values of p_a and τ _ex differed from their true values, whereas the optimized values of δω values did not. A theoretical analysis using the Carver–Richards equation explains these results, and yields simple, general equations for estimating the magnitudes of the errors in the optimized parameters, as a function of ( R₂^0a − R₂^0b).     

Effectiveness of Ethylenediamine-N(o-hydroxyphenylacetic)-N′(p-hydroxyphenylacetic) acid (o,p-EDDHA) to Supply Iron to Plants

The Fe chelate o,p-EDDHA/Fe³⁺, in addition to o,o-EDDHA/Fe³⁺, was found recently to be a component of commercial EDDHA/Fe³⁺ chelates. The European Regulation on fertilisers has included o,p-EDDHA as an authorized chelating agent. The efficacy of o,o-EDDHA/Fe³⁺, o,p-EDDHA/Fe³⁺ and EDTA/Fe³⁺ chelates as Fe sources in plant nutrition was studied. Iron-chelate reductase (FC-R) in young cucumber plants (Cucumis sativus L.) roots reduced o,p-EDDHA/Fe³⁺ faster than o,o-EDDHA/Fe³⁺, EDTA/Fe³⁺ and a commercial source of EDDHA/Fe³⁺. The o,p-EDDHA/Fe³⁺ chelate was also more effective than the o,o-EDDHA/Fe³⁺ in decreasing the severity of Fe-deficiency chlorosis in leaves of young soybean (Glycine max L.) plants grown hydroponically. The o,p-EDDHA ligand was more effective in the short-term than the EDTA and o,o-EDDHA ligands at dissolving Fe from selected Fe minerals and soils. However, the ultimate quantity of dissolve Fe was greatest with the o,o-EDDHA ligand.     

Spike morphology alternations in androgenic progeny of hexaploid triticale (× Triticosecale Wittmack) caused by nullisomy of 2R and 5R chromosomes

Induction of androgenesis, followed by chromosome doubling, is a crucial method to obtain complete homozygosity in one-generation route. However, in vitro androgenesis can result in various genetic and epigenetic changes in derived triticale plants. In this study, we evaluated chromosome alternations and we associated them with the changes of spike morphology in androgenic progeny of triticale. We karyotyped offspring plants that derived from double haploid plants using fluorescence in situ hybridization techniques. We distinguished four major groups of karyotypes: double ditelosomics, nullisomics N2R, nullisomics N5R, and triticale plants with a complete set of chromosomes. It is known that more than half of QTLs connected with androgenic response are located in R-genome of triticale but 2R, 5R, and 6R chromosomes are not included. We hypothesized that the reason why only aberrations of chromosomes 2R and 5R appear during androgenesis of triticale is that because these chromosomes are not involved in the stimulation of androgenic response and the following regeneration of plants is not disrupted. Concerning the established groups, we evaluated following quantitative traits: spike length, number of spikes per plant, number of spikelets per spike, and number of grains per spike. The nullisomy of chromosome 2R and 5R resulted in vast changes in spike architecture of triticale plants, which can be correlated with the location of major QTLs for spike morphology traits on these chromosomes. The spikes of nullisomic plants had significantly decreased spike length which correlated with the reduction of number of spikelets per spike and number of grains per spike.

     

Selective synthesis of cerebrosides: (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R and 2′S)-2′-hydroxytetracosanoyl-sphingenine

The cerebrosides were first isolated by Thudicum in 1874 and the structures were established by Carteret al. in 1950 (for review, see [2]). In 1961 Shapiro and Flowers [3] reported the first total synthesis of a cerebroside1 (Fig. 1) which was identified with the natural sample, only through comparison of their i.r. data. In order to confirm the absolute configuration at C-2 of natural cerebroside1, we describe here an unambiguous synthesis of two stereoisomeric cerebrosides1 and2, and found that the¹H-NMR spectra of the synthetic1 (Fig. 2) was completely identical with that of the natural cerebroside reported recently by Dabrowskiet al. [4].In planning the synthetic route, the target structures1 and2 were disconnected at the dotted lines to give three key synthetic intermediates3, 4 and5 or6 (Fig. 1).Abbreviations Bu butyl - Ph phenyl - t-BuPh₂SiCl t-butyldiphenylsilyl chloride - MTPA -methoxy--trifluoromethylphenylacetic acid - THF tetrahydrofuran Part 36 in the series Synthetic Studies on Cell-surface Glycans, for part 35, see [1]     

Total synthesis of cerebrosides: (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R and 2′S)-2′-hydroxytetracosanoylsphingenine

Total syntheses of both (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R)-2′-hydroxytetracosanoylsphingenine 23 and the (2′S) stereoisomer were performed in an unambiguous way by employing either (2S, 3R, 4E)-N-(2′R)-2′-(tert-butyl-diphenylsilyloxy)tetracosanoylsphingenine or its (2′S) stereoisomer as the key glycosyl acceptors. The synthetic cerebroside 23 was shown to be identical with the natural product through comparison of their 400-MHz, ¹H-n.m.r. spectra, thus providing synthetic evidence for the 2′R configuration of the natural cerebroside.     

Cloning and chromosomal assignment of the bovine interleukin-2 receptor alpha (IL-2Rα) gene

The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number U24226.     

The Structure of 2′ (R)-Mercapto-2′-deoxyneplanocin A (Nucleosides and Nucleotides 48.)

Abstract

The molecular and crystal structure of 2′(R)-mercapto-2′-deoxyneplanocin A, C₁₁H₁₃N₅O₂S M.W.=279.32, has been determined by X-ray analysis. The space group is P2₁2₁2₁ with a=10.322(1), b=22.870(2), c=5.273(1)Å and z=4. The structure was solved by direct method, and least-squares refinement using 1806 reflections with |Fo| > 30(F) led to the final R value of 0.045. The sugar C(2′) atom is displaced by 0.35Å opposite to the base N(9), i.e., C(2′)-exo conformation and the torsion angle about the N(9)-C(1) bond is 26.3(4)° (anti conformation).