Synthesis and properties of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides

Novel bicyclo nucleosides, 2'-O,4'-C-ethylene nucleosides and 2'-O,4'-C-propylene nucleosides, were synthesized as building blocks for antisense oligonucleotides to further optimize the 2'-O,4'-C-methylene-linkage of bridged nucleic acids (2',4'-BNA) or locked nucleic acids (LNA). Both the 2'-O,4'-C-ethylene- and propylene-linkage within these nucleosides restrict the sugar puckering to the N-conformation of RNA as do 2',4'-BNA/LNA. Furthermore, ethylene-bridged nucleic acids (ENA) having 2'-O,4'-C-ethylene nucleosides had considerably increased the affinity to complementary RNA, and were as high as that of 2',4'-BNA/LNA (DeltaT(m)=+3 approximately 5 degrees C per modification). On the other hand, addition of 2'-O,4'-C-propylene modifications in oligonucleotides led to a decrease in the affinity to complementary RNA. As for the stability against nucleases, incorporation of one 2'-O,4'-C-ethylene or one 2'-O,4'-C-propylene nucleoside into oligonucleotides considerably increased their resistance against exonucleases to an extent greater than 2',4'-BNA/LNA. These results indicate that ENA is more suitable as an antisense oligonucleotide and is expected to have better antisense activity than 2',4'-BNA/LNA.     

2'-O,4'-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug.

To develop antisense oligonucleotides, novel nucleosides, 2'-O,4'-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2'-O,4'-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.     

Synthesis of 3'-O2-(azaheterocycle)-thymidines

The synthesis of 3'-O2-(azaheterocycle)-thymidines is presented from 1-thia-3-aza- 1,3-butadiene precursors (N-thioacylamidines). A variety of heterocycles is accessible using the dienic, the electrophilic or the nucleophilic reactivity of these thia-azabutadiene systems. 3'-O2-(azaheterocycle)-thymidine analogues are regarded as potential substrates to interfere with the DNA-polymerization process.     

Synthesis of 1′,2′-methano-2′,3′-dideoxynucleosides as potential antivirals

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1′,2′- methano-2′,3′-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.     

Synthesis of CMP-9'-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5'-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate and 2-cyanoethyl 2',3'-O,N4, triacetylcytidine-5'-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9'-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9'-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver alpha-(2-->6)-sialyltransferase and Photobacterium alpha-(2-->6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9'-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.     

Substrate analogues and divalent cations as inhibitors of glutamate decarboxylase from Escherichia coli

To examine the idea that glutamate decarboxylase from E. coli can be a convenient source for the study of the effects of compounds on GABA synthesis in the nervous system, a series of substrate analogues and divalent cations were tested as potential inhibitors of the bacterial enzyme. Those analogues exhibiting inhibitor activity did so in a competitive manner. The most effective inhibitors were 3-mercaptopropionic acid, 4-bromoisophthalic acid and isophthalic acid which exhibited Ki values of 0.13 mM, 0.22 mM and 0.31 mM, respectively. Eight other analogues produced lesser degrees of inhibition. In addition, seven divalent metal cations were tested as inhibitors of the enzyme. However, only Hg2+, Cd2+, Cu2+ and Zn2+ were effective at a concentration of 0.1mM. When these results were compared to the patterns of inhibition of glutamate decarboxylase from mouse brain, certain differences in the manner in which the enzymes responded to the inhibitors, emerged. Consequently, the bacterial decarboxylase may not be a good model for the study of drug action on brain GABA synthesis.     

Preparation and properties of 2',5'-linked oligonucleotide analogues containing 3'-O,4'-C-methyleneribonucleosides

Bicyclic nucleoside analogues, 3'-O,4'-C-methyleneuridine and -5-methyluridine, were successfully incorporated into oligonucleotides via connection with 2',5'-phosphodiester linkage, and hybridization behavior and nuclease stability of the modified oligonucleotides were investigated.     

Synthesis of 5-(2,2'-bipyridinyl and 2,2'-bipyridinediiumyl)-2'-deoxyuridine nucleosides: precursors to metallo-DNA conjugates

The synthesis of 2,2'-bipyridinyl-2'-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2'-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2'-deoxyuridine nucleoside (Bipy(2+)-dU) with an ethylenyl linker. The Bipy(2+)-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

Hexose transport in L6 rat myoblasts. I. Rate-limiting step, kinetic properties, and evidence for two systems

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2-Deoxy-D-glucose (2-DOG) and 2-deoxy-2-fluoro-D-glucose (2FG) were used as analogues to investigate the rate-limiting step of hexose uptake into the cell. Virtually all of the 2-DOG or 2FG taken up into the cell was found to be in the phosphorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate-limiting step. The inhibitory effect of various glucose analogues on 2-DOG and 3-O-methyl-D-glucose (3-OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2-DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3-OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.     

Achievements and prospects of the directed search for antiviral agents in the nucleoside series and their derivatives]

Recent advances of antiviral drug design among nucleosides and their derivatives have been summarized. The first chapter deals with the history of nucleic acids components and further developments in this area. Next part discusses the mechanism of action of biologically active nucleosides: 2',3'-dideoxynucleosides, acyclic analogues, phosphonate derivatives and nucleoside antibiotics. The third chapter describes planning of complicated synthesis of nucleoside analogues from branched-chain sugars and stereo-specific formation of glycosidic bond upon synthesis of ribonucleoside and 2'-deoxyribonucleoside. The last part outlines further perspectives, i. e. preparation of antiviral compounds and use of nucleoside analogues in oligonucleotide synthesis.     

Synthesis and Biological Activity of Chloroethyl Pyrimidine Nucleosides

The synthesis and biological activity of chloroethyl pyrimidine nucleosides is presented. One of these new nucleosides analogues significantly inhibited cell proliferation, migration and invasion as tested in vitro on the A431 vulvar epidermal carcinoma cell line.     

Synthesis of cis-disubstituted cyclobutenyl nucleoside analogues.

cis-Disubstituted cyclobutene nucleosides analogues were prepared by a linear synthesis starting from cis-cyclobutene dicarboxylic anhydride. This strategy involved mild reaction conditions with intent to restrict the thermal electrocyclic ring opening into (Z,E)-dienes.     

Synthesis and biological activity of chloroethyl pyrimidine nucleosides

The synthesis and biological activity of chloroethyl pyrimidine nucleosides is presented. One of these new nucleosides analogues significantly inhibited cell proliferation, migration and invasion as tested in vitro on the A431 vulvar epidermal carcinoma cell line.     

Synthesis and biological evaluation of 2',4'- and 3',4'-bridged nucleoside analogues

Most nucleosides in solution typically exist in equilibrium between two major sugar pucker forms, N-type and S-type, but bridged nucleosides can be locked into one of these conformations depending on their specific structure. While many groups have researched these bridged nucleosides for the purpose of determining their binding affinity for antisense applications, we opted to look into the potential for biological activity within these conformationally-locked structures. A small library of 2',4'- and 3',4'-bridged nucleoside analogues was synthesized, including a novel 3',4'-carbocyclic bridged system. The synthesized compounds were tested for antibacterial, antitumor, and antiviral activities, leading to the identification of nucleosides possessing such biological activities. To the best of our knowledge, these biologically active compounds represent the first example of 2',4'-bridged nucleosides to demonstrate such properties. The most potent compound, nucleoside 33, exhibited significant antiviral activity against pseudoviruses SF162 (IC(50)=7.0 μM) and HxB2 (IC(50)=2.4 μM). These findings render bridged nucleosides as credible leads for drug discovery in the anti-HIV area of research.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.     

SYNTHESIS OF 5-(2,2′-BIPYRIDINYL AND 2,2′-BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: PRECURSORS TO METALLO-DNA CONJUGATES

ABSTRACT

The synthesis of 2,2′-bipyridinyl-2′-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2′-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2′-deoxyuridine nucleoside (Bipy²⁺-dU) with an ethylenyl linker. The Bipy²⁺-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

Nucleolar necklaces in chick embryo fibroblast cells. I. Formation of necklaces by dichlororibobenzimidazole and other adenosine analogues that decrease RNA synthesis and degrade preribosomes

A number of chemicals, mostly adenosine analogues, cause the nucleolus of the chick embryo fibroblast to lose material and unravel over a period of several hours into beaded strands termed nucleolar necklaces (NN). The results of analyses of the fibroblasts, treated with the NN-forming chemical dichlororibobenzimidazole (DRB), suggests that the following biochemical alterations occur: DRB almost completely prevents the increase in both messenger RNA (mRNA) and heterogeneous nuclear RNA. It interferes with ribosome synthesis by decreasing the rate of 45S ribosomal RNA (rRNA) accumulation by 50%, slowing the rate of 18S rRNA appearance by 50%, and causing an extensive degradation (80%) of the 32S and 28S rRNA-containing preribisomes. Most of this preribosome degration probably occurs at or before the 32S rRNA preribosome stage. The degradation of these preribosomes appears to be due to the formation of defective 45S rRNA preribosomes rather than to a direct DRB interference with preribosome processing enzyme action. DRB inhibits total cellular RNA synthesis in less than 15 min, suggesting a direct interference with RNA synthesis. DRB also inhibits the uptake of nucleosides into the cell. DRB in the concentrations used does not appear to directly interfere with the translation of mRNA (i.e., protein synthesis). Other NN-forming adenoside analogues and high concentrations of adenosine (2 mM) cause biochemical alterations similar to those produced by DRB. To explain the preribosome degradation, we propose the hypothesis that DRB inhibits the synthesis of mRNA; as a consequence, some of the preribosomal proteins that normally coat the 32S rRNA portion of the 45S precursor RNA become limiting, and this defective portion is then subject to degradation by nucleases.     

The synthesis of oligoribonucleotides containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines via post-synthetic modification of precursor oligomers

The N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines are the most common modified adenosine nucleosides and transfer ribonucleic acids (tRNA) are particularly rich in these modified nucleosides. They are present at position 37 of the anticodon arm and the contribution of these hypermodified nucleosides to codon–anticodon interactions, as well as translation, are significant, although not fully understood. Herein we described a new chemical synthesis method of the oligoribonucleotides containing N⁶-alkyladenosines and 2-methylthio-N⁶-alkyladenosines via post-synthetic modifications of precursor oligoribonucleotides. To obtain oligoribonucleotides containing N⁶-alkyladenosines, the precursor oligoribonucleotide carrying 6-methylthiopurine riboside residue was used, whereas for the synthesis of oligoribonucleotides containing 2-methylthio-N⁶-alkyladenosines the precursor oligoribonucleotide carrying the 2-methylthio-6-chloropurine riboside was applied. Among the modified oligoribonucleotides of different length and secondary structures, there were several containing naturally occurring modified nucleosides such as: N⁶-isopentenyladenosine (i⁶A), N⁶-methyladenosine (m⁶A), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A), and 2-methylthio-N⁶-methyladenosine (ms²m⁶A), as well as several unnaturally modified adenosine derivatives.