Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.     

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease.     

Activation of the ppp(A2'p)nA system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(A2'p)nA-dependent RNase

High doses (100-1000 reference units/ml) of alpha or beta interferons are required to inhibit the growth of herpes simplex virus types I and II (HSV-I and HSV-II) in human Chang cells. In contrast, much lower doses (10-100 reference units/ml) of interferon inhibit replication of encephalomyocarditis virus (EMCV) in these cells. In the HSV-infected cells these high doses did not prevent the virus-induced shut off of host protein synthesis. The interferons were more effective in reducing the virus yield of HSV-I than of HSV-II. At the above concentrations they inhibited HSV-I protein synthesis but had little apparent effect on that of HSV-II. Similar amounts of (2'-5')oligo(adenylate)s were synthesised in response to HSV-I, HSV-II and EMCV infection of Chang cells after treatment with alpha or beta interferons. No (i.e. less than 1 nM) (2'-5')oligo(adenylate)s were found in control cells or on virus infection alone. Only low levels of ppp(A2'p)nA-specific rRNA cleavage were observed in the interferon-treated HSV-infected cells. In contrast, high levels were found in response to EMCV, despite the fact that ppp(A2'p)nA accumulated to similar levels with each of the three viruses in these cells. High-performance liquid chromatographic analysis of material from interferon-treated Chang cells 18 h after infection with HSV-I or HSV-II, combined with radiobinding, radioimmune and rRNA cleavage assays, confirmed the presence of ppp(A2'p)2A and ppp(A2'p)3A at greater than nanomolar concentration. In addition, apparently equivalent amounts of two other putative (2'-5')oligo(adenylate) derivatives which compete in the radiobinding and radioimmune assays, were present. These compounds were only weak activators of the ppp(A2'p)nA-dependent RNase and under appropriate conditions were capable of inhibiting the activation of this RNase by authentic ppp(A2'p)nA. The presence of these potentially inhibitory compounds provides a possible explanation for the relatively low levels of activation of the ppp(A2'p)nA-dependent RNase in interferon-treated, HSV-infected Chang cells.     

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of ribonuclease L by (2'-5')(A)4-poly(L-lysine) conjugates in intact cells

Molecular hybrids were synthesized by coupling (2'-5')(A)n oligoadenylates or 2-5A, an intracellular mediator involved in antiviral activity of interferons (IFNs), with poly(L-lysine) used as a membrane carrier. (2'-5')(A)n in its free form was not taken up by cells, probably because of its ionic character. Conjugation with the polypeptide carrier overcame this problem and enabled its pharmacological properties to be developed. The alpha-glycol group of individual (2'-5')(A)n oligomers was oxidized by periodate oxidation and conjugated by an amino reductive reaction to poly(L-lysine), Mr 14 000, in a molar ratio of 5:1. These hybrid molecules left the biologically active 5' end moiety of the (2'-5')(A)n molecule unchanged, and in particular its triphosphate group, and stabilized the molecule by increasing its resistance to phosphodiesterase hydrolysis. A dose-dependent inhibition of virus growth was observed on concomitant incubation of (2'-5')(A)n-poly(L-lysine) conjugates with vesicular stomatitis virus infected L1210 cell cultures. This was a result of the activation of the (2'-5')(A)n-dependent endoribonuclease (RNase L) by intracellularly delivered (2'-5')(A)n as in some IFN-treated virus-infected cells. Indeed, (2'-5')(A)n-poly(L-lysine) conjugates bind RNase L effectively as can be seen from their ability to compete with authentic (2'-5')(A)n in a cell-free radiobinding assay. Moreover, (2'-5')(A)n-poly(L-lysine) conjugates promote transient inhibition of protein synthesis and a characteristic cleavage pattern of ribosomal RNAs in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-enzymatic, template-directed ligation of 2'-5' oligoribonucleotides. Joining of a template and a ligator strand.

Decauridylate containing exclusively a 2'-5' phospho-diester bond ([2'-5']U10) served as a template for the synthesis of oligoadenylates [oligo(A)s] from the 5'-phosphorimidazolide of 2'-5' diadenylate (ImpA-2'p5'A). Joining of [2'-5']U10and ImpA2'p5'A also took place in substantial amounts to yield long-chain oligoribonucleotides in the template-directed reaction. An unusual CD spectrum ascribed to helix formation between [2'-5']U10and [2'-5'](pA)2was observed under the same conditions as that of the template-directed reaction. The 3'-5' linked decauridylate ([3'-5']U10) also promoted the template-directed synthesis of oligo(A)s from ImpA2'p5'A, but more slowly compared with [2'-5']U10. The results indicate that short-chain RNA oligomers with a 2'-5' phosphodiester bond could lead to longer oligoribonucleotides by template-directed chain elongation.     

Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.     

Anti-mitogenic function of interferon-induced (2'-5')oligo(adenylate) and growth-related variations in enzymes that synthesize and degrade this oligonucleotide

Addition of (2'5')ApApA to concanavalin-A-stimulated mouse spleen lymphocytes strongly inhibits the large increase in RNA and protein synthesis which takes place 24-48 h after stimulation. The inhibitory effect on protein synthesis precedes the effect on RNA synthesis and takes at least 6 h to be detected. Histone synthesis is preferentially inhibited at 48 h. No effect on protein synthesis was detected in unstimulated resting lymphocytes, or in stimulated lymphocytes during the first 24 h after concanavalin A treatment. The anti-mitogenic effect of the (2'-5')oligo(adenylate) seems to result, therefore, from inhibition of protein synthesis taking place before initiation of DNA replication. The mitogenic stimulus produced by the lectin enhances, in lymphocytes, the level of the 2'-phosphodiesterase which degrades (2'-5')oligo(adenylate). Enhancement of the 2'-phosphodiesterase was also observed after serum stimulation of confluent monkey kidney cells. Furthermore, the ratio of (2'-5')oligo(adenylate) synthetase to 2'-phosphodiesterase is ten-times lower in fast-growing kidney cells than in quiescent serum-starved cells. A model for the role of (2'-5')oligo(adenylate) synthesis and degradation in the regulation of cell proliferation by interferon and by mitogens is presented.     

Lipid derivatives of (2'-5')oligo A

The synthesis of adenylyl-(2'-5')-adenylyl-(2'-5')-2', 3'-O-(1-methoxyhexadecylidene)-adenosine (III) and its 5'-phosphorylated analogue (V) is described. Phosphorylation was achieved by (2-cyanoethyl)-phosphodichloridite agent followed by iodine oxidation.     

Antiviral effects of 2',5'oligoadenylates (2-5As), and related compounds

Dephosphorylated "core" of 2',5'-oligoadenylate (2-5A) dimer (A2'p5'A), exogenously added to nonpermeabilized FL cells, inhibited the multiplication of Sindbis virus and vesicular stomatitis virus (VSV). The compound was shown to inhibit viral protein synthesis. The addition of A2'p5'A at the early stage of viral replication was more effective than that at the late stage. In contrast with the core, phosphorylated 2-5A (p5'A2'p5'A and ppp5'A2'p5'A) and 2-5A analogs containing cordycepin (3'-deoxyadenosine) did not show such antiviral effects. The rate of uptake of [3H]ppp5'A 2'p5'A into acid-soluble and acid-insoluble fractions, especially into the acid-insoluble fraction, was faster than that of [3H]A2'p5'A. These results suggest that the difference of antiviral activity between A2'p5'A and ppp5'A2'p5'A does not result from the different rate of uptake by cells, but from the different rate of from acid-soluble to acid-insoluble fractions.     

Antiviral activity in L1210 cells of liposome-encapsulated (2'-5')oligo(adenylate) analogues

Evidence is available for a role of a (2'-5')(A)n-activated endoribonuclease (RNase L) in the antiviral activity of interferon for several RNA viruses. (2'-5')(A)n and their analogues might thus provide an interesting alternative to exogenous interferons or their inducers in antiviral chemotherapy. In addition, the evaluation of the activity of (2'-5)(A)n as mediators of interferon's biological activities or as cell growth regulators requires biochemical studies using agonists or antagonists of the system. Non-disruptive techniques for the introduction of (2'-5')(A)n and their analogues into cell lines or tissues are required for these studies since these highly charged compounds are cell impermeable. (2'-5')(A)n oligomers and analogues of increased stability towards phosphodiesterases were derived by chemical modification of their 2' end and encapsulated in protein-A-bearing liposomes. The specific delivery of liposome contents into L1210 mouse leukemic cells was achieved with the help of monoclonal antibodies directed against the appropriate class I major histocompatibility complex-encoded proteins expressed by these cells. This intracellular delivery led to transient inhibition of protein synthesis and an antiviral activity, both compatible with activation of RNase L. This activity was enhanced for the analogues designed to resist degradation, with respect to the natural product.     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.     

Synthesis and properties of 5'-triphosphoryl 2'-5' oligoadenylates (2-5A), and a general method for synthesis of 3', 5'-bisphosphorylated oligonucleotides.

2'-5'-Linked oligoadenylic acid 5'-triphosphates (2-5A) having chain lengths of 2-4 have been synthesized by polymerization of 3'-O-(o-nitrobenzyl)-N-benzoyladenosine 5'-phosphate followed by 5'-triphosphorylation via the imidazolidates. A large scale preparation of 5'-O-phosphoryladenylyl-(2'-5')-adenylyl-(2'-5')-adenosine was performed by the phosphotriester method using 5'-O-monomethoxytrityl-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate and 5'-O-phosphorodianilido-3'-O-(o-nitrobenzyl)-N-benzoyladenosine 2'-O-p-chlorophenylphosphate as intermediates. The trimer was also triphosphorylated by the imidazolide method. CD spectra for 5'-mono and triphosphorylated 2'-5' adenylates were measured as well as their UV hypochromicities. This triester method was also applied to the synthesis of 3',5'-bisphosphorylated protected oligoadenylic acids with natural 3'-5' linkages which could be used for further condensations to yield 5'-phosphorylated polynucleotides.     

Detection of (2'-5')oligoadenylate binding proteins by nondenaturing polyacrylamide gel electrophoresis and affinity blotting onto nitrocellulose

A latent endoribonuclease, RNase L, binds to and is activated by (2'-5')oligoadenylates ((2'-5')(A)n, n = 2-15). Binding to a labeled derivative of (2'-5')(A)n, [32P](2'-5')(A)3pCp, is detected as a protein-ligand complex observed following nondenaturing polyacrylamide gel electrophoresis. One major binding complex and two minor binding complexes are readily seen in cytoplasmic extracts from Ehrlich ascites tumor cells, murine tissue extracts and rabbit liver tissue extracts. At least one of the more rapidly migrating complexes appears to be a proteolytic degradation product of the larger [32P](2'-5')(A)3pCp binding protein. Cell and tissue extracts containing [32P](2'-5')(A)3pCp binding activity can be immobilized onto nitrocellulose filters and [32P](2'-5')(A)3pCp binding activity detected using a simple, rapid, economical affinity blot assay. Detection of [32P](2'-5')(A)3pCp binding proteins following electrophoresis on nondenaturing polyacrylamide gels and the affinity blot assay significantly improve and simplify the analysis of (2'-5')(A)n binding proteins.     

The human 2'-5'oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2'-5' instead of 3'-5' phosphodiester bond formation

The demonstration by Kerr and colleagues that double-stranded (ds) RNA inhibits drastically protein synthesis in cell-free systems prepared from interferon-treated cells, suggested the existence of an interferon-induced enzyme, which is dependent on dsRNA. Consequently, two distinct dsRNA-dependent enzymes were discovered: a serine/threonine protein kinase that nowadays is referred to as PKR and a 2'-5'oligoadenylate synthetase (2'-5'OAS) that polymerizes ATP to 2'-5'-linked oligomers of adenosine with the general formula pppA(2'p5'A)(n), n>or=1. The product is pppG2'p5'G when GTP is used as a substrate. Three distinct forms of 2'-5'OAS exist in human cells, small, medium, and large, which contain one, two, and three OAS units, respectively, and are encoded by distinct genes clustered on the 2'-5'OAS locus on human chromosome 12. OASL is an OAS like IFN-induced protein encoded by a gene located about 8 Mb telomeric from the 2'-5'OAS locus. OASL is composed of one OAS unit fused at its C-terminus with two ubiquitin-like repeats. The human OASL is devoid of the typical 2'-5'OAS catalytic activity. In addition to these structural differences between the various OAS proteins, the three forms of 2'-5'OAS are characterized by different subcellular locations and enzymatic parameters. These findings illustrate the apparent structural and functional complexity of the human 2'-5'OAS family, and suggest that these proteins may have distinct roles in the cell.     

Interaction of 3'-fluoro-3'-deoxyanalogs of a trimer of (2'-5')oligoadenylic acid with (2'-5')A3-antibodies: stereochemical aspect

Mouse antibodies to (2'-5')oligoadenylates were obtained by the immunization of animals with the (2'-5')oligoadenylic acid trimer conjugated with bovine serum albumin through a 2',3'-levulinic acid residue. Using radioimmunoassay, the reactivity of mouse polyclonal antibodies to the (2'-5')oligoadenylic acid trimer was studied for the trimer analogues containing 9-(3-deoxy-3-fluro-beta-D- xylofuranosyl)adenine and 3'-deoxy-3'-fluoro-adenosine in various positions of the chain. It was found that (a) the three-dimensional structure of short oligonucleotides is an important factor in the antibody recognition; (b) antibodies are more sensitive to modifications of the 5'-terminal and central ribose fragments of the (2'-5')oligoadenylic acid trimer; (c) the 3'-hydroxyl group plays a secondary role in the formation of the antigen determinant.     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

3'-Fluoro-3'-deoxy analogs of 2-5A 5'-monophosphate: binding to 2-5A-dependent endoribonuclease and susceptibility to (2'-5')phosphodiesterase degradation

Analogs of 2-5A trimer 5'-monophosphate (2'-5')pA3,p5'A2'p5'A2'p5'A containing 9-(3-fluoro-3-deoxy-c-D-xylofuranosyl)adenine (AF) or 3'-fluoro-3'- deoxyadenosine (AF) at different positions of the chain have been synthesized. All of them were compared with (2'-5')pA3 and (2'-5')pA2 (3'dA) by (i) their ability to bind to 2-5A-dependent endoribonuclease(RNase L) of mouse L cells and of rabbit reticulocyte lysates and (ii) their susceptibility to the degradation by the (2'-5')phosphodiesterase activity. The results of this study suggest that the oligonucleotide conformation is important for its biochemical properties.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).