Bacterial Synthesis of Nucleotides

Inosine-5′, 2′(or 3′)-diphosphate was prepared by incubating 5′-IMP and p-nitrophenyl-phosphate with the bacteria characterized to phosphorylate at C_{3′ (&2′)}, or, on the contrary, by incubating 2′-IMP and a donor with the others capable of synthesizing 5′-nucleotide, via their phosphoryl transfer reactions.

Formation of the 5′, 2′(or 3′)-diphosphates of guanosine, cytidine, and uridine was also demonstrated to be carried out under the same relationship between nucleotide isomer as an acceptor and specificities of bacterial phosphotransferases, as observed in the phosphorylation of adenylic acid isomers, while 5′-dTMP was phosphorylated by both groups of bacteria.     

Bacterial Synthesis of Nucleotides

General properties of bacterial nucleoside phosphotransferase were demonstrated. Nucleoside phosphotransferase activity was observed somewhere in cells, and the activity and the specificity for donor and product in this reaction are described to be due to the basic character of strains. Such aromatic phosphates as p-nitrophenylphosphate, phenylphosphate, benzylphosphate and the nucleotides were apparent to be useful for nucleotide synthesis, and the ability as donor did not always depend upon the energy consideration. The product specificity of this reaction was confirmed to correlate with nucleotide isomer added as donor; that is, the bacteria characterized to phosphorylate at 5′-position of nucleoside catalyzed the interconversion of phosphoryl or phosphate radical between 5′-nuclotides and those characterized to do at 3′(& 2′)-position of nucleoside catalyzed the interconversion of that between 3′(& 2′)-nucleotides. The phosphoryl or phosphate transfer reaction using nucleotide as donor is reversible but that using p-nitrophenylphosphate as donor is irreversible. The factors to get a good yield on the synthesis of 5′-inosinic acid were discussed, then the maximum yield was accounted to 80%.     

Bacterial Synthesis of Nucleotides

5′-Phosphoribosyl 5-amino-4-imidazole carboxamide was prepared by incubating 5-amino-4-imidazole carboxamide riboside and a phosphate compound with the bacteria characterized to phosphorylate at C_5′ via the phosphoryl transfer reaction. Aromatic phosphate compounds and 5′-nucleotides were able to act as the phosphate donor. This material was isolated chromatographically and its properties were studied. The other bacteria characterized to phosphorylate at C_{3′ (or 2′)} also phosphorylated a little probably at C_{3′ (or 2′)} of 5-amino-4-imidazole carboxamide riboside.

The phosphoryl interconversion between nucleotides and nucleosides was studied to be carried out via the phosphoryl transfer reaction observed in bacteria. The phosphotransferase activity of Ps. trifolii mediated reversibly the phosphoryl transfer between 5′-nucleotides and nucleosides, and its optimal pH was at around 8.5, whereas that of Prot. mirabilis did transfer the phosphoryl radical from 2′- and 3′-nucleotide to nucleoside at its optimal pH, around 5.0.

These donor- and product-isomer specificities of both bacteria were evident to be invariable, regardless of reaction pH and cultural conditions. These reactions, especially using the bacteria characterized to phosphorylate at C_5′ of nucleoside, were demonstrated to catalyze the phosphoryl interconversion between 5′-purine nucleotides and pyrimidine nucleosides or vice versa.     

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.     

Novel Acyclic Derivatives of Aica-Riboside (1-(β-D-Ribopuranosyl)5-Amino-4-Imidazolecarboxamide), Potential Antiviral Agents

Abstract

A synthetic approach is described to obtain from AICA-riboside acyclic analogues of 2′-deoxyribosides in which the C(2′)-C(3′) bond is cleaved and the natural configuration (E) at the anomeric C (1′)-position is retained.     

Synthesis and Potent Anti-HCMV Activity of 2′,5′,5′-trifluoro-3′-hydroxy-apiosyl Nucleoside Phosphonic Acid Analogues

As antiviral nucleosides containing a fluorine atom at 2′-position are endowed with increased stabilization of glycosyl bond, it was of interest to investigate the influence of three fluorine atoms at 2′- and 5′-positions of apiosyl nucleoside phosphonate analogues. Various pyrimidine and purine 2′,5′,5′-trifluoro-3′-hydroxy-apiose nucleoside phosphonic acid analogues were synthesized from 1,3-dihydroxyacetone. Electrophilic fluorination of lactone was performed using N-fluorodibenzenesulfonimide. Difluorophosphonation was performed by direct displacement of triflate intermediate with diethyl(lithiodifluoromethyl) phosphonate to give the corresponding (α,α-difluoroalkyl) phosphonate. Condensation successfully proceeded from a glycosyl donor with persilylated bases to yield nucleoside phosphonate analogues. Deprotection of diethyl phosphonates provided the final phosphonic acid sodium salts. The synthesized nucleoside analogues were subjected to antiviral screening against various viruses.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

2′(3′)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di-O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)-O-(α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[¹⁴C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10^?5 M puromycin) was achieved at 9.5·10^?6 M of compound 1c and 9·10^?5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[¹⁴C]Phe to the A site of peptidyltransferase in a system containing tRNA^Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.     

Preparation and stability of the helical form of poly 2'-O-ethyluridylic acid

2′ (3′)-O-ethyl-CMP was prepared by alkylation of CMP with diethylsulphate in alkaline medium and deaminated to give 2′(3′)-O-ethyl-UMP, which was phosphorylated to 2′(3′)-O-ethyl-UDP. About 90% of the product consisted of the 2′ isomer. The 2′(3′)-O-ethyl-UDP was readily polymerized by $\text{E}$. $\text{coli}$ polynucleotide phosphorylase in the presence of Mn⁺⁺, but not Mg⁺⁺. The 3′-isomer did not seriously interfere with polymerization nor did it act as a chain terminator. The resulting poly 2′-O-ethyluridylic acid formed a helical structure with a stability much higher then that of poly (rU) or poly 2′-O-methyluridylic acid. It also complexed readily with poly (rA). Implications with regard to the role of the 2′-hydroxyl in nucleic acid conformation are discussed.     

Bacterial Synthesis of Nucleotides

The synthesis of inosinic acid from inosine and p-nitrophenylphosphate by the partially purified enzyme, nucleoside phosphotransferase, prepared from Escherichia coli (B-25) is described.

The results presented in this paper represent that the nucleotide, inosinic acid, synthesized by the nucleoside phosphotransferase of E. coli, used as an example of bacterial enzymes, is not always 5′-isomer and that most of inosinic acid synthesized are 3′(& 2′)-isomer, together with a small amount of 5′-isomer. It was pointed out that cupric ion accelerated both the synthesis of inosinic acid and the liberation of p-nitrophenol, and that the nucleoside phosphotransferase and the phosphatase may be different from each other.     

Synthesis of Novel 2′,3′-Difluorinated 5′-Deoxythreosyl Phosphonic Acid Nucleosides as Antiviral Agents

A novel route for the synthesis of 2′,3′-difluorinated 5′-deoxythreosyl phosphonic acid nucleosides from glyceraldehyde using the Horner-Emmons reaction in the presence of triethyl α-fluorophosphonoacetate is described. The second fluorination at the 2′-position was an electrophilic reaction performed using N-fluorodibenzenesulfonimide. Glycosylation reactions between the nucleosidic bases and glycosyl donor 9 generated nucleosides that were further phosphonated and hydrolyzed to produce the desired nucleoside analogues. The synthesized nucleoside analogues 13, 16, 20, and 23 were tested for anti- human immunodeficiency virus (HIV) activity as well as cytotoxicity. Adenine derivative 16 showed significant anti-HIV activity up to 100 μM.     

Insight into dihalogenation of E-ring of podophyllotoxins,and their acyloxyation derivatives at the C4 position as insecticidal agents

Unexpected sequential E-ring dihalogenation of podophyllotoxin analogues is reported. It demonstrated that a chlorine/bromine atom was prior introduced at the C2′ position of podophyllotoxin, and the corresponding free rotation of E-ring around the C1–C1′ bond of 2′-chloro or 2′-bromopodophyllotoxin was restricted. When 2′-chloro or 2′-bromopodophyllotoxin reacted with N-chlorosuccinimide (NCS), the chlorine atom was regioselectively introduced at their C6′ position on the E-ring. Whereas 2′-chloro or 2′-bromopodophyllotoxin reacted with NBS, the bromine atom was regioselectively introduced at their C5 position on the B-ring. When 2′-chloropodophyllotoxin reacted with different carboxylic acids in the presence of BF₃·Et₂O, the steric effect of its E-ring for stereoselective synthesis of 4β-acyloxy-2′-chloropodophyllotoxin derivatives was observed. The insecticidal activity of 2′(2′,6′)-(di)halogen-substituted podophyllotoxin derivatives were evaluated with Mythimna separata Walker.     

The First Stereocontrolled Synthesis of Thiooligoribonucleotide: (RpRp)- and (SpSp)-UpsUpsU

Abstract

An approach to the stereocontrolled synthesis of P-homochiral thiooligoribonucleotide: (Rp,Rp)- and (Sp,Sp)-diastereomers of uridinylyl′(3′, 5′)uridinylyl(3′,5′)uridine di (0,0-phosphorothioate) (9) is decribed. The influence of 2′-protection on the efficiency and stereochemistry of the coupling reaction is discussed.     

Lithiation-Stannylation Chemistry of Nucleosides

Abstract

Two examples of anionic stannyl migration practically useful for nucleoside synthesis are presented. One involves the migration from the 8- to 2-position of 6-chloropurine derivatives, which provided a new entry to 2-substituted purine nucleosides. The other is that from the 6- to 2′-position of 1′,2′-unsaturated uridine. The latter enabled the preparation of a hitheroto unknown class of nucleoside analogues, 2′-substituted 1′,2′-unsaturated uridines.     

2-Bromoadenosine-Substituted 2′,5′-Oligoadenylates Modulate Binding and Activation Abilities of Human Recombinant RNase L

Abstract

2-Bromoadenosine-substituted analogues of 2–5A, p5′A2′p-5′A2′p5′(br²A), p5′(br²A)2′p5′A2′p5′A, and p5′(br²A)2′p5′(br²A)2′p-S′(br²A), were prepared via a modification of a lead ion-catalyzed ligation reaction and were subsequently converted into the corresponding 5′-triphosphates. Both binding and activation of human recombinant RNase L by various 2-bromoadenosine-substituted 2–5A analogues were examined. Among the 2-bromoadenosine-substituted 2–5A analogues, the analogue with 2-bromoadenosine residing in the 2′-terminal position, p5′A2′p5′A2′p-5′(br²A), showed the strongest binding affinity and was as effective as 2–5A itself as an activator of RNase L. The CD spectrum of p5′A2′p-5′A2′p5′(br²A) was superimposable on that of p5′A2′p5′A2′p5′A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2–5A.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

A Universal Glass Support for Oligonucleotide Synthesis

Abstract

A single type of controlled pore glass derivatized with 3-anisoyl-2′(3′)-O-benzoyluridine 5′-O-succinyl residues can be used as the support in solid phase syntheses of either oligoribo- or oligodeoxyribonucleotides.     

Some Reactions of 4′-Thionucleosides and Their Sulfones

Abstract

We report interesting and novel reactions of 4′-thionucleosides and their sulfone derivatives when a good leaving group is present in the 5′-position. The results have important implications for the phosphorylation of these nucleoside analogues by standard chemical procedures. Possible mechanisms for these reactions are discussed.

     

2′(3′)-O-l-phenylalanyl derivatives of N2,5′-anhydroformycin and N4,5′-anhydroformycin: new substrates for ribosomal peptidyltransferase with a fixed anti and syn conformation of the base

The $\text{2′(3′)-O-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^2\text{,5′-anhydroformycin}$ (1c) and $\text{2′(3′)-O-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^4\text{,5′-anhydroformycin}$ (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 · 10^?4 M determined by the release of N-Ac-Phe residue from the 70 S $\text{ribosome}\text{-}\text{poly}\text{(}\text{U}\text{)-N-}\text{Ac}\text{-[}{}^{14}\text{C]}\text{Phe-tRNA}$ complex. The reaction product, $\text{2′(3′)-O-(N-}\text{acetyl}\text{)-}\text{l}\text{-}\text{phenylalanyl}\text{-}\text{l}\text{-}\text{phenylalanyl}\text{-N}{}^2\text{,5′-}\text{anhydroformycin}$ (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 · 10^∮4 M and essentially none in the concentration region between 1 · 10^?6 and 1 · 10^?4 M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart, Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates.     

The enzymatic synthesis of ATP analogues.

The enzymatic synthesis of selected adenosine 5′-triphosphate analogues from their respective 5′-monosphosphates has been achieved using phosphoenolpyruvate synthetase. Adenosine 5′-monophosphate analogues altered at positions 1,6,7,8 or 9 of the purine ring, or at the ribose 2′- or 3′-positions are substrates with 30% conversion to the nucleoside 5′-triphosphate.