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.     

Adenylyl-(5′, 2′)-adenosine 5′-Phosphate ((2′-5′) pA-A) in Crude Crystals of 5′-Adenylic Acid Isolated from Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Adenylyl (5′,2′)-adenosine 5′-phosphate ((2′-5′)pA-A) was detected in crude crystals of 5′-AMP prepared from Penicillium nuclease (nuclease P₁) digest of a technical grade yeast RNA. While (3′–5′)A-A was split by nuclease P₁, spleen phosphodiesterase, snake venom phosphodiesterase or alkali, (2′–5′)A-A was not split by a usual level of nuclease P₁ or spleen phosphodiesterase. Nuclease P₁ digests of 12 preparations of technical grade yeast RNA tested were confirmed to contain (2′–5′)pA-A. Its content was about 1 to 2% of the AMP component of each RNA preparation. As poly(A) was degraded completely by the Penicillium enzyme into 5′-AMP without formation of any appreciable amount of (2′–5′)pA-A, the technical grade RNA is supposed to contain 2–5′ phosphodiester linkages in addition to 3′–5′ major linkages.     

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.     

Effects of 3′-C-Methylation on the Hydrolytic Stability and Hydroxyl pK a Values of Dinucleoside 2′,5′- and 3′,5′-Monophosphates

Abstract

The first-order rate constants for hydrolysis of 3′-C-methyluridylyl(2′,5′)- and -(3′,5′)adenosine and the corresponding native dinucleoside monophosphates (2′,5′- and 3′,5′-UpA) have been determined as a function of hydroxide-ion concentration (0.025 - 7 M) at 25°C. In addition to the effects on the hydrolytic stability of the compounds, the effects of the 3′-C-methyl substitution on the kinetically determined pK _a values for the sugar hydroxyls of the undine moiety are discussed.     

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.     

A 2′←5′-Adenylate Trimer With a Positively Charged 2′(3′)-Terminal 8-(4-Aminobunl)Aminoadenosine Residue: Synthesis,Conformation and RNase L Binding

Abstract

An analogue of the 2-5A core trimer containing an 8-(4-aminobutyl)-aminoadenosine (1; A) residue at the 2′(3′)-terminus [2; (2′,5′)A₂A^?] was synthesized. The conformation of (2′,5′)A₂A^? was studied by ₁H, ¹³C-NMR, and CD spectroscopy. The (2′,5′)A₂A^? exhibits very low binding ability to the RNase L of mouse L cells, but slightly enhanced resistance to digestion by SVPD compared to the parent trimer.     

Nucleosides and Nucleotides. 104. Radical and Palladium-Catalyzed Deoxygenation of the Allylic Alcohol Systems in the Sugar Moiety of Pyrimidine Nucleosides§,1

Abstract

New methods for the synthesis of 2′,3′-didehydro-2′,3′-dideoxy-2′ (and 3′)-methyl-5-methyluridines and 2′,3′-dideoxy-2′ (and 3′)-methylidene pyrimidine nucleosides have been developed from the corresponding 2′ (and 3′)-deoxy-2′ (and 3′)-methylidene pyrimidine nucleosides. Treatment of a 3′-deoxy-3′-methylidene-5-methyluridine derivative 8 with 1,1′-thiocarbonyldiimidazole gave the allylic rearranged 2′,3′-didehydro-2′,3′-dideoxy-3′-[(imidazol-1-yl)carbonylthiomethyl] derivative 24. On the other hand, reaction of 8 with methyloxalyl chloride afforded 2′-O-methyloxalyl ester 25. Radical deoxygenation of both 24 and 25 gave 26 exclusively. Palladium-catalyzed reduction of 2′,5′-di-O-acetyl-3′-deoxy-3′-methylidene-5-methyluridine (32) with triethylammonium formate as a hydride donor regioselectively afforded the 2′,3′-dideoxy-3′-methylidene derivative 35 and 2′,3′-didehydro-2′,3′-dideoxy-3′-methyl derivative 34 in a ratio of 95:5 in 78% yield. These reactions were used on the corresponding 2′-deoxy-2′-methylidene derivatives. An alternative synthesis of 2′,3′-dideoxy-2′-methylidene pyrimidine nucleosides (43, 52, and 54) was achieved from the corresponding 1-(3-deoxy-β-D-thero-pentofuranosyl)pyrimidines (44 and 45). The cytotoxicity against L1210 and KB cells and inhibitory activity of the pathogenicity of HIV-1 are also described     

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%.     

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.     

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.     

Acceptor Specificity Observed with Crude Preparation of Nucleoside Phosphotransferases

The acceptor specificities of bacterial nucleoside phosphotransferase were further investigated by phosphorylating various kinds of nucleoside analogues. The bacteria belonging to A group(5′-nucleotide former) specifically phosphorylated the primary alcohol at 5′-position of nucleosides and their analogues, such as adenine xyloside, psicofuranine and pseudouridine, while the others belonging to B group (3′(2′)-nucleotide former) the secondary alcohol at 3′(2′)-position. The phosphorylation at 5′-primary alcohol with the bacteria belonging to A group, however, was prohibited mainly by phosphoryl-or amino-radical at 3′-position, as observed in the case of 3′-nucleotide or amino-nucleoside (or puromycin), depending on the steric conformation around the 3′-position of acceptor. Besides, both types of nucleoside phosphotransferases were also able to phosphorylate nucleoside having a C-C-linkage between base and sugar moieties.     

Studies on Synthesis and Structure of O-β-D-Ribofuranosyl(1″→2′)-ribonucleosides and Oligonucleotides♣

Abstract

Minor nucleosides found in several eukaryotic initiator tRNAs_i ^Met, O-β-D-ribofuranosyl(1″→2′)adenosine and -guanosine (Ar and Gr), as well as their pyrimidine analogues, were obtained from N-protected 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)ribonucleosides and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in the presence of tin tetrachloride in 1,2-dichloroethane. A crystal structure has been solved for 2′-O-ribosyluridine. The 3′-phosphoramidites of protected 2′-O-ribosylribonucleosides were prepared as the reagents for 2′-O-ribofuranosyloligonucleotides synthesis. O-β-D-Ribofuranosyl(1″→2′)adenylyl(3′→5′)guanosine (ArpG) was obtained and its structure was analysed by NMR spectroscopy.

     

THE NMR CONFORMATION STUDY OF THE COMPLEXES OF DEOXYCYTIDINE KINASE (dCK) AND 2′-DEOXYCYTIDINE/2′-DEOXYADENOSINE

The structures of the bound ¹³C/²H double-labelled 2′(R/S), 5′(R/S)-²H₂-1′,2′,3′,4′,5′-¹³C₅-2′-deoxyadenosine and the corresponding 2′-deoxycytidine moieties in the complexes with human deoxycytidine kinase (dCK) have been characterized for the first time by the solution NMR spectroscopy, using Transferred Dipole-Dipole Cross-correlated Relaxation and Transferred nOe experiments. It has been shown that the ligand adopts a South-type sugar conformation when bound to dCK.     

Mitsunobu Reactions of 5‐Fluorouridine with the Terpenols Phytol and Nerol: DNA Building Blocks for a Biomimetic Lipophilization of Nucleic Acids

The cancerostatic 5‐fluorouridine (5‐FUrd; 1 ) was sequentially sugar‐protected by introduction of a 2′,3′‐O‐heptylidene ketal group (→ 2 ), followed by 5′‐O‐monomethoxytritylation (→ 3 ). This fully protected derivative was submitted to Mitsunobu reactions with either phytol ((Z and E)‐isomer) or nerol ((Z)‐isomer) to yield the nucleoterpenes 4a and 4b . Both were 5′‐O‐deprotected with 2% Cl₂CHCOOH in CH₂Cl₂ to yield compounds 5a and 5b , respectively. These were converted to the 5′‐O‐cyanoethyl phosphoramidites 6a and 6b , respectively. Moreover, the 2′,3′‐O‐(1‐nonyldecylidene) derivative, 7a , of 5‐fluorouridine was resynthesized and labelled at C(5′) with an Eterneon‐480 fluorophor^® (→ 7b ). The resulting nucleolipid was studied with respect to its incorporation in an artificial bilayer, as well as to its aggregate formation. Additionally, two oligonucleotides carrying terminal phytol‐alkylated 5‐fluorouridine tags were prepared, one of which was studied concerning its incorporation in an artificial lipid bilayer.     

SYNTHESIS of 2′-DEOXY-2′-FLUOROGUANYL-(3′,5′)-GUANOSINE

ABSTRACT

The protected analogue of 2-amnio-6-chloropurine arabinoside (3b) was subjected to reaction with diethylaminosulfur trifluoride (DAST) and subsequently treated with NaOAc in Ac₂O/AcOH to give N ²,O ^3′,O ^5′-triacetyl-2′-deoxy-2′-fluoroguanosine (5a). After deacetylation of the sugar moiety and protection of 5′-OH by a 4,4′-dimethoxytrityl group, this nucleoside component was converted to 2′-deoxy-2′-fluoroguanyl-(3′,5′)-guanosine (6c, GfpG).     

Crystal Structure and Molecular Conformation of 4-Thiouracil-2′-Trifluorothioacetamide -3′, 5′-Diacetyl-β-D-Riboside

Abstract

4-thiouracil-2′-trifluorothioacetamide-3′, 5′-diacetyl-β-D-riboside is one of the modified thiouracil analogs synthesized in our institute. The determination of the crystal and molecular structure of this compound was carried out with a view to study the conformation of the molecule in the solid state as well as to investigate the conformations of the trifluoroacetamide and the acetyl substituents of the ribose and their effects on the conformation of the ribose ring. Crystals of 4-thiouracil-2′-trifluorothioacetamide-3′,5′- diacetyl-β-D-riboside are orthorhombic, space group P2₁ 2₁ 2₁, with cell dimensions a= 15.351 (2), b= 15.535 (1), c= 8.307 (1) Å, V=1981.0 (7) ų, Z=4, D_m= 1.53, D_c=1.527 g/c.c. and μ=30.1cm -¹. The structure was determined using CuKα (λ, =1.5418 Å) at a temperature T of 297K, with 2333 reflections, which were collected on a Enraf-Nonius CAD-4 diffactometer, out of which 2249 (I ≥20) were considered observed. The structure was determined by direct methods using MULTAN and refined by full matrix least squares method to a final reliability factor of 0.054 and a weighted R factor of 0.079. The nucleoside is in the anti conformation [X_CN =51.4 (5)°], the ribose has the unusual C (2′) endo -C (1′) exo (²T₁), and a g+ conformation [ψ=47.5 (4)] across C(4′)-C(5′) bond. The pseudorotation angle P is 152.8 (4) ° and the amplitude of pucker τ_m of 42.7 (3)°. The average C-F bond distance is 1.308 Å. There is no base pairing and the typical base-base hydrogen bonded interactions are not present in this structure. On the other hand, a hydrogen bonded dimer is formed involving C(3′) - H(3′)… O (2) and N(3) -H (N3) … O (Al) hydrogen bonds joining the base, ribose ring and the acetyl group. The trend towards longer exocyclic bonds at the acetyl centers in compounds with strongly electronegative aglycones, is also exhibited in this compound, with C(3′)-O(3′) and C(5′)-0(5′) being much longer than C(1′)-O(4′). The acetyl groups also take part in C-H…O hydrogen bonding with the acetyl oxygen atom OA2.     

Phenolic lipids from related marine algae of the order dictyotales

2-(1′-Oxo-dodeca-5′, 8′, 11′, 14′, 17′(all Z)-pentaenyl)-5-methoxy-1, 3-dihydroxybenzene, 2- (1′-oxo-dodeca-5′, 8′, 11′, 14′, 17′(all Z)-pentaenyl)-1, 3, 5-trihydroxybenzene, 2-(17′-hydroxy-1′-oxo-dodeca-5′, 8′, 11′, 14′(all Z)-tetraenyl)-1, 3, 5-trihydroxybenzene and 2-(1′oxo-hexadecyl)-1, 3, 5-trihydroxybenzene have been isolated from the related brown algae Zonaria farlowii, Z diesingiana and Lobophora papenfussii. The structures of these new metabolites are based on extensive spectral analyses and comparisons with model compounds. The isolation of (+)-7, 8-dimethyltocol, from L. papenfussii, is also reported.     

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.     

Synthetic Nucleosides and Nucleotides. 40. Selective Inhibition of Eukaryotic DNA Polymerase α by 9-(β-D-Arabinofuranosyl)-2-(p-n-butylanilino)adenine 5′-Triphosphate (BuAaraATP) and Its 2′-Up Azido Analog: Synthesis and Enzymatic Evaluations†

Abstract

Starting from 2′,3′,5′-tri-O-acetyl-2-iodoadenosine, 9-(β-D-arabinofuranosyl)-2-(p-n-butylanilino)adenine and its 2′(S)-azido counterparts were synthesized in seven steps. These exhibited only moderate growth-inhibitory effects against mouse leukemic P388 cells (IC₅₀ = 13–24 μM), although 5′-triphosphate derivatives showed strong and selective inhibitory action on calf thymus DNA polymerase α, but not on β- and ?-polymerases from eukaryotes.

     

Conformational study of ribonucleotides, 2′-deoxyribonucleotides,and arabinonucleotides by carbon-13 nuclear magnetic resonance

The geminal and vicinal ¹³C-³¹P coupling constants have been monitored, as a function of pH, for a series of uracil and cytosine 3′- and 5′-nucleotides with a ribose, arabinose, or 2′-deoxyribose sugar. Data were also obtained for two 3′,5′-diphosphates in the ribose and arabinose series. The geminal J(C5′-P5′) and J(C3′-P3′) couplings show only a small dependence on the ionization state of the phosphate, decreasing by < 0.5 Hz in the pH 5–7 range. For the ribose and arabinose 3′-nucleotides, the vicinal J(C4′-P3′) increase (up to 1.5 Hz) on secondary phosphate ionization in the pH 5–7 range, whereas their J(C2′-P3′) couplings decrease (up to 1.5 Hz) over the same pH range. In contrast for the 2′-deoxyribose molecules, both couplings decrease (～0.5 Hz) on phosphate ionization. The titration curves provide information about the influence of the sugar on the conformation about the C3′? O3′ bond. Some conformational trends could be rationalized by consideration of the sugar-puckerdependent contact interactions between the 3′-phosphate and the substituents on the furanose ring.