1-Deaza-3′-O-methyladenosine: A Nucleoside with the Syn-conformation in the Solid State and in Solution

Abstract

The 2′-O-methyl (2) and the 3′-O-methyl (3) derivatives of 1-deazaadenosine (1) were prepared. Single crystal X-ray analysis as well as ¹H and ¹³C NMR studies were performed on the 3′-O-methyl-1-deazaadenosine 3. In the solid state, the glycosyl torsion angle (χ = 64.7°) is in the syn-range which is caused by an intramolecular (5′)CH2OH…N(3) hydrogen bond. The ribofuranose moiety adopts a ² E (C-3′-exo; S) conformation and the orientation of the exocyclic C(4′)-C(5′) bond is + sc (γ^(+)g). The conformation in solution was found to be very similar to that in solid state. Whereas the 2′-O-methyl derivative of 1 is a strong inhibitor of adenosine deaminase the 3′-O-methyl derivative is neither inhibitor nor substrate.     

Configurational effects on conformational properties of cyclic nucleotides. I. Theoretical calculations of conformer preferences in α-nucleoside 3′,5′ cyclic monophosphates

A comparative study has been made of the configurational effects on the conformational properties of α- and β-anomers of purine and pyrimidine nucleoside 3′,5′,-cyclic monophosphates and their 2′-arabino epimers. Correlation between orientation of the base and the 2′-hydroxyl group have been studied theoretically using the PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The effect of change in ribose puckering on the base-hydroxyl interaction has also been studied. The result show that steric repulsions and stabilizing effects of intramolecular hydrogen bonding between the base and the 2′-hydroxyl (OH) group are of major importance in determining configurations of α-anomers and 2′-arabino-β-epimers. For example, hydrogen bonding between the 2′-hydroxyl group and polar centers on the base ring is clearly implicated as a determinant of syn-anti preferences of the purine (adenine) or pyrimidine (uracil) bases in α-nucleoside 3′,5′-cyclic monophosphates. Moreover, barrier heights for interconversion between conformers are sensitive to ribose pucker and 2′-OH orientations. The result clearly show that a change in ribose-ring pucker plays an essential role in relieving repulsive interaction between the base and the 2′-hydroxyl group. Thus a C2′-exo-C3′-endo (₂T³) pucker is favored for α-anomers in contrast with the C4′-exo-C3′-endo (₄T³) from found in β-compounds.     

18O Labeled Nucleosides. 2. A General Method for the Synthesis of Specifically Labeled Pyrimidine Ribonucleosides1

Abstract

A facile method for the synthesis of highly enriched ¹⁸O labeled pyrimidine ribonucleosides is described using uridine as a model compound. The isotopic label may be selectively incorporated into the base moiety at O² or into the ribose portion of the molecule at the 5′ position. In addition, both positions may be labeled and this is the first report of a method for labeling of both the base and sugar moieties of pyrimidine ribonucleosides. The site and level of isotope incorporation may be determined mass spectrometrically.     

Photo-Induced P-Methylbenzylation of 1,3-Dimethylthymine and 1, 3-Dimethyluracil in the Presence of Trifluoroacetic Acid

Abstract

Photolysis of a solution of a pyrimidine (i. e., 1, 3-dimethylthymine and 1, 3-dimethyluracil) in p-xylene in the presence of trifluoroacetic acid afforded mainly the 5, 6-dihydropyrimidine derivative together with the 5-p-methylbenzylated product and the 6-isomer as well. It is suggested that the first two products result from the C⁶-protonated pyrimidine electron adduct (III), while the 6-isomer is derived from the O⁴-protonated isomer (II).     

REACTION OF N3-BENZOYL-3′,5′-O-(DI-TERT-BUTYLSILANEDIYL)URIDINE WITH HINDERED ELECTROPHILES: INTERMOLECULAR N3 To 2′-O PROTECTING GROUP TRANSFER

ABSTRACT

The compound N³-benzoyl-3′,5′-O-(di-tert-butylsilanediyl)uridine 2 was alkylated with various alkyl iodides in CH₃CN in the presence of base. Normal 2′-O-alkylated products were obtained with methyl or benzyl iodide. if hindered alkyl iodides with β-branching such as 2-ethylbutyl iodide were used as electrophiles under the same conditions, N³-alkyl-2′-O-benzoyl uridine derivatives were produced. This unexpected transformation is usually dormant with reactive alkylating agents, but expressed with sterically hindered, less reactive electrophiles. This unwanted reaction gives isomeric products whose spectra differ in only subtle ways from target compounds.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Studies on Nucleosides: Part XXVIII1. Synthesis of 4-Amino (or Hydroxy)-6-Methylthio-1-(3′-Deoxy-β- D-ribofuranosyl)-1-H-pyrazolo[3, 4-d]Pyrimidines

Abstract

4-Amino-6-methylthio-1-(3′-deoxy-β-D-ribofuranosyl)-1H-pyrazolo-[3, 4-d]pyrimidine (11) and 6-methylthio-4(5H)-oxo-1-(3′-deoxy-β-D-ribofuranosyl)-1H-pyrazolo[3, 4-d]pyrimidine (12) have been synthesized from 1, 2-di-O-acetyl-5-O-benzoyl-3-deoxyribofuranose (5) and 4, 6-bis (methylthio)-1H-pyrazolo-[3, 4-d]pyrimidine (6). in a convergent fashion. Structural proofs are based on MS, IR, ¹H NMR, ¹³C NMR and elemental analyses.     

The Nature of Conformational Correlations in α- and β-Anomers of Nucleic Acid Components in Aqueous Solution by Nuclear Magnetic Resonance

Abstract

The solution distribution of combinations of the sugar ring puckering domains, C2′endo(S), C3′endo(N), and C4′-C5′ rotamers, +sc(g⁺), ap(t), -sc(g^?), in α and β-anomers in ribo- and deoxyribo- pyrimidine nucleic acid components can be determined from vicinal coupling constants (M. Remin, J. Biomol. Str. Dyn. 2, 211 (1984). A general correlation pattern with a conformational constant λ, reflecting an intrinsic physical property of the sugar - side chain ensemble, is developed and expressed in terms of four principles:

I) The +sc rotamer contributes to the C3′endo population to a higher extent (1 - Y_t) than to C2′endo,(l-Y_t-Y_g-/X_s).

II) The ap rotamer contributes to both C2′endo and C3′endo populations to the same extent (Y_t).

III) The—sc rotamer contributes only to the C2′endo population, (Y_g-/X_s).

IV) The molar fractions X_s, Y_t and Y_g- of conformations C2′endo, ap and—sc, respectively, are strongly correlated, λ = (Y_g-/X_s)/Y_t ≈ 0.5, and therefore Y_t is a basic variable parameter which determines all others in the correlation pattern.

In α-anomers, regardless of the type and conformation of the sugar ring and base, the molar fraction Y_t = 0.37 ± 0.02. This finding means that different α-anomers show one correlation pattern free of the influence of the base. In β-anomers, structure and conformation of the base are important factors which modulate (through Y_t) the correlation pattern, conserving its fundamental features. Y_t is considerably increased by a syn-oriented pyrimidine base, but decreases when the base is anti. The transition from anti to syn orientation of the base is followed by destabilization of (C2′endo, +sc) in favor of (C3′endo, ap). The principles of conformational correlations rationalize a variety of correlations observed in the past.     

Molecular structure of the cis-syn photodimer of d(TpT) (cyanoethyl ester)

The three-dimensional structure was determined by x-ray crystallography for d(T[p](CE)T), a uv photoproduct of the cyanoethyl (CE) derivative of d(TpT), having the cis-syn cyclobutane (CB) geometry and the S-configuration at the chiral phosphorus atom. The crystals of C₂₃H₃₀N₅O₁₂P · 2H₂O belong to the orthorhombic space group P2₁2₁2₁ (Z = 4), with cell dimensions a = 11.596 Å, b = 14.834 Å, and c = 15.946 Å, containing two water molecules per asymmetric unit. The CB ring is puckered with a dihedral angle of 151°. The two pyrimidine bases are rotated by –29° from the position of direct overlap of their corresponding atoms. This represents a major distortion of DNA, since in DNA adjacent thymines are rotated by +36°. The pyrimidine rings are puckered with Cremer–Pople parameters for T[p] and in parentheses [p]T: Q: 0.24 Å (0.31 Å); θ: 123° (120°); ?: 141° (86°). These represent half-chairs designated as ⁶H₁ (T[p]) and ₆H⁵ ([p]T). The CB and pyrimidine ring conformations are interrelated, and we postulate that they execute a coupled interconversion in solution. The T[p] segment has the syn glycosyl conformation, a ²T₃ sugar pucker, and gauche^? conformation at C4′-C5′; the [p]T segment is anti, ³T₄, trans. The C5′-O5′ torsion of the [p]T unit is –124.5°, and the C3′-O3′ torsion of the T[p] unit is –152.9°. Bond angles and bond lengths involving the phosphorus atom are similar to those of other phosphotriesters. The P-O3′ and P-05′ torsion angles are –138.1° and 58.6°, respectively. Several intermolecular (but no intramolecular) hydrogen bonds are found in the crystal.     

Photochemistry of the O-Nitrobenzyl Protecting Group in RNA Synthesis

Abstract

UV irradiation of 2′-O (o-nitrobenzyl)adenylyl(3′-5′)uridine in the presence of O₂ yields the corresponding nitrobenzoyl derivative in addition to the expected A-U. A mechanism proposed for this oxidation involves the successive removal of the two benzylic protons with a hydroperoxide as the intermediate between the two steps.     

Biotransformation of the antitumor intermediate 5-fluorouridine by recombinant Escherichia coli with high pyrimidine nucleoside phosporylase activity

Abstract

5-Fluorouridine (5-FUrd) is a precursor of the widely used antitumor drug doxifluridine. We have produced 5-FUrd by biotransformation by cloning the gene encoding pyrimidine nucleoside phosphorylase (PyNPase) from Enterobacter aero-genes CMCC (B) 45103 and expression in Escherichia coli BL21 (DE3), resulting in recombinant E. coli BL21 (DE3)/ pET28a-PyNPase. After medium optimization, the PyNPase activity in the fermentation broth was 1613 U mg^–1, which was 54-fold that of E. aerogenes. Under optimal conditions (cell concentration, 0.5 g L^–1; uridine, 10 mM; 5-fluorouracil, 45 mM; temperature, 50°C; pH, 7.8), more than 90% of uridine was converted to 5-FUrd, suggesting that this is a valuable tool for application in the preparation of antiviral and antitumor drugs.     

Pathways of Pyrimidine Salvage in <Emphasis Type="Italic">Pseudomonas</Emphasis> and Former <Emphasis Type="Italic">Pseudomonas</Emphasis>: Detection of Recycling Enzymes Using High-Performance Liquid Chromatography

Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H₂O → uracil + NH₃], cytidine deaminase [Cdd: cytidine + H₂O → uridine + NH₃], uridine phosphorylase [Udp: uridine + P_i ↔ uracil + ribose – 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H₂O → purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H₂O → uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 – PSG5 based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes.     

Determination of the activation energy for pseudorotation of the furanose ring in nucleosides by 13-C nuclear-magnetic-resonance relaxation.

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 degrees C and minus 60 degrees C. For the purine ribosides the average activation energy was found to be 4.7 plus or minus 0.5 kcal x mol-1 (20 plus or minus 2 kJ x mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13-C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.     

Efficient Synthesis of 2′-Bromo-2′-Deoxy-3′,5′-O-TPDS-pyrimidine Nucleosides by Boron Trifluoride Catalyzed Reaction of O2,2′-Anhydro-(1-β-D-Arabinofuran0Syl)pyrimidine Nucleosides with Lithium Bromide

Abstract

Efficient syntheses of 2′-bromo-2′-deoxy-3′,5′-O-TPDS-uridine (5a) and 1-(2-bromo-3,5-O-TPDS-β-D-ribofuranosyl)thymine (5b) from uridine and 1-(β-D-ribofuranosyl)thymine are described, respectively. The key step is a treatment of 3′,5′-O-TPDS-O²,2′-anhydro-1-(β-D-ardbinofuranosyl)uracil (4a) and -thymine (4b) with LiBr in the presence of BF₃-OEt₂ in 1,4-dioxane at 60°C to give 5a and 5b in 98%, and 96% yield, respectively.

     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.     

Anti-Syn Conformational Range of Pyrimidines with Deoxyribofuranose

Abstract

The ability of pyrimidine bases to adopt the syn conformation in DNA has been investigated. The distances between atoms on the sugar and base and the resulting steric energies have been calculated as a function of glycosidic torsion angle for the principal sugar puckers of the deoxyribose of cytosine. The results indicate that pyrimidines can assume both the anti and syn conformations for the ³E, ₄E, ₁E, ²E, ₃E sugar puckers and syn for the ₂E sugar pucker. For these sugar puckers the difference between the minimum energies of the anti and syn conformations is in the range of 0.1–2.0 kcal/mole, with the minimum syn energy being lower in the case of the ₄E, ₁E and ²E sugar puckers. It is particularly significant that cytosine can assume the syn conformation for the ³E sugar pucker commonly observed for the syn nucleotides in Z-DNA with both alternating pyrimidine/purine (APP) and non-APP sequences. The results of this investigation confirm that steric interactions resulting from putting a pyrimidine nucleotide in the syn conformation are not a major factor in the preference for APP base sequences in Z-DNA.     

SYNTHESIS OF A NEW 5′-O-TRIPHOSPHATE ANALOG OF 5-METHYL 2′-O-DEOXYCYTIDINE. PRELIMINARY IN VITRO LABELING FOR NON-RADIOACTIVE DETECTION OF DNA

We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2′-O-deoxycytidine 3A . We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-{n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl}-2′-deoxycytidine compounds, and confirmed their presence by ¹H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.     

Loop Stereochemistry and Dynamics in Transfer RNA

Abstract

The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology. Both loops are seven residues long and both present sharp turns after the second residue, U33 and ψ55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively. In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48. In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3′-end. At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule. The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet. Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed.     

Partial characterization of 5-fluoropyrimidine-resistant mutants of Neurospora crassa

Summary The primary lesion in a number of 5-fluoropyrimidine resistant mutants of Neurospora crassa has been identified. ud-1 mutants, previously designated fdu-2, are deficient in nucleoside uptake and show extensive intragenic complementation. uc-4 mutants lack uracil phosphoribosyl transferase with no complementation between 23 alleles. udk mutants lack uridine kinase activity. fdu-2 mutants affect the repression of the first two de novo pyrimidine biosynthetic enzymes, have no detectable uridine kinase activity and show decreased uridine uptake. Accordingly, fdu-2 may be involved in the regulation of pyrimidine uptake, salvage and de novo synthesis.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Synthesis and conformational analysis of 1,2-cis fused bicyclic α-d-galactofuranosyl thiocarbamate from per-O-tert-butyldimethylsilyl-β-d-galactofuranosyl isothiocyanate

Per-O-tert-butyldimethylsilyl-α,β-d-galactofuranosyl isothiocyanate (4) was synthesized by the reaction of per-O-TBS-β-d-galactofuranose (1) with KSCN, promoted by TMSI. Upon O-desilylation (1,2-dideoxy-α-d-galactofuranoso)[1,2d]-1,3-oxazolidine-2-thione (6), the cis-fused bicyclic thiocarbamate was obtained as the only product. Conformational analysis, aided by molecular modelling, showed two stable twist forms (³T₄ and ⁴T_O) for the five-membered sugar ring in 6. In aqueous solution, the equilibrium favours the first conformation (3:1 ratio). On the other hand, this ratio decreases for less polar solvents.