Igor A. Mikhailopulo

The High‐Anti Conformation of 7‐Halogenated 8‐Aza‐7‐deaza‐2′‐deoxy‐guanosines: A Study of the Influence of Modified Bases on the Sugar Structure of Nucleosides

Introduction. – The conformational flexibility of nucleic acids can be more complexthan that of peptides because the sugar-phosphate backbone conformation is descibedby five single-bond rotations in addition to five sugar torsions [1a]. It is expected that inmany cases, the conformational changes among nucleoside or nucleotide structuresobey a certain conformational pathway. This means that the movement of one torsion islinearly coupled with another one [2][3]. In general, the molecular structures ofnucleosides and nucleotides have been regarded as conformationally ˝rigid˛ [4]probably due to the small variances of torsion angles. Besides gauche and anomericeffects [5], important determinants of the overall structure of a nucleoside are the stericand stereoelectronic effects of the nucleobases [6]. In a series of influential reports, atfirst Gassen and coworkers [7], and later Chattopadhyaya and coworkers [8] havedemonstrated the importance of such effects on both the structure of a nucleoside aswell as on the secondary structure of a DNA molecule. Recently, our interest wasfocussed on the stereoelectronic influence of modified nucleobases – and here inparticular of aza- and deazapurines – on the structure and stability of oligonucleotidescontaining such compounds [9–13]. We have observed that the incorporation of 8-aza-7-deazapurine 2’-deoxynucleotides into oligodeoxynucleotides exerts an extraordinaryinfluence on their duplex stability which is significantly enhanced over that of theparent unmodified oligomers [14–16]. This may be traced to an altered secondarystructure of the base-modified oligomers with probably enhanced stacking interactions

A Novel Route for the Synthesis of Deoxy Fluoro Sugars and Nucleosides

The reaction of (diethylamino)sulfur trifluoride (DAST) with methyl 5-O-benzoyl-β-D-xylofuranoside (1) followed by column chromatography afforded the riboside 2 (62%) and the ribo-epoxide 3 (18%) (Scheme 1). Under similar reaction conditions, the α-D-anomer 4 gave the riboside 5 and the difluoride 6 in 60 and 9% yield, respectively. Treatment of the β-D-xyloside 10 with DAST gave, after chromatographic purification, the riboside 11 as the principal product (48%; Scheme 2). These results suggest that the C(3)−O−SF2NEt2 derivatives were initially formed in the case of the xylosides studied. The distinctive feature of the reaction of DAST with the β-D-arabinoside 12 consists in the formation of a 3- or 5-benzylideneoxoniumyl-substituted intermediate on one of the consecutive transformations, which finally give rise to the inversion of the configuration at C(3) affording the xylosides 17 (18%) and 18 (55%); the lyxoside 14 was also isolated from the reaction mixture in a yield of 25% (Scheme 3). In the presence of the non-participating 5-O-trityl group, i.e., from the reaction products of 21 with DAST, the compounds 23 and 24 were isolated in 16 and 52% yield, respectively (Scheme 4). It may be thus reasonable to conclude that, in the case of the β-D-arabinosides 12 and 21, the principal route of the reaction is the formation of the intermediate C(2)−O−SF2NEt2 derivative. Unlike 21, the α-D-arabinoside 26 was converted to the lyxo-epoxide 25 (53%) and the lyxoside 27 (14%), which implies the intermediate formation of the C(3)−O−SF2NEt2 derivative (Scheme 5).

Oligonucleotides Containing 9-(2-Deoxy-2-Fluoro-β-D-Arabinofuranosyl)-Adenine and -Guanine: Synthesis, Hybridization and Antisense Properties

Abstract Synthesis of 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-adenine (7, ara-A2′F) and -guanine (12 ara-G2′F) was accomplished via the condensation of 2, 6-dichloropurine (1) with 2-deoxy-2-fluoro-1, 3, 5-tri-O-benzoyl-α-D-arabinofuranose (2) as a key chemical step. Condensation of silylated N 6-benzoyladenine (6) with 2 gave, after deblocking and chromatographic separation, ara-A2,F (7) (14%), its α-anomer 8 (14%) and N 7-α-isomer 9 (25%). The PSEUROT analysis of N 9-β-D-arabinosides 7 and 12 manifested slight preference for the S rotamer (64%) for the former, and an equal population of the N and S rotamers for the latter. The arabinosides 7 and 12 were used for the preparation of the respective phosphoamidite building blocks 13 and 14 for automated oligonucleotide synthesis. Four 15-mer oligonucleotides (ONs) complementary to the initiation codon region of firefly luciferase mRNA were prepared: unmodified 2′-deoxy-ON (AS1) and containing (i) ara-A2,F instead of the only A (AS2), (ii) ara-G2,F vs. 3-G from the 5′-terminus (AS3), and (iii) both arabinosides at the same positions (AS4). All these ONs display practically the same (i) affinity to both complementary DNA and RNA, and (ii) ability to inhibit a luciferase gene expression in a cell-free transcription-translation system.

Synthesis of 2-Chloro-2′-Deoxyadenosine by Microbiological Transglycosylation

Abstract The title compound have been synthesized by an enzymatic trans-2′-deoxyribosylation of 2-chloroadenine using the whole cells of E. coli BMT-1D/1A as a biocatalyst and 2′-deoxyguanosine as a donor of glycosyl moiety.

Oxidation-reduction sequence for the synthesis of peracylated fluorodeoxy pentofuranosides☆

Abstract The oxidation of methyl 5-O- benzyl-3(2)-deoxy-3(2)-fluoro- α- d - pentofuranosides with dimethyl sulfoxide-acetic (trifluoroacetic) anhydride was accompanied by epimerization at the carbon atom bearing a fluoro function, resulting in the formation of the corresponding 2- or 3-keto derivatives as mixtures of two epimers in high combined yield. Reduction of a mixture of the erythro / threo epimeric 2-keto sugars (isolated as stable hydrates) with sodium borohydride in benzene-ethanol proceeded stereoselectively leading to the formation of 3-deoxy-3-fluoro ribo and lyxo-furanosides, respectively. In the case of the ribo and arabino epimers of the 3-keto sugar (isolated as free ketones), reduction stereoselectivity of the former was > 95% for the 2-deoxy-2-fluoro ribo sugar, whereas a ca. 3:1 lyxo / arabino ratio of products was obtained for the latter. Treatment of a mixture of the 2-epimeric 3-keto sugars with triethylamine in carbon tetrachloride at room temperature for 3–5 h afforded the 2-deoxy-2-fluoro ribo ketone (ca. 90%). The synthesis of 1-O- acetyl-2,5-di -O- benzoyl-3-deoxy-3-fluoro- α,β- d - lyxofuranose (8) and 1-O- acetyl-3,5-di -O- benzoyl-2-deoxy-2-fluoro -β- d - ribofuranose (16) and their use as glycosylating agents for bis-trimethylsilylated N6-benzoyladenine is described.

Different conformations of 1-deazaadenosine and its 2′-deoxyribonucleoside in the solid state and in solution

Abstract Crystallization of 1-deaza-2′-deoxyadenosine (c1Ad, 1) from propanol-2 gives two forms of crystals: type A formed firstly as plates, then, on the surface of the plates, type B appeared as needles. Single crystal X-ray analyses shows that the crystals A and B differ mainly in the sugar ring conformation: A adopts the S-type (P= 179.8°; C-2′-endo-C-3′-exo; 2T3) conformation associated with an high-anti base orientation (χ=−90.7°) and the γ = 152.3° across the exocyclic C(4′)C(5′) bond; B shows N-type (P = 21.2; C-3′-endo; 3E) conformation accompanied by a somewhat different anti base orientation (χ = −116.5°) an eclipsed orientation of the exocyclic C(4′)C(5′) bond (γ = 84.5°). No intramolecular hydrogen bonds in both types of crystals can be detected. Unlike c1Ad, the ribonucleoside (c1A, 2) is in the syn conformation (χ = 56.1°) in the solid state which is caused by an intramolecular (5′)CH2OH…N(3) hydrogen bond. The compounds 1 and 2 display very similar conformation in D2O solution with a strong preference for the S-type conformation of the furanose ring accompanied by the intramolecular (5′)CH2OH…N(3) hydrogen bond.

The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

Summary Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (1, clofarabine) were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, 2FAra-1P) via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9) into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine), their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine) and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P). Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues) and stereochemical features (2FAra-1P vs Ara-1P) of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate), phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required) (12a), and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate activity [90% yield of 2-chloroadenosine (8) in 30 min], D-arabinose reached an equilibrium at a concentration of ca. 1:1 of a starting base and the formed 2-chloro-9-(β-D-arabinofuranosyl)adenine (6) in 45 min, the formation of 2-chloro-9-(β-D-xylofuranosyl)adenine (7) proceeded very slowly attaining ca. 8% yield in 48 h.

Synthesis of a modified 2′, 5′-Adenylate trimer with a 2′,3′-Di-O-(2-Carboxyethyl)-ethylidene terminal group

Abstract The trimer of 2′, 5′-oligoadenylic acid with a (2-carboxyethyl)ethylidene group ({=1}{=6}) at the 2′-terminal adenosine moiety and its 3′-deoxyadenosine analog ({=1}{=7}) have been synthesized by the phosphotriester method.

A New Strategy for the Synthesis of Nucleosides: One-Pot Enzymatic Transformation of D-Pentoses into Nucleosides

A possibility of the one-pot synthesis of purine and pyrimidine nucleosides employing pure recombinant ribokinase, phosphopentomutase and nucleoside phosphorylases in a caskade transformation of D-pentoses into nucleosides is demonstrated. Preliminary results of this study point to reliability to develop practical methods for the preparation of a number of biologically important nucleosides.

An Enzymatic Transglycosylation of Purine Bases

An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2 carbon atoms. The glycosylation of N6-benzoyladenine and N2-acetylguanine and its O6-derivatives is not accompanied by deacylation of bases.