Nucleosides and Nucleotides. Part 13. Synthesis and spectral properties of 1- (3′-O-Phosphoryl-2′-deoxy-β -D-ribofuranosyl)-2(1H)-pyridone-(3′-5′)-1-(2′-deoxy-β-D-ribofuranosyl)-2 (1H)-pyridone

The dinucleoside phosphate Π_dpΠ_d ( 4 ) was synthesized from the monomers 1-(5′-O-monomethoxytrityl - 2′ - deoxy - β - D - ribofuranosyl) - 2 (1 H) - pyridone ((MeOTr) Π_d, 2 ) and 1-(5′-O-phosphoryl-3′-O-acetyl-2′-deoxy-β-D -ribofuranosyl)-(1H)-pyridone (pΠ_d(Ac), 3 ). Its 6.4% hyperchromicity and an analysis of the ¹H-NMR. spectra indicate that the conformation and the base-base interactions in 4 are similar to those in natural pyrimidine dinucleoside phosphates.     

Nucleotides. Part LIV. Synthesis of condensed N1-(2′-deoxy-β-D-ribofuranosyl)lumazines,New fluorescent building blocks in oligonucleotide synthesis

Various condensed areno[g]lumazine derivatives 2 , 3 , and 5 – 7 were synthesized as new fluorescent aglycones for glycosylation reactions with 2-deoxy-3, 5-di-O-(p-toluoyl)-α/β-D -erythro-pentofuranosyl chloride ( 10 ) to form, in a Hilbert-Johnson-Birkofer reaction, the corresponding N¹-(2′-deoxyribonucleosides) 15 – 21 . The β-D -anomers 15 , 17 , 19 , and 21 were deblocked to 24 – 27 and, together with N¹-(2′-deoxy-β-D -ribofuranosyl)lumazine ( 22 ) and its 6, 7-diphenyl derivative 23 , dimethoxytritylated in 5′-position to 28–33. These intermediates were then converted into the 3′-(2-cyanoethyI diisopropylphosphoramidites) 34 – 39 which function as monomeric building block in oligonucleotide syntheses as well as into the 3′-(hydrogen succinates) 40 – 45 which can be used for coupling with the solid-support material. A series of lumazine-modified oligonucleotides were synthesized and the influence of the new nucleobases on the stability of duplex formation studied by measuring the T_m values in comparison to model sequences. A substantial increase in the T_m is observed on introduction of areno[g]lumazine moieties in the oligonucleotide chain stabilizing obviously the helical structures by improved stacking effects. Stabilization is strongly dependent on the site of the modified nucleobase in the chain.     

Nucleoside und Nucleotide. Teil 16. Verhalten von 1-(2′-Desoxy-β-D-ribofuranosyl)-2(1 H)-pyrimidinon-5′-triphosphat, 1-(2′-Desoxy-β-D-ribofuranosyl)-2(1 H)pyridinon-5′-triphosphat und 4-Amino-1-(2′-Desoxy-β-D-ribofuranosyl)-2(1 H)-pyridinon-5′-triphosphat gegenüber DNA-Polymerase

Nucleosides and Nucleotides. Part 16. The Behaviour of 1-(2′-Deoxy-β-D -ribofuranosyl)-2(1H)-pyrimidinone-5′-triphosphate, 1-(2′-Deoxy-β-D -ribofuranosyl-2(1H))-pyridinone-5′-triphosphate and 4-Amino-1-(2′-desoxy-β-D -ribofuranosyl)-2(1H)-pyridinone-5′-triphosphate towards DNA Polymerase The behaviour of nucleotide base analogs in the DNA synthesis in vitro was studied. The investigated nucleoside-5′-triphosphates 1-(2′-deoxy-β-D -ribofuranosyl)-2(1 H)-pyrimidinone-5′-triphosphate (pppM_d), 1-(2′-deoxy-β-D -ribofuranosyl)-2(1 H)-pyridinone-5′-triphosphate (pppII_d) and 4-amino-1-(2′-deoxy-β-D -ribofuranosyl)-2(1 H)-pyridinone-5′-triphosphate (pppZ_d) can be considered to be analogs of 2′-deoxy-cytidine-5′-triphosphate. However, their ability to undergo base pairing to the complementary guanine is decreased. When pppM_d, pppII_d or pppZ_d are substituted for pppC_d in the enzymatic synthesis of DNA by DNA polymerase no incorporation of these analogs is observed. They exhibit only a weak inhibition of the DNA synthesis. The mode of the inhibition is uncompetitive which shows that these nucleotide analogs cannot serve as substrates for the DNA polymerase.     

Nucleotides. Part XXXI. Modified Oligomeric 2′–5′ A Analogues: Synthesis of 2′–5′ oligonucleotides with 9-(3′-azido-3′-deoxy-β-D-xylofuranosyl)adenine and 9-(3′-amino-3′-deoxy-β-D-xylofuranosyl)adenine as modified nucleosides

A series of new 2′–5′ oligonucleotides carrying the 9-(3′-azido-3′deoxy-β-D-xylofuranosyl)adenine moiety as a building block has been synthesized via the phosphotriester method. The use of the 2-(4-nitrophenyl)ethyl (npe) and 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) blocking groups for phosphate, amino, and hydroxy protection guaranteed straightforward syntheses in high yields and easy deblocking lo form the 2′–5′ trimers 21 , 22 , and 25 and the tetramer 23 . Catalytic reduction of the azido groups in [9-(3′-azido-3′-deoxy-β-D-xylofuranosyl)adenine]2′-yl-[2′-(O^p-ammonio)→ 5′]-[9-(3′-azido-3′-deoxy-β-D-xylofuranosyl)adenin]-2′-yl-[2′-(O^p-ammonio)→ 5′]-9-(3′-azido-3′-deoxy-β-D-xylofuranosyl)adenine ( 21 ) led to the corresponding 9-(3′-amino-3′-deoxy-β-D-xylofuranosyl)-adenine 2′–5′ trimer 26 in which the two internucleotidic linkages are formally neutralized by intramolecular betaine formation.     

Nucleoside und Nucleotide. Teil 10. Synthese von Thymidylyl-(3′-5′) -thymidylyl-(3′-5′)-1-(2′-desoxy-β-D-ribofuranosyl)-2(1 H)-pyridon

Nucleosides and Nucleotides. Part 10. Synthesis of Thymidylyl-(3′-5′)-thymidylyl-(3′-5′)-1-(2′-deoxy-β-D - ribofuranosyl)-2(1 H)-pyridone The synthesis of 5′-O-monomethoxytritylthymidylyl-(3′-5′)-thymidylyl-(3′-5′)-1-(2′-deoxy-β-D -ribofuranosyl)-2(1H)-pyridone ((MeOTr)T_dpT_dp∏_d, 5 ) and of thymidylyl-(3′-5′)-thymidylyl-(3′-5′)-1-(2′-deoxy-β-D -ribofuranosyl)-2(1 H)-pyridone (T_dpT_dp∏_d, 11 ) by condensing (MeOTr) T_dpT_d ( 3 ) and p∏_d(Ac) ( 4 ) in the presence of DCC in abs. pyridine is described. Condensation of (MeOTr) T_dpT_dp ( 6 ) with Π_d(Ac) ( 7 ) did not yield the desired product 5 because compound 6 formed the 3′-pyrophosphate. The removal of the acetyl- and p-methoxytrityl protecting group was effected by treatment with conc. ammonia solution at room temperature, and acetic acid/pyridine 7 : 3 at 100°, respectively. Enzymatic degradation of the trinucleoside diphosphate 11 with phosphodiesterase I and II yielded T_d, pT_d and p∏_d, T_dp and Π_d, respectively, in correct ratios.     

Synthesis of 1-(β-D-ribofuranosyl)indol-3-acetic acid

By condensation of ethyl indolin-3-acetate ( 4 ) and 2,3,5-tri-O-benzoylribofuranosyl-1-acetate ( 5 ), ethyl 1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)indolin-3-acetate ( 6 ) was obtained in good yield. The indoline nucleoside 6 was aromatized to ethyl 1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)indol-3-acetate ( 7 ) with DDQ. The treatment of the indole nucleoside with barium hydroxide and methanol gave the methyl ester 8 , which was further treated in water to give the desired 1-(β-D-ribofuranosyl)indol-3-acetic acid ( 9 ).     

Nucleosides. 117. Synthesis of 4-oxo-8-(β-D-ribofuranosyl)-3H-pyrazolo[1,5-a]-1,3,5-triazine (OPTR) via 3-amino-2N-carbamoyl-4-(β-D-ribofuranosyl)pyrazole (ACPR) derivatives

Reaction of 2-formyl-2-(2,3-O-isopropylidene-5-O-trityl-D-ribofuranosyl)acetonitrile (VII) with semicarbazide hydrochloride followed by sodium ethoxide treatment afforded an α,β-mixture of 3-amino-2N-carbamoyl-4-(2,3-O-isopropylidene-5-O-trityl-D-ribofuranosyl)pyrazole (IX). Conversion of IX to 4-oxo-8-(2,3-O-isopropylidene-5-O-trityl-D-ribofuranosyl)-3H-pyrazolo[1,5-a]-1,3,5-triazine (XIII) was achieved by treatment of IX with ethylorthoformate. The β-isomer IXb gave only the β-isomer XIIIb, and the α-isomer IXa was converted exclusively into the α-isomer XIIIa. Upon deprotection with 3% n-butanolic hydrogen chloride, both IXa and IXb gave the same mixture of the α- and β-isomers of 3-amino-2N-carbamoyl-4-(D-ribosyl)pyrazole, which were separated by chromatography. The syntheses of the hitherto unknown compounds, 3-amino-2N-carbamoylpyrazole (IVa) and its 4-methyl analog (IVb) are also reported. Experimental details of the synthesis of 3-amino-4-(2,3-O-isopropylidene-5-O-trityl-β-D-ribofuranosyl)pyrazole (XIIb), an important intermediate for “purine-like” C-nucleosides, are also described.     

Nucleosides CIII. Anhydropyrimidine-C-nueleosides. Synthesis of 4,2′-anhydro-5-(β-D-arabinofuranosyl)- and 5-(β-D-arabinofuranosyl)pyrimidine C-nucleosides

4,2′-Anhydro-5-(β-D -arabinofuranosyl)isocytosine and 4,2′-anhydro-5-(β-D -arabinofuranosyl)-uracil were synthesized. Treatment of Ψ-isocytidine with either α-acetoxyisobutyryl chloride or salicyloyl chloride in acetonitrile afforded the acylated anhydronucleoside. Deacylation of the product with methanol-hydrogen chloride afforded 4,2′-anhydro-5-(β-D -arabinofuranosyl)isocylosine hydrochloride in crystalline form. Analogous reaction of Ψ-uridine with the acyl chloride reagents, however, always gave a mixture of the acylated anhydronucleoside and 2′-chloro-2′-deoxy-Ψ-uridine. Treatment of these products either singly or as a mixture with sodium methoxide in methanol afforded 4,2′-anhydro-5-(β-D -arabinofuranosyl)uracil in crystalline form in good yield. 5-(β-D -Arabinofuranosyl)isocytosine was obtained upon treatment of the corresponding 4,2′-anhydronucleoside with 10% sodium hydroxide under reflux for 30 minutes. Treatment of the anhydro uracil nucleoside with a small amount of Dowex 50(H⁺) in water at 50° gave 5-(β-D -arabinofuranosyl)uracil.     

Nucleotides. Part XXVI.. A new synthesis of 9-(′-D-ribofuranosyl)uric acid and its 5′-monophosphate

Syntheses for 9-(β-D -ribofuranosyl)uric acid ( 16 ) and its 5′-monophosphate 14 have been achieved starting from guanosine and applying the 2-(p-nitrophenyl)ethyl group for protection of the aglycon moiety as well as the phosphate function. A more efficient and direct approach to 14 uses O⁶, O⁸-dibenzyl protection and phosphorylation by the Yoshikawa procedure. The various protected intermediates have been characterized by spectroscopic means and elemental analysis.     

Synthesis of 5-(β-D-ribofuranosyl)-1,2,4-oxadiazole-3-carboxamide

The title compound was synthesized in three steps from ethoxycarbonylformamide oxime (5) and 3,4,6-tri-O-acetyl-2,5-anhydroallonyl chloride (4b) in 62% overall yield. An acid catalyzed de-esterification was required to prevent a facile base catalyzed elimination reaction.     

Synthesis of oligonucleotides containing 7-(2-deoxy-β-d-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine

The synthesis of oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine was accomplished. The viable intermediate N²-isobutyryl-7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine ( 6 ) was prepared via a four step deoxygenation procedure from 7-β-D-ribofuranosylguanine ( 1 ). The 5′-hydroxyl group of 6 was protected as 4,4′-dimethoxytrityl ether and then converted to the target phosphoramidite ( 8 ) via conventional phosphitylation procedure. The amino groups of 8-amino-2′-deoxyguanosine ( 9 ) were protected in the form of N-(dimethylainino)methylene functions to give the protected nucleoside 10 , which was subsequently converted to the target phosphoramidite 12 via dimethoxytritylation followed by phosphitylation. The phosphoramidites 8 and 12 were incorporated into a 26-mer and a 31-mer G-rich oligonucleotide using solid-support, phosphoramidite methodology. Analysis of antiparallel triplex formation by the oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofura-nosyl)guanine in place of 2′-deoxyguanosine showed no enhancement in triple helix formation.     

5-(α-Fluorovinyl)tryptamines and 5-(α-Fluorovinyl)-3-(N-methyl-1′,2′,5′,6′,-tetrahydropyridin-3′- and -4′-yl)indoles—5-(α-Fluorovinyl)tryptamines

5-(α-Fluorovinyl)tryptamines 4a, 4b and 5-(α-fluorovinyl)-3-(N-methyl-1′,2′,5′,6′-tetrahydropyridin-3′- and -4′-yl) indoles 5a, 5b were synthesized using 5-(α-fluorovinyl)indole ( 7 ). The target compounds are bioisosteres of 5-carboxyamido substituted tryptamines and their tetrahydropyridyl analogs.     

C-Nucleosides. 8. Synthesis of 5-hydroxy-2-(β-D-ribofuranosyl)pyridine

The synthesis of 5-hydroxy-2-(β-D-ribofuranosyl)pyridine ( 12 ) from 2-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)furan ( 1 ) is described. Treatment of 1 with α-methoxycarbamate in the presence of p-toluenesulfonic acid in benzene at reflux temperature afforded furfurylcarbamate ( 2 ) and its α-isomer in a 5/1 ratio. The anomerization was circumvented by treatment of 1 with α-methoxycarbamate in the presence of boron trifluoride in benzene at room temperature. Compound 2 was electrochemically oxidized to give dihydrofuran 4 . However, conversion of 4 into 11 was unsuccessful. Treatment of azide 8 with bromine and methanol afforded 9 . Reduction of 9 with zinc powder gave dihydrofurfurylamine 10 , in 80% yield. Treatment of this with concentrated hydrochloric acid in methanol yielded 11 , which on deblocking with 5% sodium hydroxide aqueous solution gave 12.     

Nucleosides. Part L.. Structure of lumazine N1-(2′-deoxy-D-ribonucleosides) ( = 1-(2′-deoxy-D-ribofuranosyl)pteridine-2,4(1H,3H)-diones): A revision of the anomeric configuration

The anomeric configuration of the glycosidic bond in lumazine N¹-(2′-deoxy-D -ribonucleosides) 1–6 was investigated by NOE difference spectroscopy. The former configurational assignment of the α - and β -D -anomers 1 and 2, 3 and 4 , and 5 and 6 , respectively, has to be reversed to be in agreement with the physical data. Additional proof is presented by X-ray analysis of 3 and 6 . Chemical interconversions of 1-(2′-deoxy-β-D -ribofuranosyl)-6,7-diphenyllumazine ( 6 ) into 2,3′ -anhydrolumazine 2′-deoxyribonucleosides 16 and 17 are also in agreement with the revised anomeric configuration.     

Synthesis of α-2′-deoxynucleosides

α-Thymidine (4) was synthesized from thymidine (1) in 3 steps in 36% overall yield without using chro-matography and with the possibility of increasing the yield to 85% by reusing the remaining α,β-mixture. 1-(2-Deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl)thymine (3) was further converted to 1-(2-deoxy-α-D-erythro-pentofuranosyl)-5-methylcytosine (5) .     

Pterin Chemistry. Part 92.. Loading experiments with 6α,β-tetrahydro-L-[3′-2H1] biopterin

6α,β-Tetrahydro-L -[3′-²H₁]biopterin ([3′-²H₁]- 1 ) was administered orally to two primapterinuric patients in order to investigate the biosynthetic pathway of 7-substituted pterins in humans. L -Primapterin ( 2 ) and L -biopterin were isolated from urine after loading and measured by GC/MS. L -Biopterin and L -primapterin were labelled with ²H to an equal extent. From this result, one can conclude that L -primapterin is formed from tetrahydro-L -biopterin, very probably via an intramolecular rearrangement.     

Synthesis of 2-(β-D-ribofuranosyl)[1,3,5]triazines

The synthesis of the first [1,3,5]triazine carbon linked nucleosides are reported. 4-Amino-6-(β-D-ribofuranosyl)[1,3,5]triazin-2(1H)-one ( 8 ), an analog of 5-azacytidine and pseudoisocytidine was prepared. 2,5-Anhydro-D-allonamidine hydrochloride ( 3 ) was condensed with dimethyl cyanoiminodithiocarbonate ( 4 ) to give 4-methylthio-6-(β-D-ribofuranosyl)[1,3,5]triazin-2-amine ( 5 ). Compound 5 was reacted with m-chloroperbenzoic acid to give 4-methylsulfinyl-6-(β-D-ribofuranosyl)[1,3,5]triazin-2-amine ( 6 ). Displacement of the methyl sulfinyl with the appropriate nucleophile gave 6-(β-D-ribofuranosyl)[1,3,5]triazine-2,4-diamine ( 7 ), 4-amino-6-(β-D-ribofuranosyl)[1,3,5]triazin-2(1H)-one ( 8 ), and 4-amino-6-(β-D-ribofuranosyl)[1,3,5]triazine-2(1H)-thione ( 9 ). Dethiation of compound 5 with Raney nickel gave 4-(β-D-ribofuranosyl)[1,3,5]triazin-2-amine ( 10 ). The crystal structure of 7 was determined by single crystal X-ray.     

Nucleotide. X. Synthese und Eigenschaften von Dinucleosidmonophosphaten mit 2′-Desoxyadenosin und 1-(2′-Desoxy-β-D-ribofuranosyl)-lumazinen als Bausteine

Nucleotides. X. Synthesis and properties of dinucleoside monophosphates with 2′-deoxyadenosine and 1-(2′-deoxy-β-D -ribofuranosyl)-lumazines as building blocks The synthesis of various dinucleoside monophosphates 16--20 consisting of 2′-deoxyadenosine and 1-(2′-deoxy-β-D -ribofuranosyl)-lumazines via the triester approach is described. The fully protected phosphotriesters 6--10 as well as the partially deblocked intermediates 11--15 have also been isolated and characterized by physical means. Intramolecular interactions in 16--20 have been investigated by the determination of the hypochromicities and CD. spectra revealing a more or less distinct stacking effect in dependence of the 6,7-substituents in the lumazine moiety as well as the polarity of the internucleotidic linkage. Enzymatic degradations of the dinucleoside monophosphates with snake venom and spleen phosphodiesterase are depending strongly on various structural features indicating a much lower substrate specificity especially in presence of 6,7-diphenyl-lumazine as an aglycone with the latter enzyme.     

Synthesis of β-D-ribo- and 2′-deoxy-β-D-ribofuranosyl derivatives of 6-aminopyrazolo[4,3-c]pyridin-4(5H)-one by a ring closure of pyrazole nucleoside precursors

6-Amino-1-(2-deoxy-β-D-erthro-pentofuranosyl)pyrazolo[4,3-c]pyridin-4(5H)-one ( 5 ), as well as 2-(β-D-ribofuranosyl)- and 2-(2-deoxy-β-D-ribofuranosyl)- derivatives of 6-aminopyrazolo[4,3-c]pyridin-4(5H)-one ( 18 and 22 , respectively) have been synthesized by a base-catalyzed ring closure of pyrazole nucleoside precursors. Glycosylation of the sodium salt of methyl 3(5)-cyanomethylpyrazole-4-carboxylate ( 6 ) with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranose ( 8 ) provided the corresponding N-1 and N-2 glycosyl derivatives ( 9 and 10 , respectively). Debenzoylation of 9 and 10 with sodium methoxide gave deprotected nucleosides 14 and 16 , respectively. Further ammonolysis of 14 and 16 afforded 5(or 3)-cyanomethyl-1-(2-deoxy-β-D-erythro-pentofuranosyl)pyrazole-4-carboxamide ( 15 and 17 , respectively). Ring closure of 15 and 17 in the presence of sodium carbonate gave 5 and 22 , respectively. By contrast, glycosylation of the sodium salt of 6 with 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide ( 11 ) or the persilylated 6 with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose gave mainly the N-2 glycosylated derivative 13 , which on ammonolysis and ring closure furnished 18 . Phosphorylation of 18 gave 6-amino-2-β-D-ribofuranosylpyrazolo[4,3-c]pyridin-4(5H)-one 5′-phosphate ( 19 ). The site of glycosylation and the anomeric configuration of these nucleosides have been assigned on the basis of ¹H nmr and uv spectral characteristics and by single-crystal X-ray analysis of 16 .