Nucleotides,Part LXV,Synthesis of 2′‐Deoxyribonucleoside 5′‐Phosphoramidites: New Building Blocks for the Inverse (5′‐3′)‐Oligonucleotide Approach

The syntheses of the 3′‐O‐(4,4′‐dimethoxytrityl)‐protected 5′‐phosphoramidites 25 – 28 and 5′‐(hydrogen succinates) 29 – 32 , which can be used as monomeric building blocks for the inverse (5′‐3′)‐oligodeoxyribonucleotide synthesis are described (Scheme). These activated nucleosides and nucleotides were obtained by two slightly different four‐step syntheses starting with the base‐protected nucleosides 13 – 20 . For the protection of the aglycon residues, the well‐established 2‐(4‐nitrophenyl)ethyl (npe) and [2‐(4‐nitrophenyl)ethoxy]carbonyl (npeoc) groups were used. The assembly of the oligonucleotides required a slightly increased coupling time of 3 min in application of the common protocol (see Table 1). The use of pyridinium hydrochloride as an activator (instead of 1H‐tetrazole) resulted in an extremely shorter activation time of 30 seconds. We established the efficiency of this inverse strategy by the synthesis of the oligonucleotide 3′‐conjugates 33 and 34 which carry lipophilic caps derived from cholesterol and vitamin E, respectively, as well as by the formation of (3′‐3′)‐ and (5′‐5′)‐internucleotide linkages (see Table 2).     

Nucleotides,Part LXIX,Synthesis of Phosphoramidite Building Blocks of Isoxanthopterin N8‐(2′‐Deoxy‐β‐D‐ribonucleosides): New Fluorescence Markers for Oligonucleotide Synthesis

The chemical synthesis of isoxanthopterin and 6‐phenylisoxanthopterin N⁸‐(2′‐deoxy‐β‐D ‐ribofuranosyl nucleosides) is described as well as their conversion into suitably protected 3′‐phosphoramidite building blocks to be used as marker molecules for DNA synthesis. Applying the npe/npeoc (=2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl) strategy, we used the new building blocks in the preparation of oligonucleotides by an automated solid‐support approach. The hybridization properties of a series of labelled oligomers were studied by UV‐melting techniques. It was found that the newly synthesized markers only slightly interfered with the abilities of the labelled oligomers to form stable duplexes with complementary oligonucleotides.     

Nucleotides Part XL. Synthesis and Characterization of Modified 2′–5′ Adenylate Trimers–Potential Antiviral Agents

2′–5′ Adenylate trimers 41–44 carrying the (tert-butyl)dimethylsilyl (tbds) group at the 3′-OH position of various sugar moieties were synthesized via the phosphoramidite method. The use of the (tert-butyloxy)carbonyl (boc) and 2-(4-nitrophenyl)ethylsulfonyl (npes) groups for 2′-OH protection in neighbourhood to the 3′-O-tbds residue was compared during the synthesis of the target trimers. For other functional positions, the use of the 2-(4-nitrophenyl)ethyl (npe) and 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) blocking groups were favoured.     

9-(2′-Deoxy-β-D-xylofuranosyl)adenine Building Blocks for Solid-Phase Synthesis and Properties of Oligo(2′-deoxy-xylonucleotides)

The 9-(2′-deoxy-à-D -threo-pentofuranosyl)adenine (=9-(2′-deoxy-à-D -xylofuranosyl)adeninc, xA_d; 2) was protected at its 6-NH₂ group with cither a benzoyl ( 5a ) or a (dimethyfamino)methylidcnc ( 6a ) residue and with a dimethoxytntyl group at 5′-OH ( 5b, 6b ). Compounds 5b and 6b were then converted into the 3′-phosphonates 5c and 6c ; moreover, the 2-cyanoethyl phosphoramidite 6d was synthesized starting from fib. The DNA building blocks were used for solid-phase synthesis of d[(xA)₁₂2-A] ( 8 ). The latter was hybridized with d[(xT)12-T] (T_m = 35°); in contrast, with d(T₁₂), complex formation was not observed. Moreover, xAd and xTd were introduced into the self-complementary dodccamcr d(G-T-A-G-A-A-T-T-C-T-A-C) ( 12 ) at different positions lo give the oligomcrs 13 – 16 . All oligonucleotides were characterised by temperature-dependent CD and UV spectroscopy, and in addition, 14 by T-jump experiments. From concentration-dependent T_m measurements, the thermodynamic paraneters of the melting as well as the tendency of hairpin formation of the oligonucleotides were deduced. Oligemer 14 was hydrolyzed by snake-venom phosphodiesterase in a discontinuous way implying a fast hydrolysis of unmodified 3′- and 5′-flanks followed by a slow hydrolysis of the remaining modified tetramer. In contrast to this, oligonucleotide 16 was hydrolyzed in a continuous reaction. In both cases, calf-spleen phosphodiesterase hydrolyzed the oligomer only marginally.     

1-(2′-Deoxy-β-D-xylofuranosyl)thymine Building Blocks for Solid-Phase Synthesis and Properties of Oligo(2′-Deoxyxylonucleotides)

1-(2′-Deoxy-β-D -threo-pentofuranosyl)thymine (= 1-(2′-deoxy-β-D -xylofuranosyl)thymine; xT_d; 2 ) was converted into its phosphonate 3b as well as its 2-cyanoethyl phosphoramidite 3c . Both compounds were used for solid-phase synthesis of d[(xT)₁₂-T] ( 5 ), representing the first DNA fragment build up from 3′–5′-linked 2′-deoxy--β-D -xylonucleosides. Moreover, xT_d was introduced into the innermost part of the self-complementary dodecamer d(G-T-A-G-A-A-xT-xT-C-T-A-C)₂ (9). The CD spectrum of d[(xT)₁₂–T] ( 5 ) exhibits reversed Cotton effects compared to d(T₁₂) ( 6 ; see Fig. 1), implying a left-handed single strand. With d(A₁₂) ( 7 ) it could be hybridized to form a propably Left-handed double strand d(A₁₂) · d[(xT)₁₂–T] ( 7 · 5 ) which was confirmed by melting experiments in combination with temperature-dependent CD spectroscopy. While 5 was hydrolyzed by snake-venom phosphodiesterase, it was resistant towards calf-spleen phosphodiesterase. The modified, self-complementary duplex 9 was hydrolyzed completely by snake-venom phosphodiesterase, at a twelvefold slower rate compared to unmodified 8 ; calf-spleen phosphodiesterase hydrolyzed 9 only partially.     

Nucleotides,Part LXII,Pyridinium Salts – An Effective Class of Catalysts for Oligonucleotide Synthesis

Various pyridinium salts (see 1 – 9 ) have been tested as catalysts for the condensation step in the phosphoramidite approach of oligonucleotide synthesis. Pyridinium chloride ( 1 ) turned out to be the most effective activator, speeding up the condensation tremendously. Pyridinium bromide ( 2 ) and 4-methylbenzenesulfonate ( 4 ) can also be regarded as powerful substitutes for the commonly used 1H-tetrazole. The acidic pK_a of the pyridinium cation provides an optimal range for phosphoramidite activation, which is followed by a nucleophilic attack of the pyridine ring to give the P-pyridinio intermediate 11 as the most likely precursor of phosphite ester formation (Scheme). ³¹P-NMR Studies support this proposal indirectly.     

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 XXVII Bis[2-(p-nitrophenyl)ethyl] Phosphorochloridate,a New Versatile Phosphorylating Agent in Nucleotide Chemistry

A new, versatile phosphorylating agent, bis[2-(p-nitrophenyl)ethyl] phosphorochloridate ( 3 ), has been prepared and is used for 3′- and/or 5′-phosphorylations of nucleosides. The resulting bis[2-(p-nitrophenyl)ethyl] phosphotriesters are versatile synthons in oligonucleotide synthesis leading finally to 3′- and/or 5′-terminated monophosphates in excellent yields.     

Nucleotides. Part XLVIII. Synthesis of 2′-amino-2′-deoxyarabinonucleoside phosphoramidite building blocks

Chemical syntheses of 2′-amino-2′-deoxyarabinonucleosides of uracil, thymine, cytosine, adenine, and guanine and their conversion into suitably protected 3′-phosphoramidite building blocks 24–28 for oligonucleotide synthesis are described. The 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) group was used for protection of the aglycon and the 2′-amino functions.     

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.     

Nucleotides. Part XXXV. Synthesis of 3′-deoxyadenylyl-(2′–5′)-3′-deoxyadenyIyl-(2′–ω)-9-(ω-hydroxyalkyl)adenines

Via the phosphotriester approach, new structural analogs of (2′–5′)oligoadenyiates, namely 3′-deoxyadenylyl-(2′–5′)-3′-dcoxyadenylyl-(2′–ω)-9-(ω-hydroxyalkyl)adenines 18 – 21 , have been synthesized (see Scheme) which should preserve biological activity and show higher stability towards phosphodiesterases. The newly synthesized oligonucleotides 18 – 21 have been characterized by ¹H-NMR spectra, TLC, and HPLC analysis.     

Nucleotides. Part XXVIII. Chemical syntheses of the 2′-5′-cordycepin-trimer core

The chemical synthesis of 3′-deoxyadenyly-(2′-5′)-3′-deoxyadenylyl-(2′-5′)-3′-deoxyadenosine ( 30 ; trimeric cordycepin) is described by three different routes using various protecting groups and applying the phosphotriester approach. The intermediates have been isolated and characterized by elemental analyses and spectroscopic means. High yields of 30 have been obtained on deprotection making this biologically very active compound available in preparative scale.     

Nucleotides. Part. XXXII. Synthesis of 2′–5′ Connected oligonucleotides. Prodrugs for antiviral and antitumoral nucleosides

The cytotoxically and antivirally active compounds bvU_d ( 1 ), flU_d ( 4 ), acyclovir ( 7 ), and A_a ( 12 ) have chemically been combined with the appropriately protected (2′–5′)diadenylate 20 by the phosphotriester approach to give the 2′–5′ oligonucleotide trimers 21 – 24 . The deprotection of the various blocking groups by chemical means afforded the 2′–5′ trimers 25 – 28 , which can be regarded as new type of a potential prodrug form delivering nucleotides to the targets inside cells. In an analogous series of reactions, 9-(3′-azido-3′-deoxy-β-D-xylofuranosyl)adenine was coupled with 7 to the 2′–5′ trimer 31 . The antiviral screening of the oligonucleotides 25–27 and 31 showed biological activities closely related to the parent nucleosides, possibly indicating their release by enzymatic cleavage of the oligomers.     

Synthesis of 2-(o-chloropheny)- and 2-(2′,6′-dichlorophenyl)-4-formyl-1,2,3-triazoles

Mesoaldehyde 1,3-dioxime was treated with either o-chlorophenyl- or 2,6 dichloro-phenylhydrazine to give the corresponding 2-chlorophenylhydrazone. Hydrazones 1a and 1b were treated with acetic anhydride and cyclized to triazoles ( 3a and 6a ) with cesium carbonate. These were then hydrolized to the previously unknown chlorophenyltriazole aldehydes ( 4a and 4b ). They were also converted to a number of acid derivatives, alcohols, and amines.     

Nucleosides. Part LI The 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) and 2-(2,4-dinitrophenyl)ethoxycarbonyl (dnpeoc) groups for protection of hydroxy functions in ribonucleosides and 2′ -deoxyribonucleosides

The Common 2′ -deoxypyrimidine and -purine nucleosides, thymidine ( 4 ), O⁴-[2-(4-nitrophenyl)ethyl]-thymidine ( 17 ), 2′-deoxy-N⁴-[2-(4-nitrophenyl)ethoxycarbonyl]cytidine ( 26 ), 2′-deoxy-N⁶-[2-(4-nitrophenyl)-ethoxycarbonyl]adenosine- 39 , and 2′-deoxy-N²-[2-(4-nitrophenyl)(ethoxycarbonyl]-O⁶-[2–4-nitrophenyl)ethyl]-guanosine ( 52 ) were further protected by the 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) and the 2-(2,4-dinitrophenyl)ethoxycarbonyl (dnpeoc) group at the OH functions of the sugar moiety to form new partially and fully blocked intermediates for nucleoside and nucleotide syntheses. The corresponding 5′-O-monomethoxytrityl derivatives 5 , 18 , 30 , 40 , and 56 were also used as starting material to synthesize some other intermediates which were not obtained by direct acylations. In the ribonucleoside series, the 5′ -O-monomethoxytrityl derivatives 14 , 36 , 49 , and 63 reacted with 2-(4-nitrophenyl) ethyl chloroformate ( 1 ) to the corresponding 2′,3′-bis-carbonates 15 , 37 , 50 , and 64 which were either detriylated to 16 , 38 , 51 , and 65 , respectively, or converted by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) treatment to the 2′,3′-cyclic carbonates 66 – 69 . The newly synthesized compounds were characterized by elemental analyses and UV and ¹H-NMR spectra.     

Nucleotides,Part LXVIII,Acetals as New 2′‐O‐Protecting Functions for the Synthesis of Oligoribonucleotides: Synthesis of Monomeric Building Units and Oligoribonucleotides

For the efficient synthesis of oligoribonucleotides by the 5′‐O‐(4,4′‐dimethoxytrityl) phosphoramidite approach, the 2′‐O‐[1‐(benzyloxy)ethyl]acetals 56 – 67 were investigated. Studies with the 2′‐O‐[1‐(benzyloxy)ethyl]‐5′‐O‐(dimethoxytrityl)ribonucleoside 3′‐phosphoramidites 56 – 59 gave, however, only reasonable results. The oligoribonucleotides obtained showed some impurities since the acid stabilities of the acetal and dimethoxytrityl functions are too close to guarantee a high selectivity. A combination of new acid‐labile protected 2′‐O‐protecting groups with the 2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl (npe/npeoc) strategy for base protection was more successful. The synthesis and physical properties of the monomeric building units and their intermediates 8 – 67 and the conditions for the automated generation of homo‐ and mixed oligoribonucleotides is described. The new 2′‐acetal protecting group could be cleaved off in a two step procedure and was designed for levelling their stability with regard to the attached nucleobase as well. Therefore, we used the 1‐{{3‐fluoro‐4‐{{[2‐(4‐nitrophenyl)ethoxy]carbonyl}oxy}benzyl}oxy}ethyl (fnebe) moiety for the protection of 2′‐OH of uridine, and for that of 2′‐OH of A, C, and G, the 1‐{{4‐{{[2‐(4‐nitrophenyl)ethoxy]carbonyl}oxy}benzyl}oxy}ethyl (nebe) residue. After selective deprotection by β‐elimination induced by a strong organic base like DBU, the remaining activated acetal was hydrolyzed under very mild acidic protic conditions, which reduced 2′‐3′ isomerization and chain cleavage. Also storage, handling, and purification of the chemically and enzymatically sensitive oligomers was simplified by this approach.     

Nucleotides Part LI. Synthesis and biological activities of (2′-5′)adenylate trimer conjugates with 2′-terminal 3′-O(ω-hydroxyalkyl) and 3′-O-(ω-carboxyalkyl) spacers

An efficient strategy for the synthesis of (2′-5′)adenylate trimer conjugates with 2′-terminal 3′-O-(ω-hydroxyalkyl) and 3′-O-(ω-carboxyalkyl) spacers is reported. Npeoc-protected adenosine building blocks 37--40 for phosphoramidite chemistry carrying a 3′-O-[11-(levulinoyloxy)undecyl], 3′-O-{2-[2-(levulinoyloxy)ethoxy]ethyl}, 3′-O-[5-(2-cyanoethoxycarbonyl)pentyl], and 3′-O-{5-[(9H-fluoren-9-ylmethoxy)carbonyl]pentyl} moiety, respectively, were prepared (npeoc = 2-(4-nitrophenyl)ethoxycarbonyl). Condensation with the cordycepin (3′-deoxyadenosine) dimer 1 led to the corresponding trimers 42, 43, 47 , and 48. Whereas the levulinoyl (lev) and 9H-fluoren-9-ylmethyl (fm) blocking groups could be cleaved off selectively from the trimers 42, 43 , and 48 yielding the intermediates 44, 45 , and 49 for the synthesis of the 3′-O-(ω-hydroxyalkyl)trimers 53, 54 and the cholesterol conjugates 59--61 , the 2-cyanoethyl (ce) protecting group of 47 , however, could not be removed in a similar manner from the carboxy function. Trimer 47 served as precursor for the preparation of the trimer 55 with a terminal 3′-O-(5-carboxypentyl)adenosine moiety. The metabolically stable 3′-O-alkyl-(2′--5′)A derivatives were tested regarding inhibition of HIV-1 syncytia formation and HIV-1 RT activity. Only the conjugate 59 showed significant effects, whereas the trimers 53--55 and the conjugates 60 and 61 were less potent inhibitors, even at 100-fold larger concentrations.     

Nucleotides. Part XXXIVSynthesis of Modified Oligomeric 2′–5′A Analogues: Potential Antiviral Agents

A series of new 2′–5′-oligonucleotide trimers carrying a 9-(2′,3′-anhydro-β-D -ribofuranosyl)-( 59 ), 9-(3′-deoxy-β-D -glycero-pent-3-enofuranosyl)-( 63 ), 9-(3′-azido-3′-deoxy-β-D -xylofuranosyl)-( 62 ), and 9-(3′-halo-3′-deoxy-β-D -xylofuranosyl)adenine ( 60 and 61 ) moiety at the 2′-terminal end have been synthesized via the phosphotriester method. The properly protected, modified monomeric building blocks ( 6 , 9 , 16 , 19 , 27 , 33 , 36 , 37 , and 43 ) were obtained, in general, by a sequence of reactions, introducing the protecting groups into the right positions. Their condensations with the intermediary dimeric 2′-terminal phosphodiesters 48 and 49 led to the fully protected 2′–5′-trimers 50–58 which were deblocked to form the free 2′–5′-trimers 59 – 63 . Easy elimination of HBr on deprotection did not allow to form the trimeric (3′-bromo-3′-deoxy-β-D -xylofuranosyl)adenine analogue but only 63 carrying an unsaturated sugar moiety instead. The newly synthesized compounds have been characterized by UV and NMR spectra as well as by elemental analysis.