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.     

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.     

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.     

Nucleosides and nucleotides. Part 25.. Synthesis of a protected 1-(2′-deoxy-β-D-ribofuranosyl)-1 H-benzimidazole 3′-phosphate

1-(2′-Deoxy-5′-O-dimethoxytrityl-′-D -ribofuranosyl)-1 H-benzimidazole 3′-[(p-chlorophenyl)(2-cyanoethyl) phosphate] ( 6 ) has been synthesized from 1-(β-D -ribofuranosyl)-1H-benzimidazole ( 3b ) using regiospecific 2′-deoxygenation. The latter compound was obtained by glycosylation of benzimidazole with the D -ribose derivative 2 leading exclusively of the β-D -anomer.     

Synthesis of 8-Aza-1,3-dideaza-2′-deoxyadenosine and 5,6-Disubstituted Benzotriazole 2′-Deoxy-β-D-Ribofuranosides via Nucleobase-Anion Glycosylation

The synthesis of 8-aza-1,3-dideaza-2′-deoxyadenosine ( 3a ) as well as of 4- and 5,6-substituted benzotriazole 2′-deoxy-β-D -ribonucleosides is described (Schemes 1–3). Glycosylation of benzotriazole anions is stereoselective in all cases (exclusive β-D -anomer formation), but regioisomeric N¹, N², and N³-(2′-deoxyribofuranosides) are formed. The distribution of the regioisomers is controlled by the nucleobase substituents. Anomeric configuration as well as the position of glycosylation are determined by UV and NMR in combination with 1D-NOE-difference spectroscopy. The unprotonated forms of 4-aminobenzotriazoic 2′-deoxy-β-D -ribofuranosides 3a – c exhibit strong fluorescence.     

3-Deaza-2′-deoxyadenosine: Synthesis via 4-(methylthio)-1H-imidazo[4,5-c]pyridine 2′-deoxyribonucleosides and properties of oligonucleotides

The synthesis of 4-(methylthio)-1H-imidazo[4,5-c]pyridine 2′-deoxy-β-D -ribonucleosides 2 and 9 and the conversion of the N¹-isomer 2 into the 2′,3′-didehydro-2′,3′-dideoxyribonucleoside 3a or (via 7 ) 3-deaza-2′-deoxyadenosine ( 1 ) is described. Phosphonate building blocks of 1 were employed in solid-phase synthesis of self-complementary base-modified oligonucleotides. Their properties were studied with regard to duplex stability and hydrolysis by the restriction enzyme Eco RI.     

(6′RS,8′RS,2E)- und (6′RS,8′SR,2E)-3-Methyl-3-(2y,2′,6′-trimethyl-7′-oxabicyclo[4.3.0]non-9′-en-8′-yl)-2-propenal ([(5RS,8RS)- und (5RS,8SR)-5,8-Epoxy-5,8-dihydro-ionyloinden]acetaldehyd): Synthese und Röntgenstrukturanalyse

Synthesis and X-Ray Structure of (6′RS,8′RS,2E)- and (6′RS,8′SR,2E)-3-Methyl-3-(2′,2′,6′-trimethyl-7′-oxabicyclo[4.3.0]non-9′-en-8′-yl)-2-propenal ([(5RS,8RS)- and (5RS,8SR)-5,8-Epoxy-5,8-dihydro-ionylidene]acetaldehyde) To check our previous spectroscopic assignments of the structures of trans- and cis-substituted furanoid end groups of carotenoid-5,8-epoxides, we now have synthesized the title compounds. An X-ray structure determination of a single crystal of the trans-isomer (±)- -10A is in agreement with the ¹ H-NMR spectroscopic arguments: isomers with Δδ (H? C(7), H? C(8)) = 0.15–0.22 ppm and J > 1.4 for H? C(7) belong to the cis-series; Δδ in trans-compounds is < 0.07 ppm, and H? C(7) appears as a broad singulett.     

Synthesis and 1H-NMR spectra of halogenated spiro[isobenzofuran-1(3H),9′(9H)-6′-(methylcyclohexylamino)-3′-methyl-2′-anilinoxanthene]-3-ones

Reaction of spiro[isobenzofuran-1(3H).9′(9H)-6′-(methylcyclohexylamino)-3′-methyl-2′-anilinoxanthene]-3-one ( 1 ), which is typical leuco fluoran dye, with N-bromosuccinimide and N-chlorosuccinimide leads to halogenated derivatives 2a–2c and 3 , respectively. Their structures were established by two-dimensional proton-proton (COSY) experiment and their thermal properties examined by means of DSC and compared with commercially available 1 .     

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.     

Stereoselective Synthesis of Indazole 2′-Deoxy-β-D-ribonucleosides: Glycosylation of the Nucleobase Anion

The glycosylation of indazolyl anions derived from 4a , b with 2-deoxy-3,5-bis-O-(4-methylbenzoyl)-α-D -erythro-pentofuranosyl chloride ( 5 ) is described. The reaction was Stereoselective – exclusive β-D -anomer formation – but regioisomeric N¹- and N²-(2′-deoxy-β-D -ribofuranosides) (i.e. 6a and 7a , resp., and 6b and 7b , resp.) were formed in about equal amounts. They were deprotected to yield 8a , b and 9a , b . Compound 1 , related to 2′-deoxyadenosine ( 3 ), and its regioisomer 2 were obtained from 8b and 9b , respectively, by catalytic hydrogenation. The anomeric configuration as well as the position of glycosylation were determined by 1D NOE-difference spectroscopy. The first protonation site of 1 and 2 was found to be the NH₂ group. The N-glycosylic bond of 1H-indazole N¹-(2′-deoxyribofuranosides) is more stable than that of the parent purine nucleosides. Compound 1 is no substrate for adenosine deaminase.     

8-Aza-2′-deoxyguanosine and Related 1,2,3-Triazolo[4,5-d]pyrimidine 2′-Deoxyribofuranosides

The synthesis of 8-azaguanine N⁹-, N⁸-, and N⁷-(2′-deoxyribonucleosides) 1–3 , related to 2′-deoxyguanosine ( 4 ), is described. Glycosylation of the anion of 5-amino-7-methoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine ( 5 ) with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 6 ) afforded the regioisomeric glycosylation products 7a/7b, 8a/8b , and 9 (Scheme 1) which were detoluoylated to give 10a, 10b, 11a, 11b , and 12a . The anomeric configuration as well as the position of glycosylation were determined by combination of UV, ¹³C-NMR, and ¹H-NMR NOE-difference spectroscopy. The 2-amino-8-aza-2′-deoxyadenosine ( 13 ), obtained from 7a , was deaminated by adenosine deaminase to yield 8-aza-2′-deoxyguanosine ( 1 ), whereas the N⁷- and N⁸-regioisomers were no substrates of the enzyme. The N-glycosylic bond of compound 1 (0.1 N HCl) is ca. 10 times more stable than that of 2′-deoxyguanosine ( 4 ).     

Angiotensin-II Analogues. I: Synthesis and incorporation of the halogenated amino acids 3-(4′-iodophenyl)alanine, 3-(3′,5′-dibromo-4′-chlorophenyl)alanine, 3-(3′,4′,5′-tribromophenyl)alanine,and 3-(2′,3′,4′,5′,6′-pentabromophenyl)alanine

The synthesis of the polyhalogenated phenylalanines Phe(3′,4′,5′-Br₃) ( 3 ), Phe(3′,5′-Br₂-4′-Cl) ( 4 ) and DL -Phe (2′,3′,4′,5′,6′-Br₅) ( 9 ) is described. The trihalogenated phenylalanines 3 and 4 are obtained stereospecifically from Phe(4′-NH₂) by electrophilic bromination followed by Sandmeyer reaction. The most hydrophobic amino acid 9 is synthesized from pentabromobenzyl bromide and a glycine analogue by phase-transfer catalysis. With the amino acids 4, 9 , Phe(4′-I) and D -Phe, analogues of [1-sarcosin]angiotensin II ([Sar¹]AT) are produced for structure-activity studies and tritium incorporation. The diastereomeric pentabromo peptides L - and D - 13 are separated by HPLC. and identified by catalytic dehalogenation and comparison to [Sar¹]AT ( 10 ) and [Sar¹, D -Phe⁸]AT ( 14 ).     

Spirocyclization of 1-(o-aminophenyl)cycloalkanols and 1-(2′-amino-3′-pyridinyl)cycloalkanols

A one-step synthesis of spiro[cycloalkane-1,4′-2H-3′,1-benzoxazin]-2′-ones and spiro[cycloalkane-1,4′-1H-pyrido[2′,3′-d][1,3′]oxazin]-2′-ones, obtained in good yield from the corresponding 1-(o-aminophenyl) and 1-(2′-amino-3′-pyridinyl)cycloalkanols is described using ethyl carbonate in presence of n-butyllithium.     

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 12. Synthese von Dinucleosidmonophosphaten mit 1-(2′-Desoxy-β-D-ribofuranosyl)-2(1H)-pyrimidon als Baustein

Nucleosides and Nucleotides. Part 12. Synthesis of Dinucleoside Monophosphates Containing 1-(2′-Deoxy-β-D -ribofuranosyl)-2(1H)-pyrimidone The connexion of the modified nucleoside 1-(2′-deoxy-β-D -ribofuranosyl)-2(1H)-pyrimidone (M_d, 2 ) with the natural nucleotides pT_d and pG_d is described. The protected dinucleoside monophosphates (MeOTr)M_dpT_d ( 6 ) and (MeOTr)M_dpG ( 9 ) were prepared by the standard phosphodiester method using DCC as condensing agent. M_dpT_d ( 7 ) was obtained by treatment of 6 with formic acid/methanol 7 : 3 at 0° 9 was converted to the free dinucleoside monophosphate M_dpG_d ( 11 ) by removing the N-isobutyryl- and p-methoxytrityl protecting group on consecutive treatment with 0.04N CH₃ONa in CH₃OH and HCOOH/CH₃OH 7:3 at 0° respectively. Enzymatic degradation of the free dinucleoside of the free dinucleoside monophosphates 7 and 11 yielded the corresponding nucleosides and nucleotides in the correct ratios.     

Nucleic-Acid Analogues with Constraint Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 1. Preparation of (3S,5′R)-2′-Deoxy-3′,5′-ethano-αβ-D-ribonucleosides (‘Bicyclonucleosides’)

We describe the synthesis of 2′-deoxy-3′,5′-ethano-D -ribonucleosides 1 – 8 (= (5′,8′-dihydroxy-2′-oxabicyclo-[3.3.0]oct-3′-yl)purines or -pyrimidines) of the nucleobases adenine, thymine, cytosine, and guanine. They differ from natural 2′-deoxyribonucleosides only by an additional ethylene bridge between the centers C(3′) and C(5′). The configuration at these centers (3S,5′R) was chosen as to match the geometry of a repeating nucleoside unit in duplex DNA as close as possible. These nucleosides were designed to confer, as constituents of an oligonucleotide chain, a higher degree of preorganization of a single strand for duplex formation with respect to natural DNA, thus leading to an entropic advantage for the pairing process. The synthesis of these ‘bicyclonucleosides’ was achieved by construction of an enantiomerically pure carbohydrate precursor 18 / 19 (Schemes 1), which was then converted to the corresponding nucleosides by known methods in nucleoside synthesis (Schemes 2 and 3). In all cases, both anomeric forms of the nucleosides were obtained in pure crystalline form, the relative configuration of which was established by ¹H-NMR-NOE spectroscopy. A conformational analysis of the nucleosides with β-configuration at the anomeric center by means of X-ray and ¹H-NMR (including NOE) spectroscopy show the furanose part of the molecules to adopt uniformly a 1′exo-conformation with the base substituents preferentially in the anti-range in the pyrimidine nucleosides (anti/syn ca. 2:1) distribution in the purine nucleosides (in solution).     

Synthesis of 3-Deaza-2′-deoxyadenosine and 3-Deaza-2′,3′-dideoxyadenosine: Glycosylation of the 4-Chloroimidazo[4,5-c]pyridinyl Anion

The convergent syntheses of 3-deazapurine 2′-deoxy-β-D -ribonucleosides and 2′,3′-dideoxy-D -ribonucleosides, including 3-deaza-2′-deoxyadenosine ( 1a ) and 3-deaza-2′,3′-dideoxyadenosine ( 1b ) is described. The 4-chloro-lH-imidazo[4,5-c]pyridinyl anion derived from 5 was reacted with either 2′-deoxyhalogenose 6 or 2′,3′-dideoxyhalogenose 10 yielding two regioisomeric (N¹ and N³) glycosylation products. They were deprotected and converted into 4-substituted imidazo[4,5-c]pyridine 2′-deoxy-β-D -ribonucleosides and 2′,3′-dideoxy-D -ribonucleosides. Compounds 1a and 1b proved to be more stable against proton-catalyzed N-glycosylic bond hydrolysis than the parent purine nucleosides and were not deaminated by adenosine deaminase.     

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.     

Saturated heterocycles. 242. Synthesis of 2-substituted-6-(6′,7′-dimethoxy-3′,4′-dihydro-1′-isoquinolyl)-5,6,7,8-tetrahydro- quinazolin-4(3H)-one Derivatives

In the reactions of the recently synthesized β-ketoesters 1-[(3′-methoxycarbonyl- and 1-[(3′-ethoxycarbonyl-4′-oxo)-1′-cyclohexyl]-3,4-dihydroisoquinoline 4, 5 with amidines or cyclic guanidines, a number of 2-substituted-6-(6′,7′-dimethoxy-3′,4′-dihydro-1′-isoquinolyl)-5,6,7,8-tetrahydroquinazolin-4(3H)-one derivatives 6–8 were prepared. The new compounds possess various pharmacological actions.