8-Aza-7-deazaadenine N8- and N8-(β-D-2′-Deoxyribofuranosides): Building blocks for automated DNA synthesis and properties of oligodeoxyribonucleotides

Oligonucleotides with alternating 8-aza-7-deaza-2′-deoxyadenosine (= c⁷z⁸A_d2) and dT residues (see 11, 14 and 16 ) or 4-aminopyrazolo [3,4-d] pyrimidine N²-(β-D -2′-deoxyribofuranoside) (= c⁷z⁸A′_d¹); ( 3 ) and dT residues (see 12 ) have been prepared by solid-phase synthesis using P(III) chemistry, Additionally, palindromic oligomers derived from d(C-T-G-G-A-T-C-C-A-G) but containing 2 or 3 instead of dA (see 18 – 22 ) have been synthesized. Benzoylation of 2 or 3 , followed by 4,4′-dimethoxytritylation and subsequent phosphitylation yielded the methyl or the cyanoethyl phosphoramidites 8a,b and 9 . They were employed in automated. DNA synthesis. Alternating oligomers containing 2 or 3 showed increase dT_m values compared to those with dA, in particular 12 with an unusual N²-glycosylic bond. The palindromic oligomers 18 - 22 containing 2 or 3 instead of dA outside of the enzymic recognition side reduced the hydrolysis rate, replacement within d(G-A-T-C) abolished phosphodiester hydrolysis.     

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines: The Influence of 7-Substituted 8-Aza-7-deaza-2′-deoxyguanosines on the Duplex Structure and Stability

Oligonucleotides containing 7-substituted 8-aza-7-deazaguanines (=6-amino-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones) were prepared by automated solid-phase synthesis. A series of 7-alkynylated 8-aza-7-deaza-2′-deoxyguanosines (see 4a – d ) were synthesized with the 7-iodonucleoside 3c as starting material and by the Pd⁰/Cu^I-catalyzed cross-coupling reaction with various alkynes. Phosphoramidites were prepared from the 7-substituted 8-aza-7-deaza-2′-deoxyguanosine derivatives carrying halogeno, cyano, and hexynyl substituents. From the melting profiles of oligonucleotide duplexes, the T_m values as well as the thermodynamic data were determined. A significant duplex stabilization by the 7-substituents was observed for the DNA⋅DNA duplexes, but not in the case of DNA⋅RNA hybrids.     

N6-(Carbamoylmethyl)-2′-deoxyadenosine,a rare DNA Constituent: Phosphoramidite Synthesis and Properties of Palindromic Dodecanucleotides

N⁶-(Carbamoylmethyl)-2′-deoxyadenosine ( 1 ), a modified nucleoside occurring in bacteriophage Mu, was synthesized by two different routes. Glycinamide was introdued by nucleophilic displacement of(2,4,6,-triisopro-pylphenyl)sulfonyloxy or ethylsulfinyl groups at C(6) of the purine moiety. Compound 1 was converted into the protected phosphoramidite 6b and employed in solid-phase synthesis of the self-complementary oligonucleotides 7–14 . Replacement of 2′-deoxyadenosine by 1 led to a strong decrease of the T_m values of the oligomers d(A-T)₆ ( 7 ) and d(A-T-G-A-A-G-C-T-T-C-A-T)( 10 ), respectively. As the oligemer 10 contains the recognition site d(A-A-G-C-T-T) of the endodeoxyribonuclease Hind III, it was subjected to sequence-specific hydrolysis experiments. Replacement of the first or second A_d by 1 prevented enzymatic phosphodiester hydrolysis (results with 11 and 12 ). In contrast, slow hydrolysis was observed if the less bulky N⁶-methyl-2′-deoxyadenosine replaced the second A _d residue (results with 14 ).     

Studies on the Base-Pairing Properties of N7-(2-Deoxy-β-D-erythro-pentofuranosyl)guanine (N7Gd)

The base-pairing properties of N⁷-(2-deoxy-β-D -erythro-pentofuranosyl)guanine (N⁷G_d; 1 ) are investigated. The nucleoside 1 was obtained by nucleobase-anion glycosylation. The glycosylation reaction of various 6-alkoxy-purin-2-amines 3a - i with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 8 ) was studied. The N⁹/N⁷-glycosylation ratio was found to be 1:1 when 6-isopropoxypurin-2-amine ( 3d ) was used, whereas 6-(2-methoxyethoxy)purin-2-arnine ( 3i ) gave mainly the N⁹-nucleoside (2:1). Oligonucleotides containing compound 1 were prepared by solid-phase synthesis and hybridized with complementary strands having the four conventional nucleosides located opposite to N⁷G_d. According to T_m values and enthalpy data of duplex formation, a base pair between N⁷G_d and dG is suggested. From the possible N⁷G_d dG base pair motives, Hoogsteen pairing can be excluded as 7-deaza-2′-deoxyguanosine forms the same stable base pair with N⁷G_d as dG.     

7-Substituted 7-Deaza-2′-deoxyguanosines: Regioselective Halogenation of Pyrrolo[2,3-d]pyrimidine Nucleosides

The synthesis of 7-chloro-, 7-bromo-, and 7-iodo-substituted 7-deaza-2′-deoxyguanosine derivatives 2b – d is described. The regioselective 7-halogenation with N-halogenosuccinimides was accomplished using 7-[2-deoxy-3,5-O-di(2-methylpropanoyl)-β-D -erythro- pentofuranosyl]-2-(formylamino)-4-methoxy-7H-pyrrolo[2,3-d]- pyrimidine ( 4 ) as the common precursor. A one-pot reaction (2N aq. NaOH) of the halogenated intermediates 5a – c furnished the desired compounds. Also the 7-hexynyl derivative 2e of 7-deaza-2′-deoxyguanosine is described.     

Oligonucleotides Containing 7‐Deaza‐2′‐deoxyinosine as Universal Nucleoside: Synthesis of 7‐Halogenated and 7‐Alkynylated Derivatives,Ambiguous Base Pairing,and Dye Functionalization by the Alkyne–Azide ‘Click’ Reaction

Oligonucleotides containing 7‐deaza‐2′‐deoxyinosine derivatives bearing 7‐halogen substituents or 7‐alkynyl groups were prepared. For this, the phosphoramidites 2b – 2g containing 7‐substituted 7‐deaza‐2′‐deoxyinosine analogues 1b – 1g were synthesized (Scheme 2). Hybridization experiments with modified oligonucleotides demonstrate that all 2′‐deoxyinosine derivatives show ambiguous base pairing, as 2′‐deoxyinosine does. The duplex stability decreases in the order C_d>A_d>T_d>G_d when 2b – 2g pair with these canonical nucleosides (Table 6). The self‐complementary duplexes 5′‐d(F⁷c⁷I‐C)₆, d(Br⁷c⁷I‐C)₆, and d(I⁷c⁷I‐C)₆ are more stable than the parent duplex d(c⁷I‐C)₆ (Table 7). An oligonucleotide containing the octa‐1,7‐diyn‐1‐yl derivative 1g , i.e., 27 , was functionalized with the nonfluorescent 3‐azido‐7‐hydroxycoumarin ( 28 ) by the Huisgen–Sharpless–Meldal cycloaddition ‘click’ reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently, oligonucleotides incorporating the derivative 1g bearing a terminal C?C bond show a number of favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the ‘click’ chemistry. ii) Space demanding residues introduced in the 7‐position of the 7‐deazapurine base does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry, molecular biology, and nanobiotechnology.     

Synthesis of 8-Aza-2′-deoxyadenosine and related 7-Amino-3H-1,2,3-triazolo[4,5-d]pyrimidine 2′-Deoxyribofuranosides: Stereoselective glycosylation via the nucleobase anion

The synthesis of 8-aza-2′-deoxyadenosine ( = 7-amino-3H-1,2,3 triazolo[4,5-d]pyrimidine N³-(2′-deoxy-β-D-ribofuranoside); 1 ) as well as the N²- and N¹-(2′-deoxy-β-D-ribofuranosides) 2 and 3 is described. Glycosylation of the anion of 7-amino-3H-1,2,3-triazolo[4,5-d]pyrimidine ( 6 ) in DMF yielded three regioisomeric protected 2′-deoxy-β-D-ribofuranosides, i.e. the N³-, N²-, and N⁴-glycosylated isomers 7 (14%), 9 (11%), and 11 (3%), respectively, together with nearly equal amounts of their α-D-anomers 8 (13%), 10 (12%), and 12 (4%; Scheme 1). The reaction became Stereoselective for the β-D-nucleosides if the anion of 7-methoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine ( 13 ) was glycosylated in MeCN: only the N³-, N², and N¹-(2′-deoxy-β-D-nucleosides) 14 (29%), 15 (32%), and 16 (23%), respectively, were formed (Scheme 2). NH₃ Treatment of the methoxynucleosides 14–16 afforded the aminonucleosides 1–3 . The anomeric configuration as well as the position of glycosylation were determined by combination of ¹³ C-NMR , ¹ H-NMR , and 1D-NOE difference spectroscopy. Compound 1 proved to be a substrate for adenosine deaminase, whereas the regioisomers 2 and 3 were not deaminated.     

8-Aza-7-deaza-2′,3′-dideoxyguanosine: Deoxygenation of its 2′deoxy-β-D-ribofuranoside

The synthesis of 6-amino-1-(2′,3′-dideoxy-β-D -glycero-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one ( =8-aza-7-deaza-2′,3′-dideoxyguanosine; 1 ) from its 2′-deoxyribofuranoside 5a by a five-step deoxygenation route is described. The precursor of 5a, 3a , was prepared by solid-liquid phase-transfer glyscosylation which gave higher yields (57%) than the liquid-liquid method. Ammonoloysis of 3b furnished the diamino nucleoside 3c . Compound 1 was less acid sensitive at the N-glycosydic bond than 2′,3′-dideoxyguanosine ( 2 ).     

1,7-Dideaza-2′-deoxyadenosine: Building blocks for solid-phase synthesis and secondary structure of base-modified oligodeoxyribonucleotides

The 1,7-dideaza-2′-deoxyadenosine (c¹c⁷A_d; 1 ) was converted into building blocks 3a , b for solid-phase oligodeoxyribonucleotide synthesis. Testing various N-protecting groups – benzoyl, phenoxyacetyl, [(fluoren-9-yl)methoxy]carbonyl, and (dimethylamino)methylidene – only the latter two were found to be suitable ( 1 → 4b, d ). Ensuing 4,4′-dimethoxytritylation of 4d and phosphitylation afforded the 3′-phosphonate 3a or the 3′-[(2-cyanoethyl)diisopropylphosphoramidite] 3b . Self-complementary oligonucleotides with alternating dA or c¹c⁷A_d and dT residues ( 7 and 8 ) as well as palindromic oligomers such as d(C-G-C-G-c¹c⁷ A-c¹c⁷ A-T-T-C-G-C-G) ( 10 ) and d(G-T-A-G-c¹c⁷ A-c¹c⁷ A-T-T-C-T-A-C) ( 12 ) were synthesized. Duplex stability was decreased because 1 cannot form Watson-Crick or Hoogsteen base pairs if incorporated into oligonucleotides. On the other hand, the structural modifications in 10 and 12 forced these palindromic oligomers to form hairpin structures.     

7-Deazaguanine DNA: Oligonucleotides with hydrophobic or cationic side chains

The phosphoramidites 6b and 9 as well as the phosphonate 6a derived from 7-(hex-1-ynyl)- and 7-[5-(trifluoroacetamido)pent-1-ynyl]-substituted 7-deaza-2′-deoxyguanosines 1 and 10 , respectively, were prepared (Scheme 1). They were employed in solid-phase oligodeoxynucleotide synthesis of the alternating octamers d(hxy⁷c⁷G-C)₄ ( 12 ), d(C-hxy⁷c⁷G)₄ ( 13 ), and d(npey ⁷c⁷G-C)₄ ( 15 ) as well as of other oligonucleotides (see 22 – 25 ; Table 2; hxy = hex-1-ynyl, npey = 5-aminopent-1-ynyl). The T_m values and the thermodynamic data of duplex formation were determined and correlated with the major-groove modification of the DNA fragments. A hexynyl side chain introduced into the 7-position of a 7-deazaguanine residue (see 1 ) was found to fit into the major groove without any protrusion. The incorporation of the (5-aminopent-1-ynyl)-modified 7-deaza-2′-deoxyguanosine 2 into single-stranded oligomers of the type 24 and 25 did not lead to change in duplex stability compared to the parent oligonucleotides. The self-complementary oligomer 15 with alternating npey⁷c⁷G_d ( 2 ) and dC units did not lead to a cooperative melting, either due to orientational disorder or interaction of the 5-aminopent-1-ynyl moiety with a base or with phosphate residues nearby or on the opposite strand.     

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 ).     

Duplex Stability of 7-Deazapurine DNA: Oligonucleotides containing 7-bromo- or 7-iodo-7-deazaguanine

The oligonucleotide building blocks, the phosphonates 1a, b and the phosphoramidites 2a, b derived from 7-iodo- and 7-bromo-7-deaza-2′-deoxyguanosines 3a, b were prepared. They were employed in solid-phase oligonucleotide synthesis of the alternating octamers d(Br⁷c⁷G-C)₄ ( 8 ) and d(I⁷c⁷G-C)₄ ( 9 ) as well as the homo-oligonucleotides d[(Br⁷c⁷G)₅-G] ( 11 ) and d[(I⁷c⁷G)₅-G] ( 12 ). The melting profiles and CD spectra of oligonucleotide duplexes were measured. The T_m values as well as the thermodynamic data were determined and correlated to the major-groove modification of this DNA. The self-complementary octamers 8 and 9 form more stable duplexes compared to the parent oligomer d(G-C)₄. The heteroduplex of d[(I⁷c⁷G)₅-G] ( 12 ) with d(C₆) is slightly destabilized (ΔT_m = ?12°) over that of d[(c⁷G)₅-G] with d(C₆). However, the complex of 12 with poly(C) is more stable than that of d[(c⁷G₅-G)] with poly(C).     

8-Aza-7-deaza-2′-deoxyguanosine: Phosphoramidite synthesis and properties of octanucleotides

Base-modified octanucleotides derived from d(G1–G2–A–A–T–T–C–C–) ( 11 ) but containing 8-aza-7-deaza-2′-deoxyguanosine ( 2 ) instead of 2′-deoxyguanosine ( 1 ) have been prepared by solid-phase synthesis employing P(III) chemistry. Isobutyrylation of 2 , followed by 4, 4′-dimethoxytritylation and subsequent phosphitylation yielded the methyl or the cyanoethyl phosphoramidites 6a or 6b , respectively. They were used as building blocks in automated DNA synthesis. The resulting octanucleotides 12–14 containing 2 showed increased T_m values compared to the parent oligomer 11 . The oligomers 11 – 14 were employed as sequence-specific probes in endo-deoxyribonuclease Eco RI oligonucleotide recognition. Whereas displacement of dG-2 (enzymic cleavage site of 11 ) abolished phosphodiester hydrolysis, replacement of dG-1 enhanced the cleavage rate compared to 11 .     

Hoogsteen-Duplex DNA: Synthesis and base pairing of oligonucleotides containing 1-deaza-2′-deoxyadenosine

Oligodeoxyribonucleotides containing 1-deaza-2′-deoxyadenosine ( = 7-amino-3-(2-deoxy-β-D -erythro-pentofuranosyl)-3H-imidazo[4, 5-b]pyridine; 1b ) form Hoogsteen duplexes. Watson-Crick base pairs cannot be built up due to the absence of N(1). For these studies, oligonucleotide building blocks – the phosphonate 3a and the phosphoramidite 3b – were prepared from 1b via 4a and 5 , as well as the Fractosil-linked 6b , and used in solid-phase synthesis. The applicability of various N-protecting groups (see 4a – c ) was also studied. The Hoogsteen duplex d[(c¹A)₂₀] · d(T₂₀) ( 11 · 13 ; T_m 15°) is less stable than d(A₂₀) · d(T₂₀) ( 12 · 13 ; T_m 60°). The block oligomers d([c¹A)₁₀–;T₁₀] ( 14 ) and d[T₁₀–(c¹A)₁₀] ( 15 ) containing purine and pyrimidine bases in the same strand are also able to form duplexes with each other. The chain polarity was found to be parallel.     

Synthesis of 2′-Deoxyribofuranosides of 8-Aza-7-deazaguanine and Related Pyrazolo[3,4-d]pyrimidines

The N(1)- and N(2)-(2′-deoxyribofuranosides) 1 and 2 , respectively, of 8-aza-7-deazaguanine were prepared via phase-transfer glycosylation in the presence or absence of Bu₄NHSO₄ as catalyst of 6-amino-4-methoxy-lH-pyrazolo[3,4-d]pyrimidine ( 7c ) with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 10 ). On a similar route, but without catalyst and employing THF as organic phase, the 6-amino-4-chloronucleosides 11b and 12b were synthesized from 7a and converted into the N(1)-and N(2)-substituted 4-thioxo analogues 17a and 18a , respectively. The ratio of N(1)- to N(2)-glycosylation was 2:1 for 7c and 1:1 for 7a , viz. depending on the nucleobase structure. The rate of the H⁺-catalyzed N-glycosyl hydrolysis was strongly decreased for the N(2)-(β-D -2′-deoxyribofuranosides) as compared to the N(1)-compounds. However, the N(1)-nucleoside 1 , which is an isostere of 2′-deoxyguanosine, is sufficiently stable to be employed later in solid-phase oligonucleotide synthesis.     

N7-DNA: Synthesis and Base Pairing of Oligonucleotides containing 7-(2-Deoxy-β -D-erythro-pentofuranosyl)adenine (N7Ad)

The synthesis of oligonucleotides containing 7-(2-deoxy-β -D -erythro-pentofuranosyl)adenine (N⁷A_d; 1 ) is described. Compound 1 was obtained from the precursor 4-amino-1H -imidazole-5-carbonitrile 2-deoxyribonucleoside 6 and was found to be much more labile than A_d. The N⁶-benzoyl protecting group (see 8 ) destabilized the N-glycosylic bond further and was difficult to remove by NH₃-catalyzed hydrolysis. Therefore, a (dimethyl-amino)methylidene residue was introduced (→ 9 ). Amidine 9 was blocked at OH? C(5′) with the dimethoxytrityl residue ((MeO)₂Tr), and phosphonate 4 as well as phosphoramidite 5 were prepared under standard conditions. Phosphonate 4 was employed in solid-phase oligonucleotide synthesis. Homooligonucleotides as well as self-complementary oligonucleotides were prepared. The oligomer d[(N⁷A)₁₁-A] ( 11 ) formed a duplex with d(T₁₂) ( 13 ). Antiparallel chain polarity and reverse Watson-Crick base pairing was deduced from duplex formation of the self-complementary d[(N⁷A)₈-T₈] ( 14 ).     

N 7-DNA: Base-Pairing Properties of N 7-(2′-Deoxy-β-D-erythro-pentofuranosyl)-Substituted Adenine,Hypoxanthine, and Guanine in Duplexes with Parallel Chain Orientation

Oligonucleotides containing N ⁷-(2′-deoxy-β-D -erythro-pentofuranosyl)adenine ( 1 ), -hypoxanthine ( 2 ), and -guanine ( 3 ) were synthesized on solid-phase using phosphonate and phosphoramidite chemistry. As part of the synthesis of compound 2 , the nucleobase-anion glycosylation of various 6-alkoxypurines with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 5 ) was investigated. The duplex stability of oligonucleotides containing N ⁷-glycosylated purines opposite to regular pyrimidines was determined, and thermodynamic data were calculated from melting profiles. Oligodeoxyribonucleotide duplexes containing N ⁷-glycosylated adenine⋅T_d or N ⁷-glycosylated guanine⋅C_d base pairs are more stable in the case of parallel strand orientation than in the case of antiparallel chains.