2‐Substituted dATP Derivatives as Building Blocks for Polymerase‐Catalyzed Synthesis of DNA Modified in the Minor Groove

Abstract

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH 2 ,CH 3 ,vinyl, ethynyl, and phenyl) at position 2were prepared and tested as substrates for DNApolymerases.The 2-phenyl-dATP was not asubstrate for DNApolymerases,but the dATPs bearing smaller substituents were good substrates in primer-extension experiments,producing DNAsubstituted in the minor groove. The vinyl-modified DNAw as applied in thiol-ene addition and the ethynylmodified DNAwas applied in aCuAACclick reaction to form DNAlabelled with fluorescent dyes in the minor groove Base-modified oligonucleotides (ONs) or DNAa re widely used as tools in chemical biology,d iagnostics,o rm aterials science.[1] Themodification is mostly attached to position 5of pyrimidines or position 7of7-deazapurines,not only because it then points out into the major groove of DNAand thus does not destabilize the duplex, but because in most cases,t he corresponding substituted 2'-deoxyribonucleoside triphosphates (dNTPs) are good substrates for DNAp olymerases and can be used in the polymerase-catalyzed synthesis of modified DNA. [2,3] Diverse modifications,i ncluding fluorophores, [4] redox [5] or spin labels, [6] reactive groups for conjugations, [7] and biomolecules (e.g., oligonucleotides [8] or proteins [9] ), have been introduced into the major groove through the enzymatic incorporation of modified nucleotides and applied in different fields.Modification or labelling of the minor groove has mostly been reported with 2'-and 4'-sugarmodified derivatives.[10-13] 2-Chloroadenine [14] and 2,6-diaminopurine [15] dNTPs are the only minor-groove base-modified nucleotides that have been reported as substrates for DNA polymerases,w hereas 2-arylamino-dATP derivatives were found to act as polymerase inhibitors.[16] Them inor groove sites of the nucleobases are difficult to modify since they are crucial both for Watson-Crick base pairing and for key minorgroove interactions with DNApolymerase that are important for extension of the chain.[17] On the other hand, 2-ethynylpyridone-C-nucleotide incorporated into DNA [18] formed as table base pair with adenine,a nd 2-(imidazolylalkylamino)purines in ONs also stabilized duplexes. [19] Because the possibility of minor-groove base labelling would be attractive for many prospective applications,for example,the mapping of DNA-protein interactions,w ee nvisaged that as mall substituent at position 2ofapurine may not fully disturb the key H-bonding interactions with the opposite base and the polymerase,a nd we report herein the first enzymatic synthesis of minor-groove base-modified DNA.Aseries of six 2-substituted dATP derivatives bearing Cl, NH 2 ,C H 3 ,v inyl, ethynyl and phenyl substituents (d R ATPs) was designed to study the effect of substituents of different bulkiness at position 2o fa denine on polymerase-mediated incorporation. While dCl ATP [14,20] and d NH2 ATP [15] were known, the others were prepared through triphosphorylation [21] of the corresponding 2'-deoxy-ribonucleosides (d R As...

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

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Convertible and Constrained Nucleotides: The 2’-Deoxyribose 5’-C-Functionalization Approach, a French Touch

“…One key feature (for better or for worse) is also the reversibility of such a bond, cleaved under reducing conditions [ 38 ]. Thiol-ene chemistry was also exemplified by the Hocek group, reaching good coupling yield without the need of UV irradiation [ 39 , 40 ].…”

Enzymatic synthesis and modification of high molecular weight DNA using terminal deoxynucleotidyl transferase

“…However, dNTPs modified with bulky hydrophobic groups such as dibenzylcyclooctyne (DBCO) or large fluorophores such as Cy3 or fluorescein often require a hydrophilic linker such as poly(ethylene glycol) (PEG) to position these groups away from the enzyme's active site. A study of how TdT tolerates modifications at position 2 of dATP found that bulky modifications such as phenyl groups have an inhibitory effect on TdT activity, while smaller modifications such as Cl, NH 2 , CH 3 , vinyl, and ethynyl groups are readily incorporated (Matyašovský, Perlíková, Malnuit, Pohl, & Hocek, 2016). Interestingly, dNTPs with bulky, hydrophobic modifications impart nuclease stability to the modified ssDNA (Gu et al, 2018).…”