Design, Synthesis, and Polymerase‐Catalyzed Incorporation of Click‐Modified Boronic Acid–TTP Analogues

Abstract

DNA molecules are known to be important materials in sensing, aptamer selection, nanocomputing, and construction of unique architectures. The incorporation of modified nucleobases affords unique DNA properties for applications in areas that would otherwise be difficult or not possible. Earlier, we demonstrated that the boronic acid moiety can be introduced into DNA through polymerase‐catalyzed reactions. In order to study whether such incorporation by polymerase is a general phenomenon, we designed and synthesized four boronic acid‐modified thymidine triphosphate (TTP) analogues. The synthesis of certain analogues was through the use of a single dialkyne tether for both the Sonogashira coupling with thymidine and the later Cu‐mediated [3+2] cycloaddition for linking the boronic acid moiety. This approach is much more efficient than the previously described method, and paves the way for the preparation of a large number of boronic acid‐modified TTPs with a diverse set of structural features. All analogues showed very good stability under polymerase chain reaction (PCR) conditions and were recognized as a substrate by DNA polymerase, and thus incorporated into DNA.

Enzymatic Synthesis of 7′,5′‐Bicyclo‐DNA Oligonucleotides

“…[4] The enzymatic polymerization of modified nucleoside triphosphates (dN*TPs)r epresents av alid and solid alternative for the inclusion of chemical diversity into nucleic acids. [5] Indeed, ap lethora of functional groups ranging from boronic acids, [6] protein-like residues, [7] and redox labels [8] to biomolecules [9] have been incorporated into DNA and RNA by this enzymaticm ethod. In addition to large functional group tolerance, the polymerization of dN*TPs offers the advantages of facile access to long sequences (> 100 nt) and amplification by the polymerase chain reaction (PCR).…”

Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids

“…Indeed, no protecting group is required and the use of trialkylphosphate solvents mainly directs the phosphorylation to the 5'-regioisomer [ 7 , 10 , 11 ]. Consequently, a vast array of dNTPs modified with functionalities such as organic polymers [ 12 ], diamondoid-like structures [ 13 ], amino acid and amino acid-like residues [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ], modified dNTPs with an sp 3 -hybridized carbon connecting the nucleobase and the linker arm [ 22 , 23 , 24 ], perfluorinated side-chains [ 25 ], unnatural bases [ 26 , 27 , 28 , 29 ], boronic acids [ 30 ], 2'-methylseleno triphosphates [ 31 , 32 ], and dual modified 4'- C -aminomethyl-2'- O -methylthymidine [ 33 ] have been reported. However, the use of a strong electrophilic phosphorous reagent is not compatible with all nucleosides [ 34 ], and modern analytical techniques have revealed the formation of a quantity of undesirable by-products [ 35 ].…”

Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

“…16,17 Due to staggering advances in organic synthesis and polymerase engineering, the co-polymerization of modified deoxynucleoside triphosphates (dN*TPs) has advanced as a versatile and convenient method for increasing the chemical repertoire of the functionality deprived nucleic acids. 18,19 Indeed, the inclusion of dN*TPs in primer extension reactions has allowed to equip nucleic acids with a variety of functional groups including amino acids, [20][21][22][23][24] redox labels, [25][26][27] side-chains capable of organocatalysis, 28 transition metals, 29,30 boronic acids, 31 and even oligonucleotides. 32 These modified nucleotides are typically inserted in oligonucleotides by primer extension reactions (PEX) or PCR, yielding dsDNA products which can then be further converted to ssDNAs.…”