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Synthesis of Deoxynucleoside Triphosphates that Include Proline, Urea, or Sulfonamide Groups and Their Polymerase Incorporation into DNA
Author(s) -
Hollenstein Marcel
Publication year - 2012
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201201662
Subject(s) - klenow fragment , dna polymerase , dna polymerase i , chemistry , polymerase , biochemistry , oligonucleotide , dna , stereochemistry , exonuclease , polymerase chain reaction , reverse transcriptase , gene
To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling of L ‐proline‐containing residues (dU t P TP and dU c P TP), a dipeptide (dU FP TP), a urea derivative (dU Bpu TP), and a sulfamide residue (dU Bs TP) to a suitably protected common intermediate, followed by triphosphorylation. These modified dNTPs were shown to be excellent substrates for the Vent ( exo − ) and Pwo DNA polymerases, as well as the Klenow fragment of E. coli DNA polymerase I, although they were only acceptable substrates for the 9° N m polymerase. All of the modified dNTPs, with the exception of dU Bpu TP, were readily incorporated into DNA by the polymerase chain reaction (PCR). Modified oligonucleotides efficiently served as templates for PCR for the regeneration of unmodified DNA. Thermal denaturation experiments showed that these modifications are tolerated in the major groove. Overall, these heavily modified dNTPs are excellent candidates for SELEX.