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Synthesis of 3′‐Sugar‐ and Base‐Modified Nucleotides and Their Application as Potent Chain Terminators in DNA Sequencing
Author(s) -
Stolze Karen,
Koert Ulrich,
Klingel Sven,
Sagner Gregor,
Wartbichler Regina,
Engels Joachim W.
Publication year - 1999
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/(sici)1522-2675(19990908)82:9<1311::aid-hlca1311>3.0.co;2-z
Subject(s) - chemistry , thiourea , nucleotide , dna , base pair , combinatorial chemistry , base (topology) , thymidine , stereochemistry , biochemistry , organic chemistry , gene , mathematical analysis , mathematics
Two 3′‐modified and three base‐modified ddNTPs were synthesized and tested with several DNA polymerases for incorporation activity. Starting from 3′‐azido‐3′‐deoxythymidine (AZT; 1 ), we were able to produce 3′‐deoxy‐3′‐isocyanato‐thymidine and 3′‐deoxy‐3′‐isothiocyanatothymidine ( 3 and 4 , resp.) in a rapid synthesis based on the solid‐support approach ( Scheme 1 ). These 3′‐functionalities could be used to attach a spacer molecule via urea and thiourea groups, respectively. Since the thus‐obtained tethered nucleotides 7 and 8 can be used to label with fluorescent dyes ( cf . Scheme 5 ), they are convenient building blocks for practical applications in DNA sequencing. Furthermore, we synthesized, via 14 ( Scheme 2 ), 17 ( Scheme 3 ), and 19 ( Scheme 4 ), the N 4 ‐modified dideoxycytidine 5′‐triphosphate dye derivatives 22 , 23 , and 24 , respectively, with different lengths of linkers between the base residue and the dye ( Scheme 5 ). Base‐specific termination for the derivatives 22 and 24 was demonstrated ( Fig. 2a and 2b ).