Polymer supported DNA synthesis using hydroxybenzotriazole activated pbosphotriester intermediates (original) (raw)
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Protection-Free One-Pot Synthesis of 2′-Deoxynucleoside 5′-Triphosphates and DNA Polymerization
Organic Letters, 2011
By differentiating the functional groups on nucleosides, we have designed and developed a onepot synthesis of deoxyribonucleoside 5′-triphosphates without any protections on the nucleosides. A facile synthesis is achieved by generating an in situ phosphitylating reagent that reacts selectively with the 5′-hydroxyl groups of the unprotected nucleosides. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. This novel approach is straightforward and cost-effective for triphosphate synthesis. Nucleoside 5′-triphosphates (dNTPs and NTPs) are the building blocks for the synthesis of nucleic acids (DNA and RNA) and are also utilized in many important biological systems, including DNA replication, RNA transcription, purinergic signaling, neurotransmission, and signal transduction. 1 To better understand the roles of triphosphates and meet the needs in nucleic acid research, the first chemical synthesis of nucleoside 5′-triphosphates was achieved over six decades ago. 2 Although numerous strategies have been continuously developed in the past decades, 3 a convenient synthesis of the nucleoside 5′-triphosphates remains as a long-standing challenge. This is primarily due to the multiple functionalities (hydroxyl and amino groups) of the nucleosides, which generally requires many synthetic steps because of the protection and deprotection of these functionalities. Moreover, the synthesis of nucleoside 5′-triphosphates generates many by-products that are very difficult to remove. Thus, there is an urgent need to develop straightforward strategies for synthesizing nucleoside 5′-triphosphates in order to reduce the cost of triphosphates (especially modified ones) significantly and meet the growing needs in signal regulation research and studies of nucleic acid structure, function, and detection. 4 Nucleoside triphosphates are currently synthesized via three major approaches: phosphitylation (or phosphoramiditylation), 3a-c, 5 mono-phosphate isolation/ activation, 3c-e, 6 and phosphorylation. 3b, c, 7 The first strategy employs a highly reactive phosphitylating agent, thereby requiring the protection of both the sugar and nucleobase moieties 3c, 5a-c, 5f, g in order to reduce unwanted by-products. Since the 5′-hydroxy groups
Nucleoside 5′-triphosphates with modified sugars as substrates for DNA polymerases
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1986
A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase a. It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates, ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase a only, but araNTP(3'NH2) was inactive with E. coil DNA polymerase I. dTTP(3'N3),
FEBS Letters, 1993
S-Triphosphates of 1-(2',3'-epithio-2'.3'-dideoxy-B_D-lyxofuranosyl)thymine, l-(2',3'-eptthio-~,~-dideoxy-~-D-ribofuranosyl)th~lne and 2',3'lyxoanhydrothymidine have been shown to be termmatton substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase 1 from E. co11 and DNA polymerase j3 from rat hver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase E from human placenta. Km values of ItTTP. rtTTP and laTTP incorporation mto the DNA chain during catalysts by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than K,,, value for dTTP Furthermore, V,,, values for modified substrates are only 2-3 times lower than b',,,',,, for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides wtth a flattened furanosyl rmg for DNA polymerase active sites.
Phosphotriester approach to the synthesis of oligonucleotides: a reappraisal
Journal of the Chemical Society, Perkin Transactions 1, 1993
The phosphotriester approach to the synthesis of oligodeoxyribo-and oligoribo-nucleotides in solution has been reinvestigated. The efficacy of mesitylene-2-sulfonyl chloride (MSCI) 15a, 2,4,6triisopropylbenzenesulfonyl chloride (TrisCI) 1 5b, 4bromobenzenesulfonyl chloride 1 5c. naphthalene-1-sulfonyl chloride 39, and 2-and 4-nitrobenzenesulfonyl chlorides 40a and 40b, respectively, as activating agents has been examined. The latter arenesulfonyl chlorides have been used in conjunction with the following nucleophilic catalysts: 1-methylimidazole, 3-nitro-I H-1,2,4-triazole 19, 5-(3-nitrophenyl)-l H-tetrazole 20a, 5-(3.5-dinitrophenyl)-l H-tetrazole 20b. 5-(1-methylimidazol-2y l)-I H-tetrazole 21, 5-[ (1-methylimidazol-2-yl)methyl]-1 H-tetrazole 22, 4-ethoxypyridine 1-oxide 14a. 4,6-dinitro-l-hydroxybenzotriazole 29a, 1-hydroxy-4-nitro-6-(trifluoromethyl) benzotriazole 29b. 1-hydroxy-5-phenyltetrazole 30a and 1-hydroxy-5-(3-nitrophenyl) tetrazole 30b. The rates of formation and yields of the fully protected dideoxyribonucleoside and diribonucleoside phosphates 37 and 47, respectively, were determined using various combinations of activating agents and nucleophilic catalysts. Although 2-and 4-nitrobenzenesulfonyl chlorides 40a and 40b. respectively, proved to be the most powerful activating agents, their use in the deoxy-series led to the formation of by-products and hence to unsatisfactory isolated yields of the dideoxyribonucleoside phosphate 37. I I +-0 ii. iii /o 3 4 Ar = 2-chlorophenyl; B and B' are protected in substrates 1, 2 and 3, and unprotected base residues in product 4
Termination of DNA synthesis by novel 3'-modifieddeoxyribonucleoside 5'-triphosphates
Nucleic Acids Research, 1994
Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-0-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-0-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stopstart DNA synthesis are initial steps in the development of a novel sequencing strategy.
The topochemical synthesis of triazole-linked homobasic DNA
Chemical Communications, 2016
Triazolyl-DNA (TLDNA), DNA wherein phosphodiester units are replaced by triazole units, is of great interest. By adopting Topochemical Azide–Alkyne Cycloaddition (TAAC) reaction, we have synthesized homobasic TLDNA oligomers. 5′-ethynyl-3′-azido-2′,3′,5′-tri-deoxycytosine, which crystallized with proximal placement of azide and alkyne units of adjacent molecules, underwent TAAC reaction to TLDNA oligomers.
Angewandte Chemie International Edition, 2010
Apart from a wide range of novel applications of functionalized DNA in chemical biology, nanotechnology, and material sciences, attachment of reactive functional groups to nucleic acids is needed for further transformations or bioconjugates. The introduction of alkyne, azide, or diene groups either by chemical phosphoramidite synthesis or by enzymatic polymerase synthesis has been achieved and the modified DNA was used for click-chemistry, Staudinger ligation, and Diels-Alder reactions. An aldehyde functional group is a very attractive group because of its high and specific reactivity with diverse reagents. However, it was considered too reactive and fragile to be incorporated directly (chemically or enzymatically) and the few successful examples were prepared indirectly by a click reaction with azide derivatives of reducing sugars, or by introduction of 2,3-dihydroxypropyl or 3,4-dihydroxypyrrolidine moieties and subsequent oxidative cleavage of the vicinal diols to (di)aldehydes. The syntheses of the nucleoside/nucleotide monomers were laborious multistep procedures and additional post-synthetic steps were required to release the aldehyde function in DNA. Metallization or interstrand cross-linking were demonstrated to be very useful applications of aldehyde-modified oligonucleotides (ONs) or DNA. Therefore we decided to develop a simple and efficient direct protocol for construction of aldehyde-modified DNA by application of our two-step (cross-coupling polymerase incorporation) method. In addition, we wished to develop a methodology for additional conjugation and staining of aldehyde-modified DNA by hydrazone formation.
Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.], 2011
This unit describes procedures for preparation of two phosphoramidite building blocks III and IV, both containing a TBDMS as 5-CH(2)OH-protecting group. Phosphoramidites III and IV allow efficient incorporation of 5-hmC into DNA and a "one-step" deprotection procedure to cleanly remove all the protecting groups. A "two-step" deprotection strategy is compatible with ultramild DNA synthesis, which enables the synthesis of 5 hmC-containing DNA with additional modifications. Methods are also presented for their incorporation into oligonucleotides by solid-phase synthesis, subsequent deprotection, and HPLC analysis.
Solid-Phase Chemical Synthesis of 5′-Triphosphate DNA, RNA, and Chemically Modified Oligonucleotides
Current Protocols in Nucleic Acid Chemistry, 2001
A chemical method for the solid-phase synthesis of 5 -triphosphate oligonucleotides is described. The full-length oligonucleotides are first constructed using standard solidphase DNA/RNA synthesis, and then efficient implementation of a sequential 4-steps synthetic procedure, executed either manually or in a fully automated fashion, affords the corresponding solid-supported 5 -triphosphate oligonucleotides. Using this synthetic procedure, the full-length 5 -hydroxyl oligonucleotides are initially transformed into the corresponding 5 -H-phosphonate mono esters, subsequently oxidized in the presence of imidazole to the activated 5 -phosphorimidazolidates, and finally reacted with pyrophosphate on the solid support. The method uses safe, stable, and inexpensive reagents, and the process is scalable and readily applicable to automated synthesis compatible with the current commercially available DNA/RNA synthesizers. After cleavage from the solid support and deprotection, a range of DNA, RNA, and chemically modified 5 -triphosphate oligonucleotides are obtained in a convenient and efficient manner and isolated in good yields after HPLC purification.