Synthesis of 2′-<i>O</i>-Propargyl Nucleoside Triphosphates for Enzymatic Oligonucleotide Preparation and “Click” Modification of DNA with Nile Red as Fluorescent Probe

Wenge, Ulrike; Ehrenschwender, Thomas; Wagenknecht, Hans‐Achim

doi:10.1021/bc300624m

Cited by 21 publications

(10 citation statements)

References 28 publications

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“…The alkyne groups as reactive precursors are attached to the oligonucleotide [ 13 ], especially at the 5-position of pyrimidines [ 13 ], the 7-position of 7-deazapurines [ 14 ], and the 2’-position of ribofuranosides [ 11 , 15 ]. These positions were chosen since they are typically accepted by DNA polymerases in primer extension experiments and PCR [ 4 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA

Walter¹,

Olshausen²,

Schepers³

et al. 2017

Beilstein J. Org. Chem.

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The arabino-configured analog of uridine with a propargyl group at the 2’-position was synthesized and incorporated into DNA by solid-phase chemistry. The fluorescence quantum yields of DNA strands that were postsynthetically modified by blue and green emitting cyanine-styryl dyes were improved due to the arabino-configured anchor. These oligonucleotides were used as energy transfer donors in hybrids with oligonucleotides modified with acceptor dyes that emit in the yellow-red range. These combinations give energy transfer pairs with blue–yellow, blue–red and green–red emission color changes. All combinations of arabino- and ribo-configured donor strands with arabino- and ribo-configured acceptor strands were evaluated. This array of doubly modified hybrids was screened by their emission color contrast and fluorescence quantum yield. Especially mixed combinations, that means donor dyes with arabino-configured anchor with acceptor dyes with ribo-configured anchor, and vice versa, showed significantly improved fluorescence properties. Those were successfully applied for fluorescent imaging of DNA after transport into living cells.

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Section: Introductionmentioning

confidence: 99%

A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA

Walter¹,

Olshausen²,

Schepers³

et al. 2017

Beilstein J. Org. Chem.

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“…Diverse modifications, including fluorophores, redox or spin labels, reactive groups for conjugations, and biomolecules (e.g., oligonucleotides or proteins), 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′‐sugar‐modified derivatives . 2‐Chloroadenine and 2,6‐diaminopurine dNTPs are the only minor‐groove base‐modified nucleotides that have been reported as substrates for DNA polymerases, whereas 2‐arylamino‐dATP derivatives were found to act as polymerase inhibitors .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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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...

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“…19 Acetylenes as reactive groups at these representative positions are accepted by DNA polymerases in primer extension experiments and PCR 14b. 20 Highly modified DNA, for instance, can be prepared by a combination of PCR with triphosphates bearing alkyne groups and subsequent Cu I ‐catalyzed modification 21…”

Section: Copper‐catalyzed Azide–alkyne Cycloaddition (Cuaac) and Tmentioning

confidence: 99%

Copper‐Free Postsynthetic Labeling of Nucleic Acids by Means of Bioorthogonal Reactions

et al. 2015

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Postsynthetic modification of nucleic acids has the advantage that the chemical development of only a few building blocks is necessary, each bearing a chosen reactive functional group that is applicable to its reactive counterpart for a variety of different labeling types. The reactive group is either linked to phosphoramidites for chemical synthesis on solid phase or attached to nucleoside triphosphates for application in primer extension experiments and PCR. Chemoselectivity is required for this strategy, together with bioorthogonality to perform these labelings in living cells or even organisms. Currently, the copper-free reactions include strain-promoted 1,3-dipolar cycloadditions, "photoclick" reactions, Diels-Alder reactions with inverse electron demand, and nucleophilic additions. The majority of these modification strategies show good to excellent reaction kinetics, an important prerequisite for labeling inside cells and in vivo in order to keep the concentrations of the reacting partners as low as possible.

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Synthesis of 2′-O-Propargyl Nucleoside Triphosphates for Enzymatic Oligonucleotide Preparation and “Click” Modification of DNA with Nile Red as Fluorescent Probe

Cited by 21 publications

References 28 publications

A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA

A postsynthetically 2’-“clickable” uridine with arabino configuration and its application for fluorescent labeling and imaging of DNA

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

Copper‐Free Postsynthetic Labeling of Nucleic Acids by Means of Bioorthogonal Reactions

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