5-(5-Formylthienyl)-, 5-(4-formylphenyl)- and 5-(2-fluoro-5-formylphenyl)cytosine 2'-deoxyribonucleoside mono- (dC(R)MP) and triphosphates (dC(R)TP) were prepared by aqueous Suzuki-Miyaura cross-coupling of 5-iodocytosine nucleotides with the corresponding formylarylboronic acids. The dC(R)TPs were excellent substrates for DNA polymerases and were incorporated into DNA by primer extension or PCR. Reductive aminations of the model dC(R)MPs with lysine or lysine-containing tripeptide were studied and optimized. In aqueous phosphate buffer (pH 6.7) the yields of the reductive aminations with tripeptide III were up to 25 %. Bioconjugation of an aldehyde-containing DNA with a lysine-containing tripeptide was achieved through reductive amination in yields of up to 90 % in aqueous phosphate buffer.
Apart from a wide range of novel applications of functionalized DNA in chemical biology, nanotechnology, and material sciences, [1] 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, [2,3] Staudinger ligation, [4] and Diels-Alder reactions.[5] 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) [6] and the few successful examples were prepared indirectly by a click reaction with azide derivatives of reducing sugars, [3] or by introduction of 2,3-dihydroxypropyl or 3,4-dihydroxypyrrolidine moieties [7,8] 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. [7,8] Metallization [7] or interstrand cross-linking [8] 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. [9,10] In addition, we wished to develop a methodology for additional conjugation and staining of aldehyde-modified DNA by hydrazone formation.The methodology of choice involved Suzuki cross-coupling of a halogenated nucleoside triphosphate (dNTP) with an aldehyde-containing boronic acid, and subsequent polymerase incorporation into DNA.[9, 10] Furthermore, we wanted to develop a general methodology for hydrazone formation in aqueous media. To test the first and last steps of our proposed route, we performed the reactions on the model compound 5-iodo-dCMP (1; dCMP = 2'-deoxycytidine-5'-O-monophosphate). Commercially available 5-formylthiophene-2-boronic acid was selected as a suitable carrier for the aldehyde group, and its aqueous-phase cross-coupling with monophosphate 1 proceeded within 40 minutes and gave aldehyde-modified dCMP 2 in 50 % yield (Scheme 1). The next task was the formation of the hydrazone species, which is usually only performed in dry organic solvents (owing to the formation of water in the reaction). To make the reaction amenable to aqueous conditions, we have adapted the protocol developed by Dawson and co-workers [11] for aqueous conjugation of peptides, which uses aqueous ammonium acetate and aniline to facilitate the condensation. To test the reactions with 2, we selected two arylhydrazines (3 and 4) that are commonly used as aldehyde-specific dyes. [12,13] The reactions of aldehydenucleotide 2 with 3 or 4 proceeded at room temperature for approximately 20 ho...
DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli. Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. coli enzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.
Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.
Apart from a wide range of novel applications of functionalized DNA in chemical biology, nanotechnology, and material sciences, [1] 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, [2,3] Staudinger ligation, [4] and Diels-Alder reactions. [5] 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) [6] and the few successful examples were prepared indirectly by a click reaction with azide derivatives of reducing sugars, [3] or by introduction of 2,3-dihydroxypropyl or 3,4-dihydroxypyrrolidine moieties [7,8] 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. [7,8] Metallization [7] or interstrand cross-linking [8] 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. [9,10] In addition, we wished to develop a methodology for additional conjugation and staining of aldehyde-modified DNA by hydrazone formation.The methodology of choice involved Suzuki cross-coupling of a halogenated nucleoside triphosphate (dNTP) with an aldehyde-containing boronic acid, and subsequent polymerase incorporation into DNA. [9,10] Furthermore, we wanted to develop a general methodology for hydrazone formation in aqueous media. To test the first and last steps of our proposed route, we performed the reactions on the model compound 5iodo-dCMP (1; dCMP = 2'-deoxycytidine-5'-O-monophos-phate). Commercially available 5-formylthiophene-2-boronic acid was selected as a suitable carrier for the aldehyde group, and its aqueous-phase cross-coupling with monophosphate 1 proceeded within 40 minutes and gave aldehyde-modified dCMP 2 in 50 % yield (Scheme 1). The next task was the formation of the hydrazone species, which is usually only performed in dry organic solvents (owing to the formation of water in the reaction). To make the reaction amenable to aqueous conditions, we have adapted the protocol developed by Dawson and co-workers [11] for aqueous conjugation of peptides, which uses aqueous ammonium acetate and aniline to facilitate the condensation. To test the reactions with 2, we selected two arylhydrazines (3 and 4) that are commonly used as aldehyde-specific dyes. [12,13] The reactions of aldehydenucleotide 2 with 3 or 4 proceeded at room temperature for approximately 20 ho...
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