Synthesis and Reaction of DNA Oligomers Containing Modified Cytosines Related to Bisulfite Sequencing

Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
Organic Letters (Impact Factor: 6.36). 04/2009; 11(6):1377-9. DOI: 10.1021/ol900195z
Source: PubMed


The synthesis of DNA oligomers containing N(4)-hydroxy-5,6-dihydrocytosine-6-sulfonate by using ligand-induced base flipping of cytosine followed by the simultaneous addition of bisulfite and hydroxylamine is reported. In contrast to C, the flipped-out 5-methylcytosine was selectively oxidized over thymines and cytosines in the duplex by potassium permanganate. Ligand-induced base flipping is a convenient and powerful strategy for the synthesis of modified cytosines and 5-methylcytosines related to bisulfite sequencing at the predetermined site of DNA.

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    • "OsO4 and reagents derived thereof were found to cis-hydroxylate dC bases with some selectivity (24, 25). Furthermore photochemical methods were reported (26, 27) that allow some discrimination and finally even compounds were reported that are added during bisulfite sequencing to trap intermediates of the bisulfite reaction (28). All reported approaches target the reactivity differences between dC and 5MedC imposed by the additional methyl group. "
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    ABSTRACT: The presence of the methylated nucleobase 5MedC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of 5MedC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and 5MedC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the 5MedC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and 5MedC with high accuracy, leading towards a novel and mild chemistry for methylation analysis.
    Full-text · Article · Nov 2010 · Nucleic Acids Research
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    ABSTRACT: The novel transformation of cytosine oxime sulfonate (C*) to uracil sulfonate (U*) proceeded by a Co(II)-assisted benzoyl peroxide reaction, eventually leading to the conversion of cytosine to uracil.
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    ABSTRACT: a b s t r a c t Modified nucleobases and nucleic acids have found many biological and pharmaceutical applications. Here we report a microwave-directed synthesis of a variety of modified uracil and cytosine nucleobases with high yields under solvent-free conditions. The reaction yields were further improved by addition of Lewis acid. The crystal structures of 5-isopropyl-6-methyluracil and 6-phenyluracil were also determined. Unnatural nucleobases and oligonucleotides have been devel-oped for many bio-molecular studies ranging from detection of genomic mutations, 1–4 disease diagnosis, 5 gene silencing, 6,7 and as molecular probes. 8–11 A number of nucleobase analogs, for example, 5-fluorouracil, anilinouracils, and cyclopentenyl cyto-sines are well-known antitumor and antibacterial drugs. 12–14 In the past decade a spectrum of non-natural nucleobases was also developed to induce altered base-pairing properties into the DNA duplex as well as for the expansion of genetic alphabets. 15,16 Syn-thesis of modified nucleobases, therefore, has drawn much interest in bio-molecular studies as well as in pharmaceutical industries. Synthesis of pyrimidine nucleobases is of particular interest to study their propensity to undergo rapid photochemical lesion for-mations under ultraviolet radiation, causing genomic damages. 17,18 The conventional methods for the synthesis of pyrimidine nucleo-bases require either multi-step reactions or harsh experimental conditions and long reflux time. 19,20 Several methods have been developed, in the past, for the efficient synthesis of natural and modified nucleobases, including metal catalyzed cross coupling reactions. 21 Microwave-directed methods were also used to syn-thesize non-nucleobase pyrimidines, using N-vinyl and N-aryl amides as substrates. 22,23 Here we report a one-pot, efficient syn-thesis of pyrimidine nucleobases with altered steric and electronic properties at C-5 and C-6 positions. We found that the use of microwave-assisted synthesis under solvent-free conditions led to high yields of uracil and cytosine derivatives (Scheme 1). The yield of the reactions as well as the rate of conversions were further enhanced, significantly, by use of BF 3 ÁEt 2 O as Lewis acid. We also demonstrate here the first crystal structure of 5-isopro-pyl-6-methyluracil (5) and 6-phenyluracil (6) which clearly show different packing and altered hydrogen bonding abilities in com-parison to 6-propyl uracil (4). A series of uracil and cytosine nucleobase derivatives were syn-thesized in a single step, using solvent-free, microwave-assisted synthesis. All reactions were carried out in a Anton Paar Synthos 3000 closed vessel microwave reactor at about 135–145 °C for var-iable durations. 27,28 Uracil nucleobases (1–10) were synthesized by treatment of the respective b-ketoesters or b-aldehydo ester with urea, whereas the cytosine derivatives (11, 12) were obtained from benzoylacetonitrile or N,N-diethylamide precursors. The substrate b-ketoesters (1a–8a, 10a), b-aldehydo ester (9a), benzoylacetonitri-le, and N,N-diethylamide (11a, 12a) were either purchased or
    No preview · Article · Aug 2012 · Tetrahedron Letters
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