Hydrazine nucleotides

Uracil reacts with hydrazine to give pyrazol-3(2if)-one (944) and urea N-methyl- and dimethyl-hydrazine behave similarly to give the 2-methyl- and 1,2-dimethyl derivatives. The reactions of hydrazines with uridine and related nucleosides and nucleotides is well studied (67JCS(C)1528). The tautomerism and predominant form of uracil are discussed in Section 2.13.1.8.4. 

One of the reactions used in determining the sequence ot nucleotides in a strand of DNA is reaction with hydrazine. Propose a mechanism for the following reaction, which occurs by an initial conjugate addition followed by internal amide formation. 

An analogous series of reactions is used to produce depyrimidinated DNA fragments. Hydrazine is used in these reactions, since both cytosine and thymine react with hydrazine. The bases are cleaved to yield urea and a pyrazole ring. The deoxyribose moiety is left as a hydrazone. Piperidine, which reacts with the hydrazone, is used to cleave the nucleotide chain. Cytosines react specifically with hydrazine in 5 mol/ L NaCl, but no specific reaction exists for thymines. Consequently, one aliquot yields labeled oligonu-cleotides 3 -terminated at cytosines, whereas a second aliquot contains nucleotides cleaved in the absence of NaCI at both cytosine and thymine residues. 

The N-levulinyl nucleosides are cleaved rapidly by hydrazine hydrate. They have been used successfully in nucleotide synthesis. 

Breaking the DNA chain next to the p3rTinudine nucleotides C andT it accomplished by treatment of DNA with hydrazine, followed ty... 

Breaking the DNA chain next to the pyrimidine nucleotides C and T is accomplished by treatment of DNA with hydrazine, H2NNH2, followed by heating with aqueous piperidine. Although no conditions have been found that are selective for cleavage next to T, a selective cleavage next to C can be accomplished by carrying out the hydrazine reaction in 5 M NaCl solution. 

HI. Hurst, R. O., The determination of nucleotide phosphorus with a stannous chloride-hydrazine sulphate reagent. Can. J. Biochem. 42, 287-292 (1964). 

Other chemical methods were also used for obtaining more-conclusive evidence. Cleavage of the isomeric p3nrimidine nucleotides with hydrazine... 

Arylamine N-acetyltransferases (NAT) are highly conserved in eukaryotes. These cytosolic enzymes transfer acetate from acetylCoA to primary amines, hydrazines, sulfonamides, and aromatic amines. They are 30 to 34 kDa in size. Humans express two functional NATs, NATl and NAT2. The genes for both isoforms encode 290 amino acids, and are found on chromosome 8. Their nucleotide sequences share 87% homology. These enzymes exhibit polymorphism, and allelic variation especially for NAT2 is associated with fast and slow acetylator phenotypes. 

The radioactively labelled DNA fragment is then treated with a reagent that specifically cleaves the DNA molecule at a particular type of nucleotide. For example, a solution of hydrazine cleaves a DNA molecule before every C-nucleotide. The cleaving reaction is carried out such that low yields are obtained. The aim is not to cut all DNA molecules at locations containing a C-nucleotide. The target is to cut each molecule only once at a randomly located C-nucleotide within the chain. 

Matz (34) separated halogenated uracils on silica 60 HPTLC plates. Solvents were chloroform-ethanol-water plus or minus acetate. Cardinaud (35) separated 27 pyr analogs. Cellulose TLC and various combinations of butanol-ammonia-ethyl acetate-formic sodium phosphate-propanol-isoamyl alcohol were used. Gommers-Ampt et al. discovered pdJ —a new nucleotide and not hydroxymeth-yl-DUMP (HmdUMP) in Trypanosoma brucei by 2D-TLC with PEI in isobutyric-water-ammonium ID and ammonium sulfate-isopropanol-sodium acetate. Hydrazine was used to destroy other pyrimidine rings. These modified nucleotides were resistant to postlabeling. 

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