Carbodiimides nucleotides

The facile and selective oxidation of both primary and secondary hydroxy groups with certain nucleotides led Pfitzner and Moffatt (48) to explore the scope of the carbodiimide-methyl sulfoxide reagent with steroid and alkaloid alcohols. Relatively minor differences were apparent in the rate of oxidation of epimeric pairs of 3- and 17- hydroxy steroids whereas the equatorial lLx-hydroxyl group in several steroids was readily oxidized under conditions where the axial epimer was unreactive [cf. chromic oxide oxidation (51)]. 

Modification of the ATPase by ATP-EDC occurred without significant incorporation of the nucleotide into the enzyme, suggesting that the inhibition of enzymatic activity is entirely due to intramolecular crosslinking by the carbodiimide moiety... 

Carbodiimide modification of the phosphomonoester end groups on DNA molecules was first used in Khorana s lab to determine nucleotide sequences (Ralph et al., 1962). That early... 

The formation of a phosphorimidazolide intermediate provides better reactivity toward amine nucleophiles than the EDC phosphodiester intermediate if EDC is used without added imidazole. The EDC phosphodiester intermediate also has a shorter half-life in aqueous conditions due to hydrolysis than the phosphorimidazolide. Although EDC alone will create nucleotide phosphoramidate conjugates with amine-containing molecules (Shabarova, 1988), the result of forming the secondary phosphorimidazolide-activated species is increased derivatiza-tion yield over carbodiimide-only reactions. 

Metz, D.H., and Brown, G.L. (1969) The investigation of nucleic acid secondary structure by means of chemical modification with a carbodiimide reagent. I. The reaction between N-cyclohexyl-N -b-(4-methylmorpholinium)ethyl carbodiimide and model nucleotides. Biochemistry 8, 2312-2328. 

The carbodiimide is also used to convert nucleotide phosphates into protected derivatives using 2-cyanoethanol or p-(triphenylmethyl)aniline. 

Carbodiimides are the diimides derived from carbon dioxide, and they are extensively used in the formation of peptide amide bonds from carboxylic acids and amines. This reaction was utilized by the Nobel laureate Sheehan in the total synthesis of penicillin. He also was the first to use water soluble carbodiimides to crosslink gelatin. Khorana, another Nobel laureate, demonstrated that carbodiimides can also be used in the synthesis of nucleotides. Today, carbodiimides are used extensively in the synthesis and modification of proteins. Proteomics is the new frontier of chemical research. 

Water-soluble carbodiimide for die modification of carboxyl groups in proteins zero-length crosslinking proteins activation of carboxyl groups for amidation reactions, as for the coupling of amino-nucleotides to mattices for DNA microarrays. 

This procedure was introduced by Halloran and Parker for use with mono- and oligonucleotides. Humayun and Jacob modified it to reduce the amount of insoluble aggregate they obtained with the original method, especially with purine nucleotides. In a reaction following the modified procedure, about 0.25 mmol of solid l-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide is added to 0.1 mmol of nucleotide in 0.5 ml of water. The pH is adjusted to 7 with dilute NaOH if necessary, and the solution is incubated at 60° for 10 min. The mixture is then added dropwise to a protein solution of 10-20 mg in 0.2-0.4 ml of 0.15 M NaCl, and this solution is kept in the dark overnight at room temperature. The product is then separated from the free nucleotide and the hydrolyzed carbodiimide reagent by dialysis or gel filtration. 

It is of interest that water-soluble carbodiimides have been used to couple nucleotides directly to proteins, presumably by formation of a P-N bond. ... 

Urease-cyclic nucleotide conjugates were not prepared by the carbodiimide method because of high loss of urease activity during the course... 

The procedures used for the synthesis of nucleotides of 2 -deoxycytidine were successfully applied to the preparation of their ptirine counterparts. 2 -Deoxyadenosine was fully benzoylated and then converted with alkali into the A -benzoyl derivative. Tritylation with (119) yielded (125), which was converted by the 2-cyanoethyl phosphate-iV,iV -dicyclohexyl-carbodiimide method, followed by removal of the protecting groups, into 2 -deoxyadenosine 3 -phosphate (126). With 2 -deoxyguanosine, direct tritylation with (119) gave (127), from which 2 -deoxyguanosine 3 -phos-phate (128) was obtained. ... 

Miller, M.M. and Boger, D.S. (2005) Polystyrene-1-ethyl-3-(3 -dimethylaminopropanej-carbodiimide Hydrochloride (PS-EDCI), in Handbook of Reagents for Organic Synthesis, Reagents for Glycoside, Nucleotide and Peptide Synthesis (ed. D. Crich), John Wiley Sons Ltd, Chichester, pp. 529-33. 

---