Carbodiimides reagents

The N-3 position of uracil also can be modified with carbodiimide reagents. In particular, the water-soluble carbodiimide CMC [l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, as the metho p-toluene sulfonate salt] can react with the N-3 nitrogen at pH 8 to give an unstable, charged adduct. The derivative is reversible at pH 10.5, regenerating the original nucleic acid base (Figure 1.47). Cytosine is unreactive in this process. 

Carraway, K.L., and Koshland Jr, D.E (1968) Reaction of tyrosine residues in proteins with carbodiimide reagents. Biochim. Biophys. Acta, 160, 272-274. 

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 general chemistry used in this approach involves the combination of a limited amount of an amine-terminated dendrimer core reagent with an excess of carboxylic acid terminated dendrimer shell reagent [31]. These two charge differentiated species are allowed to self-assemble into the electrostatically driven supramolecular core-shell tecto(dendrimer) architecture. After equilibration, covalent bond formation at these charge neutralized dendrimer contact sites is induced with carbodiimide reagents (Scheme 1). 

The utility of carbodiimide reagents was too important to consider abandoning them. Instead, it rapidly became obvious that carbodiimide activation could be used to prepare in situ active esters. Numerous nucleophilic additives were discovered and prepared for use in these reactions. The most important of these additives was 1,2,3-benzotriazol-l-ol (HOBt, 1), first reported for use in peptide synthesis by Konig and Geiger in 1970.[2(l As an example of a typical result, 1.2 equivalents of HOBt was added to the DCC coupling of Boc-Leu-Phe-OH to H-Val-OtBu in DMF as a solvent. Less than 1% of the l-d-l epimerized tripeptide was formed. When the reaction was carried out in the absence of HOBt, the amount of l-d-l product formed was 14.3%. Addition of HOBt to DCC reactions converts the intermediate G-acylisourea (2) (and any symmetrical anhydride) into the HOBt active ester 3 (Scheme 8). 

The N-3 position of uracil also can be modified with carbodiimide reagents. In... 

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. 

The carboxylic acid first adds to the carbodiimide reagent to yield a reactive acylating agent. 

Amide formation. Development of a carbodiimide reagent (1) containing at one end an isopropyl group and at the other end a chain ending in two polyfluorinated branches has been reported. ... 

Polymeric carbodiimide reagent for peptide synthesis. Japanese chemists have prepared a carbodiimide reagent attached to a styrene-divinylbenzene polymer as formulated and have used it successfully for synthesis of dipeptides. 

There has been an evolution in the development of new coupling reagents and their application to solid-phase methodology. The formerly predominant carbodiimide reagents are being replaced by onium (phosphonium and aminium) salts. As discussed later, depending on the conditions, the activation step with onium salts occurs faster than with carbodiimides and usually leads to less perishable intermediates. 

The carbodiimide reagent has also been extensively applied by Jeanloz and associates as the coupling reagent for the synthesis of oligosac-charide-L-asparagine compounds. Thus, the per-O-acetylglycosyl-amine derivatives of the disaccharides 0-(2-acetamido-2-deoxy-)8-D-glucopyranosyl)-(l- 3)- (33,34), (37), and -(l- 6)-2-... 

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