Carbodiimides water soluble

Naylor and Gilham (1966) took another route they were able to link short DNA fragments to a complementary matrix without using an enzyme. The reactions were carried out in aqueous solution, and the molecules first had to be converted into a reactive state by chemical activation the activation agent used was a water-soluble carbodiimide. 

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. 

As with pyrimidines, the water-soluble carbodiimide CMC may react with guanine derivatives to give a reversible adduct at N-l (Figure 1.52). Raising the pH to highly alkaline conditions regenerates the purine group. Adenine residues, however, display no reactivity in this process. 

The carbodiimide of choice used to couple cystamine to carboxylate- or phosphate-containing molecules is most often the water-soluble carbodiimide, EDC hydrochloride Chapter 3, Section 1.1). This reagent rapidly reacts with carboxylates or phosphates to form an active ester intermediate, which is highly reactive toward primary amines. The reaction is efficient from pH 4.7 to 7.5, and a variety of buffers may be used, providing they don t contain competing groups. 

Figure 1.71 Cystamine may be used to label protein carboxylate groups using the water-soluble carbodiimide EDC.

In most procedures, the water-soluble carbodiimide EDC hydrochloride is the most effective mediator of this reaction. Both EDC and its reaction by-products are fully soluble in aqueous buffers and can be easily separated from the modified aminoalkylphosphate (Chapter 3, Section 1.1). 

In aqueous solutions, the easiest method for forming this type of bond is to use the water-soluble carbodiimide EDC (Chapter 3, Section 1.1). For proteins and other water-soluble macromolecules, EDC reacts with their available carboxylate groups to form an intermediate, highly reactive, o-acylisourea. This active ester species may further react with nucleophiles such as a hydrazide to yield a stable imide product (Figure 1.109). 

Figure 3.4 The water-soluble carbodiimide CMC reacts with carboxylates to form an active-ester intermediate. In the presence of amine-containing molecules, amide bond formation can take place with release of an isourea by-product.

Figure 11.2 D-Biotin can be directly coupled to amine-containing molecules using the water-soluble carbodiimide EDC to form an amide bond linkage.

Figure 19.2 cBSA is formed by the reaction of ethylene diamine with BSA using the water-soluble carbodiimide EDC. Blocking of the carboxylate groups on the protein combined with the addition of terminal primary amines raises the pi of the molecule to highly basic values. 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Gilles, M.A., Hudson, A.Q., and Borders, C.L. (1990) Stability of water-soluble carbodiimides in aqueous solution. Anal. Biochem. 184, 244-248. 

Staros, J.V., Wright, R.W., and Swinghe, D.M. (1986) Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156, 220-222. 

Thelen, R, and Deuticke, B. (1988) Chemo-mechanical leak formation in human erythrocytes upon exposure to a water-soluble carbodiimide followed by very mild shear stress. II. Chemical modifications involved. Biochim. Biopbys. Acta 944, 297-307. 

Carbodiimides are, in general, useful compounds for effecting certain dehydrative condensations, e.g., in the formation of amides, esters, and anhydrides. These two crystalline water-soluble carbodiimides are especially useful in the synthesis of peptides and in the modification of proteins. The excess of reagent and the co-product (the corresponding urea) are easily separated from products with limited solubility in water. The hydrochloride is best employed in nonaqueous solvents (methylene chloride, acetonitrile, dimethylformamide). The methiodide is relatively stable in neutral aqueous systems, and thus is recommended for those media. 

FIGURE 1.16 Purified symmetrical anhydrides of iV-alkoxycarbonylamino acids obtained using a soluble carbodiimide.15 The reagent ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride,14 also known as WSCD (water-soluble carbodiimide), the NW -dialkylurea, and the iV-acyl-iV,iV -dialkylurea are soluble in water and thus can be removed from a reaction mixture by washing it with water. 

JC Sheehan, PA Cruickshank, GL Boshart. Convenient synthesis of water-soluble carbodiimides. J Org Chem 26, 2525, 1961. 

Other fixatives, such as water-soluble carbodiimides or di-imidates, have been used as fixatives, either alone or in combination with aldehydes, but except in special cases, they have no major advantage over aldehyde fixation (5) (see Chapter 8). 

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