O-Acylisoureas

Step 1 In the first stage of the reaction the carboxylic acid adds to one of the double bonds of DCCI to give an O acylisourea... 

Step 2 Structurally O acylisoureas resemble carboxylic acid anhydrides and are powerful acylatmg agents In the reaction s second stage the amine adds to the carbonyl group of the O acylisourea to give a tetrahedral intermediate... 

An O acylisourea is formed by addition of the Z protected amino acid to N N dicyclo hexylcarbodiimide as shown in Figure 27 13 This O acylisourea is attacked by p nitrophenol... 

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). 

N-substituted carbodiimides can react with carboxylic acids to form highly reactive, o-acylisourea derivatives that are extremely short-lived (Reaction 11). This active species then can react with a nucleophile such as a primary amine to form an amide bond (Reaction 12)... 

Carbodiimides are used to mediate the formation of amide or phosphoramidate linkages between a carboxylate and an amine or a phosphate and an amine, respectively (Hoare and Koshland, 1966 Chu et al., 1986 Ghosh et al., 1990). Regardless of the type of carbodiimide, the reaction proceeds by the formation of an intermediate o-acylisourea that is highly reactive and short-lived in aqueous environments. The attack of an amine nucleophile on the carbonyl group of this ester results in the loss an isourea derivative and formation of an amide bond (see Reactions 11 and 12). The major competing reaction in water is hydrolysis. 

A potential undesirable effect of DCC coupling reactions is the spontaneous rearrangement of the o-acylisourea to an inactive N-acylurea (Stewart and Young, 1984) (Figure 3.6). The rate of this rearrangement is dramatically increased in aprotic organic solvents, such as DMF. 

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group results in the formation of an amide bond (Figure 9.22). Derivatization of AMCA off its carboxylate group causes no major effects on its fluorescent properties. Thus, proteins and other macromolecules may be labeled with this intensely blue probe and easily detected by fluorescence microscopy and other techniques. 

Biotin-hydrazide also may be used to couple with carboxylate-containing molecules. Hydrazidcs can be coupled with carboxylic acid groups by using the carbodiimide reaction (Chapter 3, Section 1.1). The carbodiimide activates a carboxylate to an o-acylisourea intermediate. Biotin-hydrazide can react with this intermediate via nucleophilic addition to form a stable covalent bond. 

The reactions involved in an EDC-mediated conjugation are discussed in Chapter 3, Section 1.1 (Note EDC is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride MW 191.7 and is sometimes referred to as EDAC). The carbodiimide first reacts with available carboxylic groups on either the carrier or hapten to form a highly reactive o-acylisourea intermediate. The activated carboxylic group then can react with a primary amine to form an amide bond, with release of the EDC mediator as a soluble isourea derivative. The reaction is quite efficient with no more than 2 hours required for it to go to completion and form a conjugated immunogen. 

In a more recent study using dedicated multimode microwave reactors for chemical synthesis, which enable temperature and power control, it was demonstrated that microwave irradiation could be effectively employed to couple aromatic carboxylic acids to polystyrene Wang resin [25], if the symmetrical anhydride procedure was used, and not the three-component O-acylisourea activation method [19]. Almost quantitative loading was achieved in l-methyl-2-pyrrolidone (NMP) at 200 °C within 10 min under... 

An alternative mechanism [8] entails reaction of cyanamide (or dicyandiamide) with the dye phosphonate to give an O-acylisourea derivative (7.47). This is able to react directly with cellulose to form dye-fibre bonds, urea being released as the anticipated by-product (Scheme 7.31). In support of this mechanism, it is known that O-acylisourea derivatives of arylcarboxylic acids react readily with alcohols and this constitutes an efficient route for the preparation of carboxylic esters [44]. 

O-acylisourea generates peptide, the theoretical yield of peptide is one equivalent and one equivalent of A,/V -dialkylurea is liberated. However, a fourth and undesirable course of action is possible because of the nature the (9-acylisourea. The latter contains a basic nitrogen atom (C=NR3) in proximity to the activated carbonyl. This atom can act as a nucleophile, giving rise to a rearrangement (path J) that produces the. V-acylurea (see Section 1.12) that is a stable inert form of the acid. This reaction is irreversible and consumes starting acid without generating peptide. The exact fate of the O-acylisourea in any synthesis depends on a multitude of factors this is addressed in Section 2.3. 

NL Benoiton, FMF Chen. Not the alkoxycarbonylamino-acid O-acylisourea. J Chem Soc Chem Commun 543, 1981. 

More /V-acylurea is generated if tertiary amine is present because the latter removes any protons that might prevent the rearrangement (see Section 2.12). The two intramolecular reactions also occur to a greater extent when interaction between the O-acylisourea and the /V-nucleophile is impeded by the side chain of the activated residue. This means that more 2-alkoxy-5(4//)-oxazolone and /V-acylurea are generated when the activated residues are hindered (see Section 1.4). A corollary of the above is that the best way to prepare an /V-acylurea, should it be needed, is to heat... 

That being said, it must be recognized that the evidence that the (V-acy I isourea is the precursor of the 2-alkoxy-5(4//)-oxazolone is only circumstantial because experiments starting from the former have yet to be achieved. The oxazolone could theoretically come from the symmetrical anhydride. The latter generates 2-alkoxy-5(4//)-oxazolone in the presence of tertiary amines (see Section 4.16) even dicy-clohexylcarbodiimide (DCC) was basic enough to generate 2-t< rt-butoxy-5(4 )-oxazolone from Boc-valine anhydride. However the weight of evidence points to O-acylisourea as the precursor of the 2-alkoxy-5(4//)-oxazolone. In the absence of. V-nucleophile, such as in the preparation of esters, the major precursor of product is the symmetrical anhydride.7,8... 

The symmetrical anhydride is less reactive and consequently more selective in its reactions than the O-acylisourea. Although the latter can acylate both N- and O-nucleophiles, the symmetrical anhydride will only acylate V-nuclcophilcs. This means that the hydroxyl groups of the side chains of serine, threonine, and tyrosine that have not been deprotonated are not acceptors of the acyl group of the symmetrical anhydride. An additional feature of this approach to carbodiimide-mediated reactions is that it avoids a possible side reaction between the carbodiimide and the iV-nucleophilc, which gives a trisubstituted guanidine [(C6HuN)2C=N-CHR5CO-... 

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