Carbodiimide-mediated reactions with

Figure 1.95 Carboxylate-containing sugars may be modified with diamines using a carbodiimide-mediated reaction to create available amine groups for subsequent conjugation.

Hydrazide-containing compounds also can be coupled to carboxylate groups using a carbodiimide-mediated reaction. Using bifunctional hydrazide reagents, carboxylates can be modified to possess terminal hydrazide groups able to conjugate with other carbonyl compounds (Chapter 4, Section 8). 

Molecules containing phosphate groups, such as the 5 phosphate of oligonucleotides, also may be conjugated to amine-containing molecules by using a carbodiimide-mediated reaction (Chapter 27, Section 2.1). The carbodiimide activates the phosphate to an intermediate phosphate ester similar to its reaction with carboxylates (Chapter 3, Section 1). In the presence of an amine, the ester reacts to form a stable phosphoramidate bond (Reaction 13). 

Figure 25.2 mPEG may be derivatized with succinic anhydride to produce a carboxylate end. A reactive NHS ester can be formed from this derivative by use of a carbodiimide-mediated reaction under nonaqueous conditions. The succinimidyl succinate-mPEG is highly reactive toward amine nucleophiles. 

Dextran derivatives containing carboxyl- or amine-terminal spacer arms may be prepared by a number of techniques. These derivatives are useful for coupling amine- or carboxylate-containing molecules through a carbodiimide-mediated reaction to form an amide bond (Chapter 3, Section 1). Amine-terminal spacers also can be used to create secondary reactive groups by modification with a heterobifunctional crosslinking agent (Chapter 5). 

Figure 25.14 An amine derivative of dextran may be prepared through a two-step process involving the reac-tion of chloroacetic acid with the hydroxyl groups of the polymer to create carboxylates. Next, ethylene diamine is coupled in excess using a carbodiimide-mediated reaction to give the primary amine functional groups.

There can be a minor side reaction associated with the use of HOObt in carbodiimide-mediated reactions namely, aminolysis at the carbonyl of the activating moiety of ester 3 (Figure 2.27), giving addition product 4. The reaction is negligible in most cases. 

NL Benoiton, YC Lee, FMF Chen. Identification and suppression of decomposition during carbodiimide-mediated reactions of Boc-amino acids with phenols, hydroxy-lamines and amino acid esters. Ini J Pept Prot Res 41, 587, 1993. 

Amine-reactive biotinylation reagents contain functional groups off biotin s valeric acid side chain that are able to form covalent bonds with primary amines in proteins and other molecules. Two basic types are commonly available NHS esters and car-boxylates. NHS esters spontaneously react with amines to form amide linkages (Chapter 2, Section 1.4). Carboxylate-containing biotin compounds can be coupled to amines via a carbodiimide-mediated reaction using EDC (Chapter 3, Section 1.1). 

In view of the rapid reaction of carbodiimides with water they are often used in dehydration reactions. Major examples are the intra- and intermolecular esterification reactions of carboxylic acids, and the formation of peptides from carboxylic acids and protected amino acids. Especially, dicyclohexylcarbodiimide (DCC) or diisopropylcar-bodiimide (DIPCD) are often used in carbodiimide mediated reactions because the corresponding urea byproducts are insoluble in most organic solvents and water, and therefore are readily removed by filtration. Also, water soluble carbodiimides, such as N-ethyl-N -(3-dimethylamino)propylcarbodiimide (EDC) or its hydrochloride (EDCCl, sometimes referred to as EDAC) are often used in these reactions. EDC reacts with carboxyl groups at pH of 4.0-6.0, but loses its reactivity at lower pH. Sometimes solid phase reactions are conducted using carbodiimide terminated linear or crosslinked polymers. 

Active esters are chirally stable under the usual conditions of coupling in peptide synthesis, but with the single exception of piperidino esters they may undergo isomerization if left in the presence of tertiary amines. In addition to their role as shelf-stable reagents, active esters are postulated as intermediates in carbodiimide-mediated reactions where a substituted hydroxylanoine is added in order to suppress the side reaction of epimerization in the... 

Various preparations of antisera to atropine have been reported. A racemic hemisuccinate ester was prepared and conjugated to bovine serum albumin by the carbodiimide technique. Antisera formed to the original immunogen selectively bound the R isomer (34), but a later antiserum prepared by this approach was reported to bind both R and S forms with "equal efficiency" (35). R, S-atropine was treated with diazotized p-amino-benzoic acid, and the resulting compound (which was not further characterized) was used for conjugation to bovine serum albumin by means of a carbodiimide-mediated reaction. Antisera resulting from use of this material were quite selective for the R isomer, with a cross-reaction of only about 2% for the S isomer (36). Virtanen et al. followed this procedure with S-atropine. Their antiserum bound equally to S- and R,S-atropine, as measured by displacement of tritium-labeled R,S-atropine (37). In another study (31), both racemic atropine and the S isomer were coupled to human serum albumin by the technique of Wurtzburger et al. (36), Antisera were obtained that were selective for both the R and S isomers (33). 

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