Haptens with hydroxyl groups

This group includes sugars, nucleosides, phenols and alcohols. Direct conjugation is generally not possible. 

The conversion of alcohol to the half ester of succinic acid ( hemi-succinate ) introduces a carboxyl group available for conjugation. This method (Table 12.6) has been used for a large number of haptens (Steiner et al., 1969 Den Hollander et al., 1974 Okabayashi et al., 1977). The bifunctional reagent, sebacoyldichloride, converts alcohol to acid chloride which, at pH 8.5, reacts readily with proteins (Bailey and Butler, 1967). 

Conversion of alcoholic hydroxyl group to carboxyl group through the formation of hemisuccinate (Okabayashi et al., 1977) 

Dissolve 15 g 2,2,2-trichloroethanol, 12 g succinic anhydride and 8.7 ml triethyl-amine in 100 ml ethylacetate. 

Evaporate solvent and take up residue in 5% aqueous NaHCO,. 

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A carboxyl group can be introduced in almost any compound by various methods. In cases where a free thiol is present, the carboxyl group can be introduced easily by reaction with bromo- or iodoacetic acid. This reaction is very mild and is usually performed at pH around 8-9. When the hapten contains a hydroxyl group, carboxylic acid may be introduced by one of the following methods (a) carboxymethylation of the hydroxyl group with bromo- or iodoacetic acid (b) esterification with dicarboxylic acid anhydrides, such as succinic anhydride, to yield hemi-succinates, which are unstable above pH 9 (c) reaction with phosgene, which results in the formation of chlorocarbonates. ... 

Synthetic procedures used to prepare antigenic hapten-carrier conjugates are reviewed by Erlanger. Procedures that have proven successful with hapten molecules that possess carboxyl, amino, hydroxyl, and carbonyl groups are fairly routine. The preparation of hapten-carrier conjugates by other synthetic routes is also considered. 

The introduction of I into molecules containing tyrosyl or histidyl moieties can be carried out chemically by the Chloramine T procedure or enzymatically by lactoperoxidase." The prelabeled Bolton-Hunter reagent [A-succinimidyl 3-(4-hydroxyl-5-[ I]iodophenylpropionate] reacts under mild conditions with molecules containing amino groups (e.g., proteins and haptens) to introduce an iodinated propyltyrosyl moi-... 

If a hapten does not contain free carboxyl, such group(s) may often be introduced. Alkylation of oxygen or nitrogen substituents with halo-esters, followed by hydrolysis of the ester, is frequently used for this purpose (Kabakoff, 1980). For steroids, carboxyl groups may be introduced to the hydroxyl or keton groups, through the formation of hemisuccinate esters or (carboxymethyl)oximes, respectively. 

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