Nuclear magnetic resonance controlled method for coupling of fenoterol to a carrier and enzyme

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Abstract
Fenoterol is a phenethanolamine with β‐adrenergic agonist activity. The development of an enzyme immunoassay for fenoterol requires coupling to a carrier (bovine serum albumin, BSA) and an enzyme (horseradish peroxidase, HRP). 1,4‐Butanediol diglycidyl ether was used as the coupling agent, providing for a 12‐atom spacer. During the coupling procedure of the spacer to the protein and fenoterol to the spacer, the coupling yield was monitored by nuclear magnetic resonance (NMR). This ensured a coupling at desired hapten: protein ratios. The average coupling ratios obtained using this method were 30 moles of fenoterol to 1 mole of BSA and 1 mole of fenoterol to 1 mole of HRP. In practice, this method can be used for the coupling of compounds containing phenolic, anilinic, primary amine and thiol groups to proteins. Its major advantage is the real‐time NMR quantification of coupling efficiency and the option to interrupt the coupling reaction to obtain the desired hapten: protein ratio.

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