Studies of the amidation of porphyrin-NHS esters in dilute aqueous solution

Abstract

Effective bioconjugation of porphyrins is desirable for diverse applications in the life sciences. Two trans-AB-porphyrins equipped with a 2,6-bis[HO₂C(CH₂CH₂O)₇]phenyl group and the NHS ester of 4-benzoic acid (P3-NHS) or 4-phenylpropanoic acid (P4-NHS) were examined for the kinetics and yields of hydrolysis and amidation (with a short amino-PEG reagent, H₂N–(CH₂CH₂O)₄–Me). The reactions were carried out by combining porphyrin-NHS esters in DMSO (containing 0.05% formic acid) with aqueous sodium carbonate buffer (50 mM) in 1 : 9 ratio and use of controlled conditions (concentration, pH, temperature) followed by HPLC analysis with absorption and mass spectrometric detection. The general conditions employed dilute solution (porphyrin-NHS ester at 1.0 mM and H₂N–(CH₂CH₂O)₄–Me at 2.0 mM) at room temperature. For P3-NHS (1.0 mM), the t_1/2 for hydrolysis at room temperature was 210, 180, or 125 min at pH 8.0, 8.5, or 9.0, respectively, versus amidation with t_1/2 of 80, 20, or 10 min and yield of amide of 80–85%. For P4-NHS (1.0 mM), the rates were faster for hydrolysis (t_1/2 = 190, 130, or 110 min) and amidation (t_1/2 = 25, 10, or 5 min), with yield of amide of 87–92%. The yield of amide as a function of concentration of NHS ester (with 2 equivalents of H₂N–(CH₂CH₂O)₄–Me) was 88% (1 mM), 74% (0.316 mM), and 56% (0.1 mM) for P3-NHS, and 97% (1 mM), 89% (0.316 mM), and 73% (0.1 mM) for P4-NHS. In both cases, the yield of amide product was slightly greater at room temperature than 37 °C. Both P3-NHS and P4-NHS were stable in frozen DMSO solutions (−20 °C). The studies establish a quantitative basis for carrying out bioconjugations of water-soluble porphyrins in dilute aqueous media without a large excess of porphyrin-NHS ester or amine. A summary of other quantitative studies of NHS-ester bioconjugations provides valuable context.