Polarity-tunable and wavelength-tunable bacteriochlorins bearing a single carboxylic acid or NHS ester. Use in a protein bioconjugation model system

Abstract

To broaden the scope of near-infrared (NIR)-active chromophores available for bioconjugation with proteins, 10 new bacteriochlorins have been synthesized: six are lipophilic and bear a carboxylic acid tether; four are hydrophilic and bear four carboxylic acids and one N-hydroxysuccinimido (NHS) ester tether. Each bacteriochlorin exhibits a sharp long-wavelength absorption (Q_y) band in the NIR region (727–823 nm). The lipophilic bacteriochlorins were examined in DMF (fluorescence quantum yield Φ_f = 0.037–0.19) whereas the hydrophilic bacteriochlorins were examined in DMF (Φ_f also = 0.037–0.19) or aqueous phosphate buffer (Φ_f = 0.0011–0.13). Two bacteriochlorins were conjugated to myoglobin (Mb), which contains ∼14 accessible amino groups. Use of 2, 10, or 50 equivalents of a hydrophilic bacteriochlorin–NHS ester (BC7) gave average loadings of 0.62, 1.6, or 7.1 bacteriochlorins/Mb as determined by absorption spectral comparison with the strongly absorbing heme ligand. MALDI-MS analysis showed a distribution of 0–9 bound bacteriochlorins for the conjugate sample with average loading of 7.1. The Mb–BC7 conjugates exhibited characteristic absorption and fluorescence spectra in aqueous buffer, yet the Φ_f value was markedly low (Φ_f ∼ 0.02) regardless of loading versus that of the BC7 monomer (Φ_f = 0.12), attributed in part to heme quenching. Removal of the heme revealed a loading-dependent Φ_f, which ranged from 0.091 (0.62 loading) to 0.023 (7.1 loading). The decrease in Φ_f with increased loading is attributed to self-quenching perhaps facilitated by excited-state energy transfer among the bacteriochlorins (Förster R₀ = 59 Å). Taken together, the results show facile access to a collection of useful bacteriochlorins for NIR spectroscopic studies, along with a pigment–protein system that serves the dual purposes of a convenient testbed for evaluating protein bioconjugation processes as well as a nanosized architecture for use in photochemical studies.