Separation and elucidation of ethylidene-bridged catechin oligomers using preparative-HPLC and NMR

Abstract

During wine aging, small amounts of oxygen are absorbed and initiate a cascade of oxidation reactions. These aging reactions create many products including ethylidene-bridged oligomers and polymers of endogenous polyphenols, like flavan-3ols. This bridging is believed to reduce bitterness and astringency while enhancing color stability in aged wine [1]. The elongation reactions are products of interaction of flavonoids with acetaldehyde, an oxidation product of ethanol. While studies have been conducted confirming the reaction between acetaldehyde and various flavan-3-ols, there has not been research elucidating the structure of individual ethylidene-bridged flavan-3-ols. Previous acetaldehyde-flavan-3-ol nuclear magnetic resonance (NMR) experiments have either been conducted using the entire reaction mixture [2], or of the total amount of precipitates, which would include all of the polymers from the reaction. The goal of this experiment was to isolate various ethylidene-bridged catechin oligomers (dimer, trimer and tetramer) and confirm their proposed structure and yield using NMR. To investigate this, exogenous acetaldehyde and catechin (both 500 mg/L) were added to model wine (12.5% EtOH, pH 3.5), and incubated at 35°C for 7 days to allow for sufficient reaction. Using reverse-phase preparative-high-performance liquid-chromatography (RP-prep-HPLC) affixed with a diode array detector (DAD), the individual ethylidene-bridged catechin oligomers were isolated. Ethylidene-bridged oligomer purity and stability were determined using previously created liquid-chromatography mass-spectrometry (LC-MS) methods. After 10 hours of incubation at 20°C, the signal of the ethylidene bridged dimer was 23.3% of the initial isolate signal, demonstrating degradation after removal from the model solution. Isolates were dried using a freeze-dryer to preserve purity and stability. Interestingly, the ethylidene-bridged oligomers are stable prior to isolation from the reaction mixture. These products could potentially be in equilibrium with the reactants. All isolates (ethylidene-bridged dimer, trimer, tetramer) were redissolved in methanol-d₄ and measured using both ¹H and ¹³C NMR for elucidation of structure. NMR characterization of these compounds has increased our understanding of LC-MS data conducted on actual wine investigating the fate and kinetics of acetaldehyde mediated bridging of flavonoids.

References

[1] Sheridan, M., Elias, R. (2016). J Agric Food Chem, 64, 45, 8615-8624.

[2] Peterson, A., Waterhouse, A. (2016). J Agric Food Chem, 64, 36, 6869–6878.