A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin ( Onobrychis viciifolia ) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6-113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.

The interaction between pentagalloyl glucose (PGG) and two globular proteins, bovine serum albumin (BSA) and ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco), was investigated by isothermal titration calorimetry (ITC). ITC data fit to a binding model consisting of two sets of multiple binding sites, which reveal similarities in the mode of binding of PGG to BSA and rubisco. In both cases, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface, which promotes aggregation and precipitation of the PGG-protein complex. This model was confirmed by turbidimetry analysis of the PGG-BSA interaction. Analysis of tryptophan fluorescence quenching during the interaction of PGG with BSA suggests that binding of PGG leads to some conformational changes that are energetically closer to the unfolded state of the BSA structure, because small red shifts in the resulting emission spectra were observed.

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a prepurification step, needed more time for large scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification orH-C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.

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A convergent synthesis for the preparation of a new orthogonally protected tripodal scaffold has been developed. The scaffold was successfully coupled to a Tentagel solid support and further derivatised at the three attachment points demonstrating its possible use for combinatorial chemistry.