Chemical Transformation of B- to A-type Proanthocyanidins and 3D Structural Implications

Abstract

In nature, proanthocyanidins (PACs) with A-type linkages are relatively rare, likely due to biosynthetic constraints in the formation of additional ether bonds, to be introduced into the more common B-type precursors. However, A-type linkages confer greater structural rigidity on PACs than B-type linkages. Prior investigations into the structure-activity relationship (SAR) describing how plant-derived PACs with B- and complex AB-type linkages affect their capacity for dentin biomodification indicate that a higher ratio of double linkages leads to greater interaction with dentin type 1 collagen. Thus, the A-type PACs emerge as particularly intriguing candidates for interventional functional biomaterials. This study employed a free-radical mediated oxidation using DPPH to transform trimeric and tetrameric B-type PACs, 2 and 4, respectively, into their exclusively A-type linked analogues, 3 and 5, respectively. The structures and absolute configurations of the semi-synthetic products, including the new all-A-type tetramer 5, were determined by comprehensive spectroscopic analysis. Additionally, molecular modeling investigated the conformational characteristics of all trimers and tetramers, 1–5. Our findings suggest that the specific interflavan linkages significantly impact the flexibility and low energy conformations of the connected monomeric units, which conversely can affect the bioactive conformations relevant for dentin biomodification.

Graphical Abstract

INTRODUCTION

Whenever small molecules achieve biological activity through ligand-receptor interaction, their complementary spatial structure, including any constitutional and conformational structural features, determine affinity and potency. Unlike many synthetic leads compounds and drugs having relatively rigid structures, natural products (NPs) often possess scaffolds with greater conformational flexibility, which may in fact contribute to its drug-like properties.¹ Proanthocyanidins (PACs), also known as condensed tannins, are oligomeric to polymeric flavan-3-ols that are widely distributed in higher plants, and their diverse benefits for human health and agriculture are well documented.^2,3 The overall 3D spatial structures of PACs are influenced by numerous factors: the complex parameters of their constitutional chemistry, including the stereochemical variation of the flavan-3-ol monomers; the different interflavan linkage (IFL) types, site connectivities, and configurations; the E (equatorial) vs. A (axial)-conformations, exhibiting equatorial and axial orientations of the B and E rings in the upper and lower units.⁴ Moreover, the occurrence of atropisomerism as a result of steric hindrance around the IFL, provides a sufficient high rotation energy barrier to separate rotamers that can be distinguished by NMR and chiral HPLC. Lower rotational barriers within PACs leads to phenomena such as the broadening of ¹H NMR signals at ambient temperature.⁵ This is typically observed in B-type PACs, which therefore require acquisition of their NMR spectra at lower temperatures (<~270K).⁶

Our interdisciplinary NP research efforts are aimed at developing plant-derived PACs as functional dental biomaterials towards clinical translation. To date, more than 20 oligomeric PACs featuring both B-type only and complex A/B-type IFLs have been isolated from priority study plants, many of them new compounds, and their dentin modification capabilities have been evaluated in a variety of dental bioassays.^7–12 Whereas the structure-activity relationships (SARs) of PACs are generally poorly understood, we recently established the first set of SARs for dentin matrices: structural features including the degree of polymerization (DP), IFLs, and the configuration of the terminal units of PACs collectively regulate how PACs can enhance the biomechanical properties of the dentin matrix.^11,13

A-type PACs are a less common yet biologically relatively potent class of PACs. This can be attributed to their additional ether bond, which enhances conformational stability, which is associated with a reduction of atropisomeric effects as well as increased chemical stability over B-type PACs, as shown in some studies.^14,15 Whereas the exact biosynthetic mechanism for the formation of the additional A-type ether linkage remains to be determined,¹⁶ several attempts have been made to afford these unique structures in a controlled manner, for example, via chemical synthesis utilizing acyclic precursor monomers of the constituent flavan-3-ols, as well as through total synthesis.^17,18 Moreover, radical mediated or enzyme catalyzed reactions using reagents such as hydrogen peroxide, 1,1-diphenyl-2-picrylhydrazyl (DPPH), polyphenol oxidase, and xanthine oxidase have been reported to convert B-type to A-type PACs, expected with low yields.^19–22

Herein, trimeric and tetrameric A-type PACs were prepared via oxidative conversion from their naturally abundant precursors using DPPH to expand the PAC SAR knowledge via dentin biological and concurrent computer modeling studies. Goal of the latter was to assess the 3D structural and conformational spaces of all five investigated bioactive PACs (1–5) and determine the impact of IFL differences IFLs via in silico molecular modeling.

RESULTS AND DISCUSSION

Chemical Transformation of B-type to A-type PACs.

Procyanidin C1 (1) and cinnamtannin B1 (2) are the major trimers found in cocoa (Theobroma cacao) and cinnamon (Cinnamomum verum) barks, respectively (Figure 1).¹⁰ Chen et al. have previously investigated the conversion of the B-type PAC, 1, to the mixed AB-type 2 under various reaction conditions, observing an average conversion ratio of about 34%.²¹ In these studies, the free radical agent DPPH showed higher conversion ability than oxidases, and conversion was affected mainly by temperature and pH regardless of the reagents.²¹ By keeping the reaction in water solution at 60 °C for 3 d, the authors obtained the oxidation products of 2 in low yields (2~10%).²¹

Figure 1.

Chemical structures including PACBAR²³ representations of the investigated PACs, 1–5. The PACBAR terminology denotes epicatechin as EC, numbers indicate linkage sites.

In order to obtain the all-doubly linked trimer, we selected DPPH as reagent and 2 as substrate due to its natural abundance in cinnamon bark, but also based on the concept that the readily formed AB-type PAC would prohibit the formation of multiple isomeric products more likely than the B-type trimer could. To optimize the conversion ratio, different concentrations of reagent and substrate as well as reaction temperatures were investigated. Reaction monitoring by LC-MS (Fig. 2 ii~iv) showed that increased temperature and extended reaction time lead to C-2 epimerization,²¹ while reactions performed under room temperature and catalyzed by two equivalents of DPPH produced only the expected product (Figure 2i). Thus, conversion was scaled up under these favorable conditions (Scheme 1), the reaction mixture then purified by Sephadex LH-20 column chromatography (CC), to afford the trimer, aesculitannin C (3),²² with 40% yield and in reasonably purity (95.0% by qHNMR). Its structure was established by comprehensive NMR and ECD spectroscopic analysis, and comparison with published data.²¹

Figure 2.

The base peak chromatograms (BPC) of the LCMS analysis of the chemical conversion of trimer 2 to 3 under different reaction conditions in MeOH (r.t.- room temperature; substrate to DPPH present in 1:2 molar ratios; 3a being a designated stereoisomer of 3).

Scheme 1.

Chemical conversion of the mixed AB-type trimer, 2, to the A-type trimer, 3, using DPPH as oxidative reagent.

Similarly, an A-type tetramer was obtained by DPPH oxidation of cinnamtannin A2 (4), the major tetramer isolated from cocoa.^12,24 As the conversion was found to occur more slowly under room temperature, the reaction temperature was elevated to about 37 °C, but not higher to avoid epimerization. The reaction was monitored by TLC until the substrate was mostly consumed (Scheme 2). The mixture was then further purified by RP-C18 CC to yield the A-type tetramer, 5, in 26% yield and 84.9% purity by qHNMR. Extensive NMR and ECD spectroscopic analysis supported the assignment of this new structure.

Scheme 2.

Chemical conversion of B-type only tetramer, 4, to the A-type only tetramer, 5, using DPPH as oxidative reagent. Previous study on the conversion mechanism of B to A-type PACs has shown the oxidation of B-type PAC (2) involves forming a quinone methide intermediate (2a) that is transformed into A-type PAC (3) via intramolecular 1,6-michael addition (Scheme 3).^19,21

Structural Analysis of Newly Generated All A-type PACs.

Compound 3 was obtained as a brownish amorphous powder, the molecular formula of C₄₅H₃₄O₁₈ was established by its HRESIMS data (Figure S13), in line with the expected two less hydrogen atoms compared to the substrate 2. Comparing the ¹³C NMR spectra of 3 (Figure S2) and 2, the most significant difference was four oxymethine carbons (I/II/III-C-3 and III-C-2) rather than five in 3, which indicated the formation of an additional ether bond. All proton and carbon signals were carefully assigned by analysis of the 1D and 2D NMR spectra of 3, which were in accordance with the reported data of aesculitannin C (Table 1).²² The absolute configuration of 3 was determined by ECD spectroscopic analysis, which showed a positive cotton effect (CE) around 230 nm, and a negative CE around 270 nm, suggesting the IFLs were in β configuration, and the B-ring in α-configuration, respectively. As established previously, the absolute configuration of C-2 in the lower unit can be deduced by comparing the ¹³C NMR chemical shifts with those of all stereochemically possible A-type PAC dimers.^10,25 Accordingly, the carbon chemical shifts of the terminal unit of 3 were compared with the corresponding resonances in EC=8EC and EC=8eEC, as the chemical shifts of the middle unit were impacted mainly by the IFL. As shown in Table 2, the total absolute deviation (TAD, ∑|ΔδC|) values calculated for EC=8EC (TAD = 3.40) were smaller than those for EC=8eEC (TAD = 4.81), thus, the terminal unit was determined to be epicatechin. Therefore, 3 being the conversion product of cinnamtannin B1 (2) was determined to be aesculitannin C, i.e., epicatechin (2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-epicatechin, which can be presented by the micro-PACBAR code EC=8EC=8EC, all shown in Figure 1.

Table 1.

¹H (600 MHz) and ¹³C NMR (150 MHz) data of 3 and 5 in methanol-d₄ at 298 K.

			3		5
Unit	Ring	No.	δ C	δ H , J	δ C	δ H , J
I	C	2	99.89, C		100.02, C
		3	68.61, CH	4.000 d (3.19)	68.52, CH	4.022 d (3.19)
		4	29.77, CH	4.705 d (3.19)	29.70, CH	4.705 d (3.19)
	A	5	157.75, C		157.78, C
		6	97.78, CH	6.041 d (2.42)	97.86, CH	6.049 d (2.42)
		7	158.07, C		158.08, C
		8	96.39, CH	6.083 d (2.42)	96.38, CH	6.082 d (2.42)
		9	154.27, C		154.37, C
		10	103.59, C		103.85, C
	B	1’	132.62, C		132.64*, C
		2’	115.48, CH	7.104 d (2.20)	115.47, CH	7.092 d (2.20)
		3’	145.56a,*, C		145.62a,*, C
		4’	146.63a, C		146.68a, C
		5’	115.76, CH	6.793 d (8.36)	115.63, CH	6.781 d (8.36)
		6’	119.58, CH	7.009 dd (8.36, 2.20)	119.63, CH	6.992 dd (8.36, 2.20)
II	F	2	100.67, C		100.72, C
		3	68.01, CH	4.122 d (2.97)	68.85, CH	4.022 d (2.86)
		4	29.70, CH	4.548 d (2.97)	30.52, CH	4.780 d (2.86)
	D	5	155.89, C		156.63, C
		6	98.39, CH	6.179 s	97.63, CH	6.152 s
		7	153.30, C		153.24, C
		8	108.85, C		108.37, C
		9	149.03, C		149.41, C
		10	105.93, C		105.36, C
	E	1’	132.44, C		132.64*, C
		2’	115.38, CH	7.438 d (2.20)	115.25, CH	7.363 d (1.76)
		3’	145.56a,*, C		145.62a,*, C
		4’	146.80a, C		146.79a,*, C
		5’	115.71, CH	6.901 d (8.25)	115.75*, CH	6.859 d (8.14)
		6’	120.22, CH	7.423 dd (8.25, 2.20)	115.38, CH	7.369 dd (8.14, 1.76)
III	I	2	81.39, CH	4.934 s	100.59, C
		3	67.10, CH	4.262 brd (4.90)	68.05, CH	4.148 d (3.19)
		4	29.96, CH2	2.960 dd (16.56, 4.90), 2.824 d (16.56)	29.70, CH	4.530 d (3.19)
	G	5	156.54, C		155.83, C
		6	96.26, CH	6.113 s	98.31, CH	6.180 s
		7	152.65, C		153.54, C
		8	107.68, C		109.44, C
		9	151.74, C		148.75, C
		10	102.40, C		106.13, C
	H	1’	131.46, C		132.64*, C
		2’	115.62, CH	7.185 d (2.20)	115.46, CH	7.422 d (2.20)
		3’	145.93a, C		145.62a,*, C
		4’	146.05a, C		146.78a,*, C
		5’	116.04, CH	6.827 d (8.36)	115.75*, CH	6.876 d (8.36)
		6’	119.97, CH	7.010 dd (8.36, 2.20)	120.36, CH	7.465 dd (8.36, 2.20)
IV	L	2			81.48, CH	4.941 s
		3			67.06, CH	4.273 d (5.06)
		4			30.08, CH2	2.966 dd (16.95, 5.06), 2.827 d (16.95)
	J	5			156.63, C
		6			96.27, CH	6.101 s
		7			152.70, C
		8			107.58, C
		9			151.74, C
		10			102.57, C
	K	1’			131.47, C
		2’			115.79, CH	7.188 d (2.09)
		3’			146.04a, C
		4’			146.17a, C
		5’			115.74*, CH	6.827 d (8.14)
		6’			120.19, CH	7.035 dd (8.14, 2.09)

^aInterchangeable assignments.

^*Overlapped signals.

Table 2.

Comparison of the ¹³C NMR resonances of the terminal units in 3 and 5 with those of the corresponding carbons in the dimers, EC=8EC and EC=8eEC, established the absolute configurations of the terminal flavan-3-ol monomers.

	EC=8ECa	EC=8eECa	3 vs EC=8EC	3 vs EC=8eEC	5 vs EC=8EC	5 vs EC=8eEC
No.	δ C	δ C	ΔδC	ΔδC	ΔδC	ΔδC
2	81.77, C	80.86, C	−0.38	0.53	−0.29	0.62
3	67.01, CH	67.16, CH	0.09	−0.06	0.05	−0.10
4	29.93, CH	29.51, CH	0.03	0.45	0.15	0.57
5	156.62, C	156.75, C	−0.08	−0.21	0.01	−0.12
6	96.46, CH	96.44, CH	−0.20	−0.18	−0.19	−0.17
7	152.32, C	152.10, C	0.33	0.55	0.38	0.60
8	107.21, CH	106.94, CH	0.47	0.74	0.37	0.64
9	152.15, C	151.31, C	−0.41	0.43	−0.41	0.43
10	102.40, C	101.89, C	0.00	0.52	0.17	0.68
1’	131.21, C	131.43, C	0.25	0.03	0.26	0.04
2’	116.01, CH	115.21, CH	−0.39	0.41	−0.22	0.58
5’	115.65, CH	116.16, CH	0.39	−0.12	0.09	−0.42
6’	120.34, CH	119.40, CH	−0.37	0.57	−0.15	0.79
∑|ΔδC|			3.40	4.81	2.74	5.78

^aThe ¹³C NMR (225 MHz) data was recorded in methanol-d₄ at 298 K. C-3’/C-4’ were excluded due to their interchangeable assignments.

Compound 5 showed a molecular formula of C₆₀H₄₄O₂₄, six hydrogen atoms less than the reaction starting material, cinnamtannin A2 (4). While eight oxymethines (C-2 and C-3 of units I~IV) were present in 4, only five (I~IV-C-3 and IV-C-2) were observed from the ¹³C NMR spectrum of 5, suggesting the formation of an additional ether bond at C-2. The ¹H and ¹³C NMR data of 5 were fully assigned based on 2D NMR correlations (Table 1). The IFLs of (2→O→7, 4→8) were further confirmed by ROESY correlations between H-4 of the upper unit to H-2‘/H-6’ of the lower unit. The ECD spectrum of 5 showed a positive CE around 220 nm and a negative CE around 280 nm, establishing the β configurations of the doubly linkages, respectively. Furthermore, comparing the carbon chemical shifts with those of two A-type dimeric PACs, EC=8EC (TAD = 2.74) and EC=8eEC (TAD = 5.78), determined the absolute configuration of the terminal unit as epicatechin. Accordingly, the all-A-type tetramer, 5, was established as epicatechin (2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-epicatechin, EC=8EC=8EC=8EC in micro-PACBAR annotation (Fig. 1).

3D Structural and Conformational Analysis.

In silico conformational analysis allows the exploration of molecular potential energy surfaces and the identification of energetically favorable molecular conformers. In the case of trimeric and tetrameric PACs, conformational searching can help elucidate how slight alterations in functional groups affect the three-dimensional structure and pinpoint low-energy conformers (LECs) that may be close to bioactive molecular conformations.^26,27 Here, we assessed the effects of changes in IFLs for PACs on conformational flexibility.

To validate our conformational analysis, we utilized the CASCADE web server,²⁸ a graph neural network trained on a variety of QM and experimental NMR chemical shift data. The predicted chemical shifts based on Boltzmann-weighting of the conformers using Merck molecular force field (MMFF) energies provided a low Mean Absolute Error (MAE), indicating that our models were consistent with experimental results. PAC 3 had a ¹H MAE of 0.17 and a ¹³C MAE of 2.06. For PAC 5, we obtained a ¹H MAE of 0.23 and a ¹³C MAE of 2.37. From this, we moved forward with analyzing the MMFF conformers to further understand the structural changes from IFL differences.

From Figures 3b and 4b, we observed that the frequency of closely related conformers increases from PAC 1 and 2 to 3. The addition of the second 2β→7 linkage in PAC 3 greatly reduces the conformational flexibility of the molecule, with a maximum Root Mean Square Deviation (RMSD) of 2.5 Å when compared to the LEC. In contrast, PACs 1 and 2 both have a maximum RMSD of 5 Å compared to their LECs, with a variety of higher energy conformers in the 2–5 Å RMSD range. This may explain the dynamic NMR behavior usually observed among B-type PACs, which appeared as peak broadening due to the low energy barrier for interconversion of rotamers. Therefore, low-temperature NMR is imperative to acquire high-resolution NMR spectra of PACs in their free phenolic form, which results in one rotamer predominating and makes NMR structural elucidation possible. Atropisomeric molecules may differ in biological activity and selectivity towards a target, as well as pharmacokinetic properties, a phenomenon that has received considerable attention (see reviews^29,30).

Figure 3 –

(a) Lowest energy conformations (LECs) for trimers 1–3, illustrating intramolecular hydrogen bonding as yellow dashed lines, aromatic hydrogen bonds in cyan, and π-π stacking in blue. (b) Histograms of conformer RMSD frequency relative to the lowest energy conformer calculated by MMFF. The A/B labels in brackets represent the IFLs.

Figure 4 –

(A) Lowest energy conformations (LECs) for tetramers 4 and 5, illustrating intramolecular hydrogen bonding as yellow dashed lines and π-π stacking in blue. (B) Histograms of conformer RMSD frequency relative to the lowest energy conformer calculated by MMFF. The A/B labels in brackets represent the IFLs.

Three-dimensional PAC structures have been previously described using a variety of analytical techniques such as NMR, and multiple conformational features have been categorized.^31,32 Specifically, the Ø (C3-C4-C8-C9) dihedral angle has been used to characterize the structure as extended vs. compact, where the sign is positive for an extended rotamer and negative for a compact rotamer in B-type linkages (Figure S17). In A-type PACs, this geometry is constrained to compact (Table 3). Our LECs observed show B-type linkages to sample both extended and compact, whereas A-types are restricted to angles between −136.6° and −149.9°. Another descriptor is categorizing the LECs of the studied PACs as axial or equatorial according to the conformations of the catechol rings (B-rings) (Table 3). We observed a greater frequency of low energy equatorial rings in the A-type PACs. Additionally, the tetramers 4 and 5 were consistently equatorial, suggesting that a greater degree of polymerization may alter the energy barrier for interconversion. The equatorial conformation may reduce 1,3-diaxial interactions, as well as steric constraints between neighboring catechols in the extended rotamers. In the case of PACs 1 and 4, the equatorial E ring provided a favorable π-π stacking with the A ring. While the B-type PACs had a larger distribution of conformers, the low-energy and predominant forms are stabilized by favorable electrostatics from specific rotameric forms, as seen in this study (Figures 3a and 4a).

Table 3.

The Lowest-Energy Conformers of 1–5 and their conformation presented according to two parameters: (a) heterocyclic rings conformation (Eq-equatorial vs. Ax-axial conformation) and (b) the rotameric relationship at the interflavan linkage (compact vs. extended).

No.	Conformation	Rotamer*	Interflavan angle [Ø = C3-C4-C8-C9]
TC-BB (1)	Ax/Eq/Ax	Extended-Compact	92.9° −89.6°
CV-AB (2)	Eq/Ax/Eq	Extended (lower unit)	−143.6° 73.3°
CV-AA (3)	Eq/Eq/Eq	NA	−142.5° −149.5°
TC-BBB (4)	Eq/Eq/Eq/Eq	Extended-Compact-Compact	112° −84.9° −117.4°
TC-AAA (5)	Eq/Eq/Eq/Eq	NA	−145.1° −136.6° −138.9°

^*Extended (positive), compact (negative), NA (not applicable).

The predicted chemical-physical properties of the LECs of PACs 1–5 are listed in Table S1. Properties such as LogP (o/w) or Solvent Accessible Surface Area (SASA) allow evaluation of absorption, distribution, metabolism and excretion (ADME) properties in drug development.³³ However, oligomeric PACs belong to compounds beyond the chemical space described by Lipinski’s rule of 5, such as does the cyclic peptide cyclosporine A and the macrocyclic roxithromycin both of which possess structural flexibility. In such molecules, conformational changes can alter chemical-physical properties, including target bonding ability, cell permeability, and aqueous solubility.^34,35 Previous studies found that the changes of the chemical-physical properties of PACs potentially interfere with not only the interactions with type I collagen, but also with dental adhesives and the resin-dentin interface, due to the change of the dynamics of water within the dentin matrix.³⁶ More recent findings have shed light on the effect of the 3D spatial configuration and the IFL type of PACs on dentin biomodification potential. The B-type oligomeric PAC containing a branched (4→6) linkage yielded lower increases in the dentin modulus compared to non-branched B-type PACs.¹² Distinctly an AB-type oligomeric PAC bearing a branched unit linked via an A-type linkage displayed similar potency as other mixed AB-type PACs.¹¹ Moreover, calculated LogP values increased as double IFLs were formed (BB<AB<AA, Table S1), which may indicate favorable bonding and/or interaction between PACs and type I collagen in dentin.

CONCLUSIONS & OUTLOOK

PACs with exclusive A-type IFLs occur only rarely in nature. They possess less structural flexibility compared to all-B- and mixed A/B-type PACs, and have been insufficiently explored in terms of their biological activity. This study presents an efficient and chemically mild semisynthetic method for converting the naturally more abundant B-type or A/B-mixed into all-A-type PACs (performed for 3 and 5 as products) through free radical-mediated oxidation. The chemical structure and absolute configurations of the semi-synthesized all-A-type PACs were determined by rigorous NMR analysis, absolute stereochemical assignments via precise carbon chemical shift differentiation, as well as ECD spectroscopic analysis. Furthermore, the conformational analysis of trimers and tetramers 1–5 were conducted via in silico molecular modeling. The findings revealed that the types of IFLs impact both the flexibility and the LECs of the linked monomeric units. Specifically, this study consistently indicated that doubly linked PACs maintain a three-dimensional LEC, whereas singly linked PACs exhibit multiple LECs.

Future investigations will focus on exploring the dentin biomodification potential of these newly available all-A-type PACs in a panel of dental biomechanical bioassays and models of resin adherence. By contributing to a more comprehensive understanding of the conformational properties of the PAC linkage variants, the outcomes encourage the translational development of medium-size A-type PACs as functional biomaterials in dental therapy.

EXPERIMENTAL SECTION

General Experimental Procedures.

ECD spectra were recorded on a JASCO-815 spectrometer with a 0.1 cm quartz cuvette; the sample concentration was 0.1 or 0.2 mg/mL in MeOH. NMR spectra were acquired on a JEOL ECZ 600 MHz spectrometer equipped with a Royal Probe^™. High-resolution electrospray ionization (ESI) mass spectra were determined by a Bruker Impact II quadrupole time of flight (qTOF) mass spectrometer. Column chromatography (CC) was performed on Sephadex LH-20 gel (Pharmacia, Uppsala, Sweden) and TOYOPEARL HW-40F (TOSOH Bioscience LLC, PA, USA). The preparative fractionation was performed on a centrifugal partition chromatography (CPC) extractor, SCPE-250 (Armen Instrument, Saint Ave, France), TLC employed SIL G/UV254 plates (Macherey-Nagel, Inc., Bethlehem, PA, USA), visualized under UV light (254 nm) and spraying with vanillin-sulfuric acid reagent followed by heating. All solvents were obtained from Fisher Scientific (Fair Lawn, NJ, USA) or Sigma-Aldrich (St. Louis, MO, USA). NMR solvents were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). The NMR data was processed with MestReNova (ver. 14.2.0–26256, Mestrelab Research, S.L.).

Materials.

Cocoa polyphenol extract (60 g) was provided by Barry Callebaut, Wieze, Belgium (2014). Cinnamtannin B1 (1.0 g) was obtained from previous work.¹¹ DPPH was purchased from Tokyo Chemistry Industry.

UHPLC-MS Analysis.

Samples for UHPLC-MS analysis were prepared at 1 mg/mL. The separation was performed on the CORTECS^® C18 (2.7 μm, 3.0 × 100 mm) column with gradient elution using MeCN/H₂O/FA from (5:95:0.01) to (30:70:0.01) in 8 min. MS positive mode, general scan m/z 100 to 2000.

Extraction and Isolation.

Cocoa polyphenol extract (60 g) was dissolved in 70% aqueous acetone (800 mL) overnight, filtered, and evaporated in vacuo then freeze-dried to afford the PAC-enriched fractions (40 g). This fraction (4 g × 10) was loaded to CPC separation using EtOAc/n-BuOH/MeOH/H₂O (6:0.1:1:5, v/v) in ascending mode, with a flow rate of 10 mL/min and rotation at 3000 rpm. The CPC fractions were pooled into 6 subfractions A-F according to their TLC profiles. Fraction D (1.3 g) was subjected to Sephadex LH-20 CC in gradient elution from 90% to 100% MeOH in water, giving four subfractions D1-D4. D3 was identified as containing the nearly pure B-type tetramer, cinnamtannin A2 (4, 550 mg).

Aesculitannin C; epicatechin (2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-epicatechin; EC=8EC=8EC (3):

Brownish amorphous powder; ECD (MeOH) λ_max (Δε) 229 (116.05), 274 (−9.46) nm; HRMS (ESI) m/z [M + H]⁺ Calcd for C₄₅H₃₅O₁₈ 863.1818; Found 863.1782. ¹H and ¹³C NMR data, see Table 1.

Epicatechin-(2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-epicatechin-(2β→O→7,4β→8)-epicatechin; EC=8EC=8EC=8EC (5):

Brownish amorphous powder; ECD (MeOH) λ_max (Δε) 229 (74.16), 274 (−5.01) nm; HRMS (ESI) m/z [M + H]⁺ Calcd for C₆₀H₄₅O₂₄ 1149.2295; Found 1149.2256. ¹H and ¹³C NMR data, see Table 1.

Oxidative Conversion.

Compound 2 (1.0 g, 12 mM) and DPPH (1.0 g, 25 mM) dissolved in 100 mL MeOH were mixed and stirred under room temperature, and the reaction was monitored by TLC. When the substrate was mostly depleted for about 3 hours, the mixture was filtered and subjected to Sephadex LH-20 CC in 90% MeOH in water. The eluents were collected and pooled into four fractions based on their TLC profiles. Fraction 3 contained compound 3 (394 mg) in adequate purity (95.0%). Compound 4 (550 mg, 3.2 mM) and DPPH (1650 mg, 27.9 mM) dissolved in a total of 150 mL MeOH were mixed, and a few drops of DMSO were added to facilitate solubility of the reaction mixture. The reaction was stirred in a 37 °C water bath for 3 hours and monitored by TLC. The solution was finally filtered and the supernatant subjected to the RP-C18 CC using gradient elution from 5% to 20% MeOH in water. The 10%~15% eluent was evaporated in vacuo to afford 5 (145 mg).

3D Molecular Modeling.

Conformer generation and NMR chemical shift prediction were performed using the CASCADE web server.²⁸ CASCADE generates conformers using MMFF^32–35,37 using RDKit³⁸ with an energy cutoff of 10 kcal/mol and a subsequent final RMSD cutoff of 0.5 Å. The conformers generated by CASCADE with MMFF were analyzed in Maestro.³⁹

Scheme 3.

Conversion mechanism of B-type to A-type IFL induced by DPPH.