Proanthocyanidin Dimers and Trimers from Vitis vinifera Provide

18 mins ago - ... as well as the following biological raw data: DBMP data and explanations, dentin stiffness data, and collagenase assay data. S1–S1...
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Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Proanthocyanidin Dimers and Trimers from Vitis vinifera Provide Diverse Structural Motifs for the Evaluation of Dentin Biomodification Rasika S. Phansalkar,† Joo-Won Nam,†,§ Ariene A. Leme-Kraus,‡ Li-She Gan,⊥ Bin Zhou,† James B. McAlpine,† Shao-Nong Chen,† Ana K. Bedran-Russo,‡ and Guido F. Pauli*,†

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†

Department of Medicinal Chemistry and Pharmacognosy, and Program for Collaborative Research in the Pharmaceutical Sciences (PCRPS), University of Illinois at Chicago, College of Pharmacy, Chicago, Illinois 60612, United States ‡ Department of Restorative Dentistry, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois 60612, United States § College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Korea ⊥ College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 31005, China S Supporting Information *

ABSTRACT: Aimed at exploring the dentin biomodification potential of proanthocyanidins (PACs) for the development of dental biomaterials, this study reports the phytochemical and dental evaluation of nine B-type PACs from grape seed extract (GSE). Out of seven isolated dimers (1−7), four new compounds (2, 3, 5, and 6) involved relatively rare entcatechin or ent-epicatechin monomeric flavan-3-ol units. Lowtemperature NMR analyses conducted along with phloroglucinolysis and electronic circular dichroism enabled unequivocal structural characterization and stereochemical assignment. Additionally, one known (8) and one new (9) B-type trimer were characterized. Differential 13C NMR chemical shifts (Δδ) were used to determine the absolute configuration of 9, relative to the dimers 1 and 2 as the possible constituent subunits. Compared to the dimers, the trimers showed superior dentin biomodification properties. The dimers, 1−7, exhibited pronounced differences in their collagenase inhibitory activity, while enhancing dentin stiffness comparably. This suggests that PAC structural features such as the degree of polymerization, relative and absolute configuration have a differential influence on enhancement of dentin biomechanical and biostability. As mechanical enhancement to dentin and resistance to proteolytic biodegradation are both essential properties functional and stable dentin substrate, the structurally closely related PACs suggest a new metric, the dentin biomodification potential (DBMP) that may rationalize both properties.

G

rape (Vitis vinifera L.) seed extract (GSE) is a rich source of polyphenols, especially proanthocyanidins (PACs). The present work on GSE is directed toward the development of PAC-based dental biomaterials for improving the life-span of composite based dental restorations.1 PACs have been evaluated in many in vitro and in vivo biological assays. Notably, the monomeric flavan-3-ol precursors of PACs are also IMPs2 (invalid metabolic panaceas) and/or PAINS,3 (pan-assay interference compounds) reflecting their ability to bind nonspecifically to proteins, cause interference in UV absorption or fluorescence based in vitro assays, and due to other factors that lead to the overestimation of their therapeutic potential. The present study evaluated the structure−activity specific collagen-cross-linking ability of PACs, capable of enhancing the biomechanical properties and thereby decreasing biodegradability of dentin tissue. Representing a topical rather than a systemic application, the target protein is type I collagen, the major organic component © XXXX American Chemical Society and American Society of Pharmacognosy

of dentin. As the in vitro responses are measured mechanically (modulus of elasticity, MPa) or gravimetrically as percent mass gain/loss (resistance to proteolytic biodegradation), the present approach avoided the potential PAC bioassay interference mechanisms inherent in colorimetric, fluorescence, or UV based detection. The structural variables involved in PAC assembly, such as the degree of polymerization (DP), type of interflavan linkage (IFL), and relative and absolute configurations, give rise to a vast and frequently underappreciated structural diversity within this compound class. This provides handles for performing structure−activity relationship (SAR) studies without synthesis, provided close natural congeners can be isolated in sufficient quantities and characterized fully. Notably, determiReceived: November 10, 2018

A

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Structures of PAC dimers 1−7 and trimers 8 and 9. The pyran rings of the monomeric building blocks are color coded to indicate the stereochemistry. C catechin, ent-C ent-catechin, EC epicatechin, ent-EC ent-epicatechin, U upper unit, M middle unit, T terminal unit.

and 2 (Figure 1). This approach has been demonstrated recently in the determination of the absolute configuration of A-type PAC trimers and tetramers from pine bark.5,6

nation of the absolute configuration in B-type PACs continues to be a challenge as the number of stereocenters and the rotameric degrees of freedom increases with the DP, leading to more complex relative configuration relationships and conformationally averaged NMR coupling constants. However, on many occasions, the PAC literature has been making assumptions regarding the absolute configuration of PACs by reverting to rather tentative biosynthetic pathways. Furthermore, even where biosynthetic mechanisms apply, one cannot assume that a molecule with a particular absolute configuration can only be formed via these pathways as the use of acid (PACs are acidic), base, and/or heat during extraction or other downstream processes can lead to epimerization.4 Hence, unequivocal determination of the absolute configuration becomes desirable, or even essential if the compound is intended for biological testing and the results used in SAR studies. In the present study, seven B-type PAC dimers including three known and four new compounds were isolated. The relatively rare ent-catechin and/or ent-epicatechin units were found to be the flavan-3-ol constituent units in the dimers 2, 3, 5, and 6. In addition to the evaluation of these dimers in a dentin biomechanical assay, the isolates served as building blocks to aid in the structural characterization of a new trimer, 9. Hence, differential chemical shift (Δδ) values were used to determine the absolute configuration of the terminal unit of trimer 9 by comparing it with the fully established dimers 1

■

RESULTS AND DISCUSSION Isolation of PAC Dimers 1−7 and Trimers 8 and 9 from GSE. Previous studies on the dentin biomodification effects of PACs from various plant sources including pine bark (Pinus massoniana), cinnamon bark (Cinnamomum verum), and GSE concluded that the trimeric and tetrameric PACs have an optimum molecular size range for effective dentin matrix crosslinking.5−7 Toward the goal of establishing the most effective PAC preparation for dental biomodification, a centrifugal partition chromatography method was shown to be capable of enriching trimers and tetramers selectively.8 The process achieved a stepwise depletion of monomers and many dimers. A dimeric PAC fraction was the byproduct of the trimer/ tetramer enrichment process and the source of the isolated dimers, 1−7, which served a dual purpose: First, their relatively higher isolation yields allowed for evaluation of both dentin biomechanical (apparent modulus of elasticity) and biodegradation (collagenase digestion) assays.1,9,10 This enhanced the understanding of the effect of subtle structural differences on dentin bioactivity. Second, the dimers were used as analytical building blocks to determine the absolute configuration of the new trimeric PAC, 9. B

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 1. 1H and

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C NMR Chemical Shifts for Compounds 2 and 3a 2

3 δC (ppm)

δH (ppm), (J in Hz) major U-2 U-3 U-4 U-6 U-8 U-2′ U-5′ U-6′ U-1′ U-5 U-7 U-8a U-4a T-2 T-3 T-4α T-4β T-6 T-2′ T-5′ T-6′ T-8 T-1′ T-5 T-7 T-8a T-4a

minor

5.0404, d (0.98) 3.9620, dd (0.98, 1.93) 4.4814, d (1.93) 5.9564d, d (2.07) 5.8992d, d (2.07) 6.9006, d (2.02) 6.7415, d (8.15) 6.6704, dd (8.15, 2.02)

5.1030, d (1.09) 3.8010, dd (1.83, 1.09) 4.5759, d (1.83) 5.8567e, d (2.16) 5.6401e, d (2.16) 6.8504, d (2.03) 6.6812, d (8.22) 6.6607, dd (8.22, 2.03)

4.7641, d (7.40) 3.8511, ddd (8.24,7.40, 5.44) 2.5989, dd (−16.08, 8.24) 2.9134, dd (−16.08, 5.44)

4.3970, 3.7826, 5.32) 2.4902, 2.6192,

d (6.58) ddd (7.09, 6.58,

5.8604, 6.8659, 6.7251, 6.7254,

6.0679, 6.4510, 6.6142, 6.1453,

s d (2.05) d (8.13) dd (8.13, 2.05)

s dd (1.04, 0.95) dd (9.30, 0.95) dd (9.30, 1.04)

δH (ppm), (J in Hz)

dd (−16.25, 7.09) dd (−16.25, 5.32)

major 76.98 73.59 36.85 95.94a 95.52a 114.95 115.71b 118.97 132.60 157.73c 158.16c 158.50c 101.43 82.72 69.11 28.71

96.79 114.09 115.79b 120.05 107.20 132.31 155.79 156.54 154.07 100.70

δC (ppm) minor

4.1997, d (9.85) 4.3863, dd (9.85, 8.36) 4.3672, d (8.36) 5.7410g, d (2.37) 5.5308g, (2.37) 6.7833, dd (1.95 0.33) 6.7397, d (8.09) 6.5779, dd (8.09, 1.95)

4.3047, d (9.89) 4.3854, dd (9.89, 8.26) 4.4750, d (8.26) 5.7960h, (2.39) 5.8295h, (2.39) 6.8915, d (1.98) 6.7205, d (8.07) 6.7485, dd (8.07, 1.98)

4.0934, 3.9016, 5.83) 2.4195, 2.9018, 0.62) 6.0406, 6.7908, 6.8039, 6.6572,

4.7362, 3.9158, 5.36) 2.5398, 2.8495, 0.69) 5.9182, 6.9168, 6.7711, 6.8978,

dd (8.22, 0.62) ddd (8.79, 8.22, dd (−16.07, 8.79) ddd (−16.07, 5.83, s d (2.03) d (8.03) dd (8.03, 2.03)

dd (7.31, 0.69) ddd (8.16, 7.31, dd (−15.86, 8.16) ddd (−15.86, 5.36, s d (2.02) d (8.10) dd (8.10, 2.02)

major

minor

83.85 73.87 38.54 95.92a 95.42a 115.67c 116.07 120.47 132.31 156.68e 156.82e 158.64e 107.18 85.7 68.50

83.86 73.62 38.67 95.72b 96.70b 115.13 116.40d 120.87 132.27 157.01f 157.59f 158.65f 107.21 83.01 69.13

29.18

28.50

96.84 115.51 115.88c 121.11 108.43 131.83 155.51 155.96 154.83 101.58

97.09 115.59 115.84d 119.97 108.37 132.52 155.39 155.82 154.98 99.52

C data of 2 are only shown for the major rotamer. The δH values are obtained from HiFSA of the experimental NMR spectrum acquired at 255 K on an 800 MHz instrument. The δC extracted from a spectrum were acquired on a 900 MHz (225 MHz 13C channel) instrument at 255 K. Assignments marked a−h are interchangeable or indistinguishable. The 13C chemical shifts for C-3′ and C-4′ were not distinguishable (145.03 to 146.09 ppm for 2; 145.49 to 146.24 for 3). a13

isolates that are typically only available in low milligram quantities. Typically, the structure elucidation workflow begins with the identification of the geminal methylene hydrogens (2.5−3.0 ppm) that exhibit a dd coupling pattern. From here, the PAC structure is elucidated “upwards”, starting from the terminal unit to the upper (U) unit using correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), and rotating-frame Overhauser spectroscopy (ROESY) correlations. A challenging step is the determination of the 4→6/8 IFL based on the HMBC data, which requires the distinctive assignment of the C-5, C-7, and C-8a oxygenated tertiary carbons. The C-5 and C-7 chemical shifts are often indistinguishable due to relatively low resolution in the 13C dimension of 1H detected 2D NMR spectra. This was solved by the parallel use of HMBC and ROESY correlations to confirm the IFLs. The final challenge is the determination of the absolute configuration of the constituent monomeric units. While the assignment of the H-2/H-3 relative configuration is achieved readily based on the coupling constants, it is difficult to derive the H-3/H-4 relative configuration in cases of H-2/ H-3 cis configured isomers.16,17 Furthermore, the use of NOESY correlations is limited by the occurrence of spin

Dimers 1, 4, and 7 were identified as procyanidin B1, procyanidin B3, and procyanidin B2, respectively. Their 1H NMR, 13C NMR, and electronic circular dichroism (ECD) spectra closely matched the reported data.11,12 Compounds 2, 3, 5, and 6 were found to be new PAC dimers containing the rare ent-catechin or ent-epicatechin constituent units. The 1H and 13C NMR assignment of all new compounds 2, 3, 5, 6, and 9 (obtained via HiFSA13) are presented in Tables 1−3. Challenging Aspects in the Structure Elucidation of Proanthocyanidins. The monomeric building blocks of PAC skeletons are primarily composed of the phloroglucinol A-ring AX spin system involved in the IFLs, followed by the pyran Cring composed of the stereogenic centers C/H-2, 3, and 4, as well as the catechol B-ring attached to C-2 that presents a 1H AMX spin system. The first structure elucidation challenge is the severe peak broadening observed in the NMR spectra of Btype PACs, due to rotational isomerism that results in uninterpretable spectra obtained at room temperature.7,11,14,15 Thus, the present study used low temperature (255 K) NMR as a means of achieving high-resolution line shape and enabling signal assignments. This is preferred over the use of chemical derivatization as it allows the structural analysis of PACs in their native form, thus enabling biological assessment of the C

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 2. 1H and

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C NMR Chemical Shifts for 5 (Major Rotamer) and 6 (Major and Minor Rotamers)a 5

6 δH (ppm), (J in Hz)

δH (ppm), (J in Hz) U-2 U-3 U-4 U-6 U-8 U-2′ U-5′ U-6′ U-1′ U-5 U-7 U-8a U-4a T-2 T-3 T-4α T-4β T-6 T-2′ T-5′ T-6′ T-8 T-1′ T-5 T-7 T-8a T-4a

5.0842, d (0.68) 4.2298, dd (0.68, 1.92) 4.6276, d (1.92) 5.8975d, d (2.23) 5.9683d, d (2.23) 6.9579, dd (1.98, 0.32) 6.7413, dd (8.28, 0.32) 6.7637, dd (8.28, 1.98)

5.0315, 4.2324, 2.8470, 2.9597, 5.8488, 7.0578, 6.7488, 6.7495,

d (0.73) ddd (4.55, 2.51, 0.73) dd (−16.56, 2.51) dd (−16.56, 4.55) s dd (1.61, 0.35) dd (11.54, 0.35) dd (11.54, 1.61)

δC (ppm) 77.21 72.70 36.60 95.43a 95.96a 115.17 115.64b 119.29 132.64 157.72c 158.16c 158.64c 101.49 79.32 67.39 29.56 96.77 114.53 115.92b 118.30 107.82 132.73 156.16 156.28 154.41 99.78

major 4.3850, dd (9.88, −0.54) 4.6393, dd (9.88, 8.77) 4.4810, d (8.77) 5.7127e, d (2.20) 5.6527e, d (2.20) 6.9482, d (2.00) 6.7333, d (8.06) 6.7805, ddd (8.06, 2.00, −0.54)

4.5368, 4.1518, 2.6893, 2.8372, 6.0683, 6.7874, 6.7765, 6.9010,

dddd (1.45, −0.78, −0.57, 0.28) ddd (4.54, 3.17, 1.45) ddd (−16.71, 3.17, 0.28) ddd (−16.71, 4.54, 0.57) d (−0.57) dd (2.05, −0.57) d (8.15) ddd (8.15, 2.05, −0.78)

δC (ppm) minor

major

minor

4.3287, dd (9.89, −0.16) 4.4960, dd (9.89, 8.26) 4.5922, d (8.26) 5.8495f, d (2.26) 5.8273f, d (2.26) 6.9014, d (2.01) 6.7673, d (8.11) 6.7577, ddd (8.11, 2.01, −0.16)

84.12 72.88 38.75 96.07a 96.96a 115.55 116.49 120.74 132.17 157.04b 156.76 158.36b 106.92 79.33 66.69 29.30

84.02 73.31 38.30 96.09d 97.00d 115.29 115.69 120.87 132.42 nd

95.62 114.44 115.86 120.23 107.70 131.71 156.17c 156.18c 155.27 100.66

95.61 115.05 115.71 119.16 107.82 132.21 nd

4.9722, 4.1541, 2.8392, 2.8927, 5.9547, 7.0585, 6.7197, 6.8893,

dd (1.17, −0.28) ddd (4.20, 3.55, 1.17) dd (−16.25, 3.55) dd (−16.25, 4.20) s d (2.02) d (8.09) d (8.09, 2.02, −0.28)

107.21 80.24 67.27 29.42

155.17 99.87

Some signals of the minor rotamer were not detected [nd] as distinct resonances. The δH values are obtained from HiFSA of the experimental NMR spectrum acquired at 255 K on an 800 MHz instrument. The δC extracted from a spectrum were acquired on a 900 MHz (225 MHz 13C channel) instrument at 255 K. Assignments marked a−f are interchangeable or indistinguishable. 13C chemical shifts for C3′ and C4′ were not distinguishable (145.27 to 145.82 ppm for 5; 145.39 to 146.26 for 6). a

diffusion due to the increased rotational flexibility of these molecules. As underivatized PACs do not readily crystallize, this study used a battery of analytical techniques including ECD18 spectroscopy, coupling constant analysis, phloroglucinolysis,19 DFT based chemical shift calculations,5,20,21 DP4 analysis,22 and differential chemical shifts (Δδ)5,6 to first narrow down the stereoisomeric possibilities and then complete the assignment of the absolute configuration. It is important to note that DFT type calculations can never replace true empirical observations. In this study, even though DP4+ analysis and DFT calculations were performed, the results were considered inconclusive toward the determination of absolute configuration due to large Δδ between the calculated and observed chemical shifts. Only the specific spectroscopic observations that were essential for the structural analysis of these dimers and trimers will be discussed in detail below. The full sets of 1D and 2D NMR spectroscopic evidence is presented in the Supporting Information (SI) with appropriate annotations. Structure Elucidation of 2 in Relation to 1 (Procyanidin B1). The molecular formula of C30H26O12 was derived from the HR-ESI-MS data of 2, exhibiting an [M + H]+ at m/z 579.1451 (calcd 579.1503) and an [M − H]− at m/z 577.1319 (calcd. 577.1346). This confirmed the constitution of 2 as a dimeric PAC. As in 1, the 3J2,3 of 0.98 Hz in 2 was an indicator of a 2,3-cis configuration in the upper

unit, whereas 3 J 2,3 of 7.40 Hz indicated a 2,3-trans configuration for the terminal unit. The position of the IFL was confirmed as U-4→T-8 from the HMBC correlation between both hydrogens U-4, T-2, and carbon T-8a (Figure 5). The ROESY correlation between hydrogens U-3 and T-2′ (Figure 2) provided additional evidence supporting the 4→8 IFL. Therefore, the only difference between 1 and 2 as nonidentical molecules was the absolute configuration at carbons T-2 and T-3, establishing the two compounds as diastereomers. Electronic circular dichroism and phloroglucinolysis experiments further confirmed this interpretation. A high amplitude positive Cotton effect (CE) around 220 nm was observed in the ECD spectrum of 2, confirming the 4β orientation of the IFL, and, thus, 4R configuration analogous to 1. On the other hand, 2 exhibited a positive CE around 280 nm, unlike 1, which shows a negative CE at this wavelength (Figure 4). This suggested that the absolute configuration at U-2 and/or T-2 in 2 was the reverse of that in 1. Chiral phase chromatographic analysis of the terminal unit released after phloroglucinolysis of 2 (S8) confirmed the presence of entcatechin as the terminal unit. Hence, two stereoisomeric possibilities, 2a (epicatechin-(4β→8)-ent-catechin) and 2b (ent-epicatechin-(4β→8)-ent-catechin), were feasible. The magnitude of the positive CE around 280 nm suggested 2b as the more likely structure. However, low amplitude CE around 270−280 nm occurring due to the aromatic 1Lb D

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 3. 1H and

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C Chemical Shift Assignments of 9a

the upper unit. An all-cis (2,3-cis-3,4-cis) configuration would result in a 3J3,4 ≥ 4 Hz.24 Additionally, an upfield shift (∼4−5 ppm) at C-2 with δ13C of 76.98 ppm observed due to a γgauche effect at C-4 indicated a 2,4-trans configuration.25 Accordingly, the proposed structure for dimer 2 is epicatechin(4β→8)-ent-catechin. Structure Elucidation of 3 in Relation to 4 (Procyanidin B3). The HR-ESI-MS data ([M + H]+ m/z 579.1448, [M − H]− m/z 577.1319) supported a molecular formula of C30H26O12 and confirmed the dimeric PAC constitution of 3. The IFL for compound 3 was established using the ROESY experiment as the resolution in the indirect 13 C dimension in the HMBC was insufficient to distinguish between carbons T-5 or 7 and T-8a. The ROESY correlation observed between the hydrogens U-3 (4.38 ppm) and T-6′ (6.65 ppm) confirmed the 4→8 IFL (Figure 2), as this correlation would be absent in the case of a 4→6 linked dimer. Large 3J2,3 values of 9.85 and 8.22 Hz in the upper (U) and terminal (T) units, respectively, confirmed the 2,3-trans configuration for both units of 3 as also being present in compound 4 (procyanidin B3). However, the ECD spectrum confirmed that 3 had the opposite absolute configuration (4β or 4R) at the IFL relative to 4, which possesses a 4α or 4S configuration, as evident from the CE around 220 nm being positive in 3 vs negative in 4 (Figure 4). Moreover, the sign of the CE around 280 nm in 3 differed relative to 4: the ECD curve was almost flat in 3, as opposed to showing a low amplitude negative CE in 4. This suggested the presence of the enantiomeric form of one of the U or T monomeric units. The possibility of both U and T being ent-catechin units could be ruled out because the resulting dimer would be an enantiomer of compound 4. Moreover, 3 and 4 were separated by nonchiral phase HPLC and had distinctly different NMR spectra. If the terminal unit was ent-catechin and the upper unit was catechin (as is the case for compound 3 published in ref 16), then a 3J3,4 value of 6.5 Hz should have been observed as expected for a 3,4-cis configured upper unit.16 In contrast, the observed 3J3,4 was 8.3 Hz, indicating a 3,4-trans configured upper unit. As the IFL was confirmed to be 4β from the ECD curve, the upper unit had to be ent-catechin. Considering that the absolute configuration at position C-4 was known to be 4R from the ECD data, and the terminal unit was confirmed to be catechin from the phloroglucinolysis experiment (S8), the structure of the dimer 3 was confirmed as ent-catechin-(4β→ 8)-catechin. Structure Elucidation of 5 in Relation to 7 (Procyanidin B2). The dimeric PAC constitution of 5 was confirmed based on the HR-ESI-MS data, showing an [M + H]+ at m/z 579.1457 and an [M − H]− at m/z 577.1320. The 1 H chemical shift assignment of hydrogens U-5′, U-6′, T-5′, and T-6’ was complicated due to a combination of severe signal overlap and 1H higher order effects. Therefore, 1H iterative full spin analysis (HiFSA)13,26−29 was employed using the PERCH NMR software tool to perform a quantum mechanical deconvolution (Figure 7) for accurate assignments. As for the previous two pairs of dimers (1/2 and 3/4), 5 showed marked similarities with another isolate, compound 7, a known compound, epicatechin-(4β→8)-epicatechin (procyanidin B2), which represents the most abundant dimer in GSE. Both 5 and 7 showed the same relative configuration at C-2 and C-3 of their U and T units. The small J2,3 values of 0.68 and 0.73 Hz for the U and T units of 5, respectively, confirmed

9 δH (ppm), (J in Hz) U-2 U-3 U-4 U-6 U-8 U-2′ U-5′ U-6′ U-1′ U-4a M-2 M-3 M-4 M-6 M-8 M-2′ M-5′ M-6′ M-1′ M-4a M-8a T-2 T-3 T-4a T-4b T-6 T-2′ T-5′ T-6′ T-8 T-1′ T-8a T-4a

5.0748, d (1.09) 3.9512, dd (2.00, 1.09) 4.6936, d (2.00) 6.0055a, d (2.33) 5.9786a, d (2.33) 6.8967, d (2.02) 6.7354, d (7.92) 6.6805, dd (7.92, 2.02)

5.2332, 4.0418, 4.5651, 5.8609,

d (1.10) dd (1.96, 1.10) d (1.96) s

7.0753, d (1.82, 0.14) 6.7323, dd (8.65, 0.14) 6.7331, dd (8.65, 1.82)

4.8007, 3.8529, 2.6179, 2.9247, 5.9198, 6.9114, 6.7459, 6.7452,

d (7.33) ddd (8.74, 7.33, 5.61) dd (−16.35, 8.47) dd (−16.35, 5.61) s d (1.83) d (8.68) dd (8.68, 1.83)

δC (ppm) 76.79 73.48 36.96 95.94b 95.93b 114.90 115.71 119.01 132.54 101.79 76.87 73.26 37.25 96.92 106.76 114.79 115.81 118.54 132.65 101.92 154.74 82.68 69.17 28.69 97.03 113.88 115.93 119.98 107.61 132.31 154.05 100.77

a

Experimental 1H NMR spectra recorded at 255 K on an 800 MHz instrument were simulated using HiFSA to obtain accurate δ and J values. 13C NMR data was obtained on a 600 MHz (150 MHz carbon frequency) instrument. a and b indicate interchangeable assignments. Assignments for the 13C resonances of the non-hydrogen bearing carbons M-5/7, U-5/7/8a, and T-5/7 could not be made due to insufficient resolution in the HMBC. The 13C chemical shifts for C-3′ and C-4′ were observed in the 145.30−146.19 ppm range.

transitions that is typically used for assignment of the C-2 configuration of flavan-3-ols is completely dominated by the aryl chromophore (A-ring) attached to the C-4 in PACs. Hence, the sign or absolute intensity of the CE at 280 nm cannot be used as a stand-alone evidence in the determination of the absolute configuration at C-2 of PACs.23 DP4+ analysis of the DFT calculated 13C NMR chemical shifts (S9) was performed to determine the correct stereoisomer22 yielding DP4+ probability scores of 0% for 2a and 100% for 2b (S9). However, the large deviations between DFT calculated and experimental NMR chemical shifts and the absence of a correlation between hydrogens U-2 and U-4 in the ROESY spectrum (NOESY not considered due to spin diffusion) could not enable an unequivocal all-cis assignment of the upper unit (isomer 2b). Considering 4-aryl-flavan-3-ols as models for PACs, the observed 3J2,3 and 3J3,4 values of 0.98 and 1.98 Hz, respectively, also suggested a 2,3-cis-3,4-trans configuration of E

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 2. ROESY correlations indicating the position of the interflavan linkage. Magnified portions of the ROESY spectra of the dimers 2, 3, 5, and 6, along with their 3D structural representation (created in Avogadro 1.1.1) that indicate the observed NOEs between the upper (U) and the terminal (T) unit; confirmations for the 4→8 IFL are shown by a dashed line.

their bis-2,3-cis configuration. An HMBC cross peak from hydrogens U-4 (4.6276 ppm) and T-4α,4β (2.8470, 2.9597 ppm) to carbon T-8a at 154.41 ppm (Figure 5) indicated that 5 possessed a 4→8 linkage. This was further confirmed by the NOE observed between hydrogens U-4 (4.6276) and T-2′ (7.0578 ppm) in the ROESY experiment (Figure 2). The highamplitude positive CE around 220 nm in the ECD spectrum confirmed the 4β orientation of the IFL and, thereby, the 4R absolute configuration (Figure 4). These observations

narrowed down the difference between 5 and 7 to the relative configuration in position C-3 and C-4, and subsequently, the absolute configurations at C-2/3/4. Considering that 5 and 7 could not be enantiomers (separable by nonchiral phase HPLC, distinctly different NMR spectra), 5 had to be one of the three diastereomeric possibilities: (5a) epicatechin-(4β→8)-ent-epicatechin, (5b) ent-epicatechin-(4β→8)-ent-epicatechin, or (5c) ent-epicatechin-(4β→8)-epicatechin. The positive sign and the low F

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. ROESY spectrum of trimer 9. (A) Full ROESY spectrum; (B) magnified portion of the ROESY spectrum showing the correlations that led to the determination of the IFL (green boxes).

Similar to 2, a δ13C of 77.22 at C-2 indicated a 2,4-trans configuration due to the γ-gauche effect related upfield shift caused by the pseudoequatorial orientation of the C-4 heterocyclic ring system.25 This led to an unequivocal determination of the structure of dimer 5 as epicatechin(4β→8)-ent-epicatechin (diastereomer 5a). Structure Elucidation of Dimer 6. Compound 6 was also determined to be a dimer based on its [M + H]+ at m/z 579.1443 and [M − H]− at m/z 577.1314, obtained by HRESI-MS analysis. The relative configuration of the U and T monomeric units of 6 was determined to be 2,3-trans in the upper and 2,3-cis in the lower unit, as gleaned from the 3J2,3 values of 9.88 and 1.45 Hz, respectively. The IFL was determined to be 4→8 from the HMBC cross peak between the hydrogens T-2 (4.5368 ppm), U-4 (4.4810 ppm), and T4α,4β (2.6893, 2.8372 ppm) and the carbon T-8a at 155.27 ppm (Figure 5). This was further confirmed by the ROESY correlation (Figure 2) between the hydrogen U-3 (4.6393 ppm) and the hydrogens T-2′ and T-6′ (6.7874 and 6.9010 ppm, respectively). Next, the absolute configuration at the IFL

magnitude of the CE around 280 nm suggested the likelihood of only one ent-epicatechin (2S configured monomeric unit) being present in 5. Phloroglucinolysis was performed to confirm that the terminal unit of dimer 5 is indeed entepicatechin (S8), thus ruling out diastereomer 5c. However, unlike for PACs with 2,3-trans monomers in the upper units (compounds 3,4, and 6), the 3J3,4 values for 2,3-cis-3,4-trans vs 2,3-cis-3,4-cis configured structures like 5a−c are not sufficiently distinct for the determination of the 3,4-relative configuration solely on the basis of coupling constant analysis.17 Based on the literature on 4-aryl-flavan-3-ols, 3J2,3 and 3J3,4 values of 0.68 and 1.92 Hz respectively suggested a 2,3-cis-3,4-trans configuration.24 As is the case for 2, large deviations were observed in the DFT calculated (for 5a and 5b) and experimental 13C chemical shifts. This could be due to the difficulty in generating the lowest energy conformers for DFT calculations as the B-type PACs have many conformational possibilities due to rotational freedom around the IFL and B-rings. Also, a definitive correlation between hydrogens U-2 and U-4 was not observed in the ROESY spectrum. G

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 4. ECD spectra of dimers 1−7 and trimers 8 and 9, all recorded in MeOH.

exhibited identical relative configurations at C-2/3 as in the known trimer 8 [EC-(4β→8)-EC-(4β→8)-C]. The 3J2,3 values of 1.09, 1.10, and 7.33 Hz for the U, M, and T units, respectively, indicated a 2,3-cis-cis-trans configured trimer. Starting with the identification of the geminal methylene hydrogens (2.9247 and 2.6179 ppm) of the T unit, the remaining aliphatic hydrogens were assigned using COSY data. Hydrogens U-4 and M-4 were identified from their characteristic δC values of 36.96 and 37.25, respectively, and the aliphatic spin system of the C-rings were assigned via the COSY spectrum. HMBC correlations from both hydrogens T2 (4.8007 ppm) and M-4 (4.5651 ppm) to carbon T-8a at 154.05 ppm confirmed the 4→8 IFL between the middle and the terminal unit (Figure 5). The δ 13C at U-8a/U-5/U-7 appeared to be further deshielded to 157−159 ppm, relative to T-8a/M-8a which usually resonates between 153 and 155 ppm. This inference and based on an HMBC cross peak from hydrogen U-4 to a carbon at 154.75 ppm, 9 had to have a U4→M-8 IFL. The ROESY crosspeaks observed between the hydrogens U-4 and M-2′ as well as M-4 and T-2′ (Figure 3)

was determined to be 4R from the 4β orientation, as evidenced by the high amplitude positive CE around 220 nm in the ECD spectrum (Figure 3). Thus, the 2D structure of dimer 6 was determined to be (ent)-catechin-(4β→8)-(ent)-epicatechin. The next step involved the determination of the relative configurations of C-3/C-4 and, subsequently, the absolute configuration at C-2/3/4 of the constituent monomeric units. For the upper unit, 3J3,4 of 8.77 Hz indicated a 3,4-trans relative configuration. As the absolute configuration at U-4 was known from the ECD data, the absolute configuration at U-2 and U-3 had to be 2S and 3R, respectively, based on the relative configuration. In case of the terminal unit, however, phloroglucinolysis was required to confirm the absolute configuration as (2R,3R) (S8). Thus, the structure of 6 was defined as ent-catechin-(4β→8)-epicatechin. Structure Elucidation of the New Trimeric PAC 9 in Relation to 8. The trimeric nature of 9 was confirmed from the HRMS data where an ion with m/z of 867.2075 was observed for [M + H]+ (calcd m/z 867.2136 for C45H39O18). Trimer 9 was found to be composed of monomeric units that H

DOI: 10.1021/acs.jnatprod.8b00953 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 5. Key HMBC correlations for 2, 5, 6, and 9, with those correlations involved in the determination of the IFL highlighted. Assignment of the 13 C chemical shift of C-8a is crucial for the confirmation of 4→8 IFL via HMBC. Low resolution in the F1 dimension prevented the unequivocal assignment of the IFL for 3; hence, ROESY correlations were used (Figure 2).

Figure 6. Comparison of the 1H and 13C δ values of dimers 1 and 2 with trimer 9. The X-axis represents the carbon (A) and hydrogen (B) atom positions of the terminal unit that was used for the differential chemical shift (Δδ) calculation. The Y-axis represents the Δδ between 9 and 1 (gray) and 9 and 2 (black). The δ values for 9 were normalized to zero in order to allow comparison of the differences. The Δδ values for the dimer 2 were significantly lower than those of 1, indicating greater structural similarities between 2 and the terminal dimeric unit of 9. This observation supports an ent-catechin moiety as the terminal flavanol unit of 9.

The molecular weight of PAC trimers is above the molecular weight threshold (