molecules Article Ellagitannin–Lipid Interaction by HR-MAS NMR Spectroscopy Valtteri Virtanen * , Susanna Räikkönen, Elina Puljula and Maarit Karonen Natural Chemistry Research Group, Department of Chemistry, University of Turku, FI-20014 Turku, Finland; [email protected] (S.R.); [email protected] (E.P.); maarit.karonen@utu.fi (M.K.) * Correspondence: vtjvir@utu.fi; Tel.: +358-29-450-3205 Abstract: Ellagitannins have antimicrobial activity, which might be related to their interactions with membrane lipids. We studied the interactions of 12 different ellagitannins and pentagalloylglucose with a lipid extract of Escherichia coli by high-resolution magic angle spinning NMR spectroscopy. The nuclear Overhauser effect was utilized to measure the cross relaxation rates between ellagitannin and lipid protons. The shifting of lipid signals in 1H NMR spectra of ellagitannin–lipid mixture due to ring current effect was also observed. The ellagitannins that showed interaction with lipids had clear structural similarities. All ellagitannins that had interactions with lipids had glucopy- ranose cores. In addition to the central polyol, the most important structural feature affecting the interaction seemed to be the structural flexibility of the ellagitannin. Even dimeric and trimeric ellagitannins could penetrate to the lipid bilayers if their structures were flexible with free galloyl and hexahydroxydiphenoyl groups. Keywords: E. coli; HR-MAS-NMR; interaction; lipid membrane; tannins; UPLC-DAD-MS Citation: Virtanen, V.; Räikkönen, S.; Puljula, E.; Karonen, M. 1. Introduction Ellagitannin–Lipid Interaction by HR-MAS NMR Spectroscopy. Tannins are a group of specialized plant metabolites, which, when included in the di- Molecules 2021, 26, 373. etary feed of ruminants, have been shown to induce many beneficial effects such as increas- https://doi.org/10.3390/ ing their effective amino acid absorption, lowering their methane production, and acting as molecules26020373 anthelmintics [1–6]. Additionally, tannins have been recently shown to inhibit the growth of several bacteria more effectively than what they were previously thought capable [7]. Academic Editors: Many of these favorable effects are traditionally thought to be governed by the protein Teresa Escribano-Bailón, affinity/protein precipitation capacity of tannins or their oxidative activity, which have Ignacio García-Estévez and been extensively studied in the literature [8–10]. However, the possible interactions be- Encarna Gómez-Plaza Received: 15 December 2020 tween lipids and tannins have not been widely considered even though they might play an Accepted: 7 January 2021 important role in understanding the capability and the mechanisms with which tannins Published: 12 January 2021 inhibit, for instance, the growth of different bacteria and their possibilities as antimicrobial agents in general [11]. Publisher’s Note: MDPI stays neu- High-resolution magic angle spinning (HR-MAS) NMR spectroscopy has revolution- tral with regard to jurisdictional clai- ized and opened all new possibilities to study lipids, lipid membranes, and their potential ms in published maps and institutio- interactions with other compounds [12–15]. A notable benefit of the HR-MAS probe is nal affiliations. that it tolerates the kind of semisolid emulsion type samples that ellagitannins (ETs) and lipids form in a solution, while still enabling normal liquid-state NMR experiments with reasonable resolution. Useful experiments include methods such as nuclear Overhauser effect spectroscopy (NOESY) to detect correlations between specific parts of the lipids with Copyright: © 2021 by the authors. Li- other molecules [12]. We studied the interactions of 12 ellagitannins (Figure1) with a lipid censee MDPI, Basel, Switzerland. extract of Escherichia coli (E. coli) by HR-MAS NMR. The ETs were selected to represent This article is an open access article different branches of the ET biosynthetic pathway and based on their studied hydropho- distributed under the terms and con- bicity [16–18]. In addition, the lipid interactions of pentagalloylglucose, the biosynthetic ditions of the Creative Commons At- tribution (CC BY) license (https:// precursor of ETs, were studied. The main aims were to study whether there are interactions creativecommons.org/licenses/by/ between ETs and lipids and whether these interactions can be studied by HR-MAS NMR. 4.0/). Molecules 2021, 26, 373. https://doi.org/10.3390/molecules26020373 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 13 Molecules 2021, 26, 373 2 of 13 interactions between ETs and lipids and whether these interactions can be studied by HR- MAS NMR. R O 5 HO OH HO OH 4 O R O R1 R3O OR2 HO OH 2 O 1 R1=OH, R2=R3=G, R4~R5=(S)-HHDP 3 R1= OG, R2=R3=(S)-HHDP, R4~R5=(S)-HHDP O O O 4 R1= OG, R2=R3=G, R4~R5=(S)-HHDP GO OH 5 R O 1 GO O O O H OR OG O OR3 GO HO O OH O OG O O 5 OR4 OR2 HO OH 1 2 4 3 5 OH OH NHTP group 6 R =G, R ~R =DHHDP, R ~R =(R)-HHDP HO OH 1 2 4 3 5 7 R =G, R ~R =modified DHHDP, R ~R =(R)-HHDP OH 8 R1=R3=R5=G, R2~R4=modified DHHDP OH 9 OH HO HO OG gallagyl group HO 12 O HO O OG O HO OG O O OH O O HO O HO O OH HO O O HO O O O 1 HO GO R O O OH HO O OG GO OH HO O 10 O OH HO O O OH O OH OH O HO OH OH HO O O O O OH HO OG O HO O O OH HO O OH HO O HO OH OH OH HO O R3O HO O R2O 1 OR G= O HO O O O HO HO HO O O O GO OH O GO O HO OHHO OH HO OH GO HO O HO OH O GO O G G=HHDP= GO OH 11 R1=G, R2~R3=(S)-HHDP GO O O O OH 13 R1=G, R2~R3=(S)-HHDP chebuloyl= O O DHHDP = O O O O HOOC OH O OH O HO OH HO HO OH O O OH OH OH HO O OH O Figure 1. Chemical structures of 12 ellagitannins and pentagalloylglucose studied for their interaction with lipids: tellima- tellima- grandin I 1, vescalagin 2, casuarictin 3, tellimagrandin II 4, pentagalloylglucose 5, geraniin 6, chebulagic acid 7, chebulinic grandin I 1, vescalagin 2, casuarictin 3, tellimagrandin II 4, pentagalloylglucose 5, geraniin 6, chebulagic acid 7, chebulinic acid 8, punicalagin 9, oenothein B 10, sanguiin H-6 11, oenothein A 12, and lambertianin C 13. DHHDP = dehydrohexahy- acid 8, punicalagin 9, oenothein B 10, sanguiin H-6 11, oenothein A 12, and lambertianin C 13. DHHDP = dehydrohex- droxydiphenoyl, G = galloyl, HHDP = hexahydroxydiphenoyl, chebuloyl = modified dehydrohexahydroxydiphenoyl, ahydroxydiphenoyl,NHTP = nonahydroxytriphenoyl. G = galloyl, HHDP = hexahydroxydiphenoyl, chebuloyl = modified dehydrohexahydroxydiphenoyl, NHTP = nonahydroxytriphenoyl. Molecules 2021, 26, x FOR PEER REVIEW 3 of 13 Molecules 2021, 26, 373 3 of 13 2. Results and Discussion 2.1.2. Results Characterization and Discussion of the Lipids in E. coli Lipid Extract 2.1. CharacterizationThe protons in the of the E. Lipidscoli lipid in E. extracts coli Lipid were Extract assigned mainly based on the 2D-corre- lationThe spectra protons measured in the withE. coli thelipid 600 extractsMHz inst wererument assigned (Chapter mainly 3.3), basedas the oncorrelation the 2D- spectracorrelation measured spectra with measured the 400 with MHz the HR-MAS 600 MHz instrument instrument (Sectiondid not achieve 3.3), as thegood correlation enough resolutionspectra measured even after with parameter the 400 MHzoptimization. HR-MAS Attempts instrument were did also not made achieve to analyze good enough a sam- pleresolution of pure evenL-α-phosphatidylethanolamine after parameter optimization. (PE) Attemptslipid in D were2O to alsoverify made the toassignations analyze a madesample from of pure the L-400a-phosphatidylethanolamine MHz HR-MAS measurements, (PE) lipidbut, because in D2O to of verify its poor the solubility assignations in water,made fromthese themeasurements 400 MHz HR-MAS did not measurements,produce the desired but, because outcome. of The its poor solubility solubility of the in purewater, PE these lipid measurements was not markedly did not increased produce the even desired after outcome.the addition The solubilityof 0.1 M ofphosphate the pure bufferPE lipid or wasafter not mixing markedly the pure increased PE lipid even with after different the addition ratios of of the 0.1 E. M coli phosphate lipid extract. buffer or afterThe mixing assigned the pure lipid PE protons lipid with are different displayed ratios in Figure of the E.2 colifromlipid spectra extract. measured with both Thea 600 assigned MHz instrument lipid protons equipped are displayed with a inCryo-Probe Figure2 from (a) spectraand a 400 measured MHz instrument with both equippeda 600 MHz with instrument an HR-MAS equipped probe with (b), a and Cryo-Probe their chemical (a) and shifts a 400 are MHz as instrumentfollows: (a) equipped 1H NMR 1 (MeOD-with and HR-MAS4, 600 MHz) probe δ (0.90b), and(m, theirH-CH chemical3), 1.29 (m, shifts H-CH are as2), follows:1.60 (m, ( aH-C3),) H NMR 2.03 (MeOD-(m, H- CHCHd4, 6002), MHz) 2.33 (m,δ 0.90 H-C2), (m, H-CH3.16 (m,3), H- 1.29β), (m,4.00 H-CH(m, H-G3),2), 1.60 4.05 (m, (m, H-C3), H-α), 2.034.19 (m,(m, H-CHCHH-G1), 4.442), (m,2.33 H-G1), (m, H-C2), 4.57 3.16(s, H- (m,γ), H-5.23b), (m, 4.00 H-G2), (m, H-G3), 5.35 (m, 4.05 H-CH); (m, H- aand), 4.19 (b) (m,1H NMR H-G1), (D 4.442O, (m,400 H-G1),MHz) 1 δ δ4.57 0.95 (s, (m, H- gH-CH), 5.233), (m, 1.35 H-G2), (m, H-CH 5.35 (m,2), H-CH);1.63 (m, and H-C3), (b) 2.08H NMR (m, (DH-CHCH2O, 4002 MHz)), 2.44 (m,0.95 H-C2), (m, H- 3.34CH3 (m,), 1.35 H-β (m,), 4.29 H-CH (m,2), H-G1), 1.63 (m, 4.51 H-C3), (m, H-G1), 2.08 (m, 5.37 H-CHCH (m, H-G2/H-CH).2), 2.44 (m, H-C2),Lipid protons 3.34 (m, H-G3, H-b), H-4.29α, (m,and H-G1),H-γ could 4.51 not (m, be H-G1), accurately 5.37 (m,assigned H-G2/H-CH).