marine drugs Article Density Functional Theory (DFT)-Aided Structure Elucidation of Linear Diterpenes from the Irish Brown Seaweed Bifurcaria bifurcata Vangelis Smyrniotopoulos 1 , Daria Firsova 1, Howard Fearnhead 2, Laura Grauso 3 , Alfonso Mangoni 4 and Deniz Tasdemir 1,5,6,* 1 School of Chemistry, National University of Ireland Galway, University Road, H91 TK33 Galway, Ireland; [email protected] (V.S.); dashafi[email protected] (D.F.) 2 Pharmacology and Therapeutics, School of Medicine, National University of Ireland Galway, University Road, H91 W2TY Galway, Ireland; [email protected] 3 Dipartimento di Agraria, Università degli Studi di Napoli Federico II, 80055 Portici (NA), Italy; [email protected] 4 Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy; [email protected] 5 GEOMAR Centre for Marine Biotechnology (GEOMAR-Biotech), Research Unit Marine Natural Product Chemistry, GEOMAR Helmholtz Centre for Ocean Research Kiel, Am Kiel-Kanal 44, 24106 Kiel, Germany 6 Faculty of Mathematics and Natural Sciences, Kiel University, Christian-Albrechts-Platz 4, 24118 Kiel, Germany * Correspondence: [email protected]; Tel.: +49-431-600-4430 Abstract: Brown alga Bifurcaria bifurcata is an extraordinarily rich source of linear (acylic) diterpenes with enormous structural diversity. As part of our interest into secondary metabolites of the Irish seaweeds, here we report four new acyclic diterpenes (1–4) and seven known terpenoids (5–11) from Citation: Smyrniotopoulos, V.; the CHCl3 extract of B. bifurcata. The planar structures of the new metabolites were elucidated by Firsova, D.; Fearnhead, H.; Grauso, L.; means of 1D and 2D NMR, HRMS, and FT-IR spectroscopy. Since linear diterpenes are highly flexible Mangoni, A.; Tasdemir, D. Density compounds, the assignment of their stereochemistry by conventional methods, e.g., NOESY NMR, Functional Theory (DFT)-Aided is difficult. Therefore, we employed extensive quantum-mechanical prediction of NMR chemical Structure Elucidation of Linear shifts and optical rotation analyses to identify the relative and absolute configurations of the new Diterpenes from the Irish Brown compounds 1–4. Several compounds moderately inhibited the human breast cancer cell line (MDA- Seaweed Bifurcaria bifurcata. Mar. MB-231) with IC values ranging from 10.0 to 33.5 µg/mL. This study not only demonstrates the Drugs 2021, 19, 42. https:// 50 B. bifurcata doi.org/10.3390/md19010042 vast capacity of the Irish to produce highly oxygenated linear diterpenoids, but also highlights the potential of new methodologies for assignment of their stereogenic centers. Received: 18 December 2020 Accepted: 15 January 2021 Keywords: Bifurcaria bifurcata; brown alga; seaweed; linear diterpene; DFT; anticancer activity Published: 19 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Bifurcaria is a small genus within the brown algal family Sargassaceae (order Fucales). iations. Bifurcaria bifurcata (Velley) R. Ross, also known as brown forking weed or brown tuning fork weed [1,2] is widespread on the seashores and tidepools of the Atlantic Ocean [1,3]. In Ire- land, it is commonly found on the limestone areas of the Northwestern Ireland [1]. From a chemical point of view, B. bifurcata is reputed for being a rich source of linear diterpenes Copyright: © 2021 by the authors. (LDs) that originate from geranylgeraniol, a C20 metabolite bearing four double bonds and Licensee MDPI, Basel, Switzerland. five methyl functions [2,3]. Further substitutions and rearrangements, such as oxygenation, This article is an open access article isomerization, unsaturation or formation of terminal ring systems (e.g., furan), result in a distributed under the terms and highly diverse LD family. In general, LDs deriving from B. bifurcata have been divided into conditions of the Creative Commons 3 subfamilies: (a) those with C-12 oxygenation, (b) those with C-13 oxygenation, and (c) the Attribution (CC BY) license (https:// non-C-12/C-13 oxygenated diterpenes [3]. The diterpene composition of B. bifurcata has creativecommons.org/licenses/by/ been reported to vary with the collection site and season, and several types of chemotypes 4.0/). Mar. Drugs 2021, 19, 42. https://doi.org/10.3390/md19010042 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, 42 2 of 12 have been reported [4–6]. These diterpenes are of ecological importance and considered as chemotaxonomical markers [5,6]. They also exhibit pharmacologically-relevant activities, such as antiprotozoal, anticancer, and neuroprotective [3,7–11]. Identification of the stereogenic centers within linear, highly flexible natural products is challenging. Our previous studies on Irish B. bifurcata reported several acyclic diterpenes belonging to the C-13 oxygenated LD series, e.g., eleganediol (12)[10]. In that work, we em- ployed, for the first time for a LD, vibrational circular dichroism (VCD) spectroscopy joined with density functional theory (DFT) calculations and DP4 probability analyses to assign S absolute configuration for single (C-13) or two (C-7, C-13) stereogenic centers [10,11]. In the continuation of our chemical studies on this seaweed [10–12], we have now isolated four new LDs (1–4) along with four known linear diterpenes (5–8), a monoterpene loliolide (9), the tetraterpene carotenoid fucoxanthin (11) and a truncated fucoxanthin analogue (10). The planar structures of the isolated metabolites were elucidated by NMR, HRMS, and FT- IR spectroscopy. Due to low supply of the new compounds, VCD spectroscopy, which requires higher amounts of pure compounds [10–12], could not be used for stereochemical assignments. Instead, we employed DFT calculation of NMR chemical shifts, followed by DP4+ analysis, and DFT prediction of optical rotations. This study describes purification, structure determination and cytotoxic activity (in vitro) of the isolated compounds, as well as DFT studies for identification of the configuration of the new compounds 1–4. 2. Results and Discussion The freeze-dried algal material was successively extracted with CH2Cl2 and MeOH. The combined organic extracts were submitted to a modified Kupchan partition [10,11]. The CHCl3-soluble portion of the crude extract was fractionated by NP-Flash column chromatography (CC) to yield twenty-one fractions (C1−C21). The RP-HPLC separation of fractions C4, C9, C10, and C14 afforded the compounds 1–11 (Figure1). The new compound 1 was obtained as a colorless film. The molecular formula of + 1 was determined by its HR-ESIMS data (m/z 345.2401 [M + Na] ) as C20H34O3, consis- tent with four double bond equivalents (DBEs). Analysis of the 1H, 13C and gHSQC NMR spectra indicated the presence of one tertiary methyl δH/δC 1.30/18.3 (CH3–18) and four olefinic methyl groups δH/δC 1.70/25.7 (CH3-16); 1.66/18.2 (CH3-17); 1.61/16.0 (H3-19) and 1.65/16.2 (H3-20), an oxymethylene δH/δC 4.13/59.4 (CH2-1), three olefinic methines δH/δC 5.38/123.6 (CH-2); δH/δC 5.16/124.7(CH-6) and 5.15/127.4 (CH-14), two oxymethines δH/δC 2.98/62.1 (CH-10); and 4.44/65.5 (CH-13) and five methylenes δH/δC 2.03/39.3 (CH2-4); 2.12/26.1 (CH2-5); 2.13/36.2 (CH2-8); 1.64/26.9 (CH2-9) and 13 1.81,1.74/44.1 (CH2-12) (Tables1 and2). The C NMR spectrum (Table2) contained four 2 additional fully substituted carbons, including three sp carbons δC 134.3 (C-7); 134.7 (C-15); 139.3 (C-3) and one fully substituted oxygenated carbon δC 60.4 (C-11), which was suggestive of an acyclic diterpene skeleton. A close comparison of the NMR data of 1 with those of eleganediol (12, Figure1), a LD, which we previously isolated from the same seaweed [10] suggested that 1 was a 13-hydroxygeranylgeraniol analogue with an 1 13 additional oxygenation. The H NMR signal at δH 2.98 (t, J = 6.2 Hz) and two C signals at δC 62.1 (CH) and δC 60.4 (C), supported the oxygenation to be due to an epoxy functionality in 1. Three double bonds and an epoxy ring make up for the required four DBEs within 1. Mar. Drugs 2021, 19, 42 3 of 12 Mar. Drugs 2021, 19, x FOR PEER REVIEW 4 of 12 16 O 3 O 13 9 7 5 1 OH OH 11 OH 17 18 19 20 OH 1 2 OH OH OH OH OH OH 3 4 OH OH O O 5 6 OH OH HO OH 7 8 HO O O O O O HO 9 10 C O O O HO 11 HO O OH OH 12 Figure 1. CompoundsCompounds isolated from Bifurcaria bifurcata. The planar structure of 1 was assigned by means of 1D and 2D NMR experiments. The gCOSY spectrum included four spin systems, comprising of (i) H2-1 and H-2, (ii) H2-4, H2- 5 and H-6, (iii) H2-8, H2-9 and the oxirane proton H-10 and (iv) H2-12, the oxymethine H- 13 and the olefinic proton H-14. This data, especially the spin system iii provided clear indication that the epoxy ring resided between C-10 and C-11. The proton sequences (i– iv) were easily connected to each other with the aid of HMBC correlations. Specifically, diagnostic 1H-13C long range couplings between H-2/C-1, H2-1/C-2, and H2-1/C-3 corrob- orated the position of terminal secondary alcohol at C-1, while HMBC correlations be- tween C-11/H-13, C-13/H2-12, C-14/H2-12, and C-15/H2-12 provided further support for Mar. Drugs 2021, 19, 42 4 of 12 1 Table 1. H NMR data (600 MHz, CDCl3) of 1–4.