Structural and Stereochemical Studies of Laurokamurols A–C

Article pubs.acs.org/JAFC

Structural and Stereochemical Studies of Laurokamurols A−C, Uncommon Bis-sesquiterpenoids from the Chinese Red Alga Laurencia okamurai Yamada Xiao-Lu Li,†,‡ Tibor Kurtán,§ Jun-Chi Hu,†,‡ Attila Mándi,§ Jia Li,† Xu-Wen Li,*,† and Yue-Wei Guo*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No. 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China § Department of Organic Chemistry, University of Debrecen, Egyetemtér 1, P.O. Box 400, 4002 Debrecen, Hungary S Supporting Information *

ABSTRACT: Three novel heterodimeric laurane-type sesquiterpenoids, laurokamurols A−C (1−3), along with eight known related monomeric ones (4−11) were isolated from the East China Sea red alga Laurencia okamurai Yamada. The absolute configurations of the new bis-sesquitepenoids, especially their axial chirality, were determined by extensive spectroscopic analyses and TDDFT-ECD method. All of the new compounds showed promising PTP1B inhibitory activities with IC50 values comparable to the positive control, indicating them as potential food additives or pharmaceutical drug leads toward obesity or diabetes. KEYWORDS: sesquiterpenoid, laurane, red alga, axial chirality, PTP1B

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INTRODUCTION Red algae are widely distributed over the world’s oceans as rich sources of proteins, fibers, vitamins, physiologically important fatty acids, and macro and trace elements, which could usually be used as food additives or healthcare products.1 Red algae of the genus Laurencia, belonging to the family Rhodomelaceae of the order Ceramiales, abound in secondary metabolites featuring predominately terpenes and C15-acetogenins.2 These metabolites were often reported to display various biological activities, ranging from antibacterial3 to antitumoral4 to insecticidal activities, etc.5−7 Laurane and cyclolaurane-type sesquiterpenoids, for example, laurinterol (6), characterized by a benzyl group linked to a 1,2,3-trisubstituted cyclopentane moiety,2a are typical metabolites frequently encountered in Laurencia, especially in the species Laurencia okamurai. Due to the intriguing scaffolds and complex chemical diversities, as well as wide biological activities, searching for structurally interesting and biologically active compounds from Laurencia algae has attracted the intense attention of natural product chemists and pharmacologists in the past decades. Our group has long been engaged in the search for bioactive natural products from Chinese algae, and numerous secondary metabolites spanning a wide range of structural classes and various biogenetic origins have been isolated and characterized.2b,7,8 For example, in the course of the chemical investigation of the title alga, L. okamurai,8a−d a series of novel laurane-type sesquiterpenoids, exemplified by laurokamurenes A−C,8b comprising an unusually rearranged laurane skeleton, were isolated.7,8a−d Particularly, some of such sesquiterpenoids showed significant PTP1B (protein-tyrosine phosphatase 1B: a recognized target for diabetes and obesity) inhibitory activities with great food and pharmaceutical potentials.7 Stimulated by this discovery and to obtain sufficient © XXXX American Chemical Society

amounts of bioactive laurane-type sesquiterpenoids for morein-depth pharmacological study, the title alga was recently recollected and chemically reinvestigated. In the course of this study, we were able not only to obtain the expected sesquiterpenoids (4−11) but also to isolate three unprecedented heterodimeric bis-sesquiterpenoids, namely, laurokamurols A−C (1−3) (Figure 1). It is worth pointing out that although three laurane-type bis-sesquiterpenoids had been previously reported,9−11 compounds 1−3 are only examples of bis-sesquiterpenes derived formally from two different laurane monomers. Herein, we report the isolation, structural elucidation, and PTP1B inhibitory activities of the isolated compounds. A plausible biosynthetic pathway has also been presented.

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 241MC polarimeter. ECD spectra were obtained on a Jasco J-810 spectropolarimeter. NMR spectra were measured on a Bruker DRX-400 spectrometer (Bruker Biospin AG, Fällanden, Germany) with the residual CHCl3 (δH 7.26; δC 77.0) as an internal standard. EIMS and HREIMS spectra were recorded on a Finnigan-MAT-95 mass spectrometer (FinniganMAT, San Jose, CA, USA). Commercial silica gel (Qingdao Haiyang Chemical Group Co., Ltd., Qingdao, China, 200−300 and 400−600 mesh) was used for column chromatography, and precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yantai, China; G60 F-254) were used for analytical TLC. Sephadex LH-20 (GE-Healthcare, Piscataway, NJ, Received: Revised: Accepted: Published: A

November 23, 2016 January 19, 2017 February 7, 2017 February 7, 2017 DOI: 10.1021/acs.jafc.6b05238 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

896, 845 cm−1; for 1H and 13C NMR spectral data (CDCl3) see Table 1; HREIMS m/z 508.1972 [M]+ (calcd for C30H37O2Br, 508.1977). Synthesis of the Dimers 12 and 13. To a dry dichloromethane (3 mL) solution of the monomeric compounds (30 mg, respectively) was added MnO2 (30 mg) at room temperature. The reaction was vigorously stirred over 30 min; it was then filtered via Celite, washed by dichloromethane, concentrated in vacuo, and purified via silica gel CC (petroleum ether/CH2Cl2 solvent system). The reaction with 5 and 6 gave only compound 13 (12 mg) as the dimer of 6, with the yield of 20%; no expected compound 1 was detected, whereas the reactions with 6 and 7 both gave only 12 (7.1 mg) and 13 (7.8 mg) in a ratio of 0.9:1 (overall yield = 25%) and no reaction between 6 and 7 toward compound 3. PTP1B Inhibitory Activity Assay. The recombinant PTP1B catalytic domain was expressed and purified according to previous studies.12 The enzymatic activities of the PTP1B catalytic domain were determined at 30 °C by monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP generated the product pNP, which was monitored at an absorbance of 405 nm with an EnVision multilabel plate reader (Perkin-Elmer Life Sciences, Boston, MA, USA). In a typical 100 μL assay mixture containing 50 mmol/L 3-morpholinopropanesulfonic acid (MOPs), pH 6.5, 2 mmol/L pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously monitored and the initial rate of hydrolysis was determined by using the early linear region of the enzymatic reaction kinetic curve. The IC50 was calculated with Prism 4 software (Graphpad, San Diego, CA, USA) from the nonlinear curve fitting of the percentage of inhibition (% inhibition) versus the inhibitor concentration [I] by using the following equation: % inhibition =1/(1 + [IC50/[I]]k), where k is the Hill coefficient; IC50 ≥ 50 μM was considered inactive. General Method for ECD Calculation. Mixed torsional/lowfrequency mode conformational searches were carried out by means of the Macromodel 9.9.223 software using the Merck Molecular Force Field (MMFF) with an implicit solvent model for CHCl3.13 Geometry reoptimizations were carried out at the B3LYP/6-31G(d) level in vacuo and at the B3LYP/TZVP and B97D/TZVP14 levels with the PCM solvent model for MeCN and CHCl3. TDDFT ECD and OR calculations were run with various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set with the same or no solvent model as the DFT optimizations as implemented in the Gaussian 09 package.15 ECD spectra were generated as sums of Gaussians with 3000−2100 cm−1 half-height widths (corresponding to ca. 12−8 at 200 nm), using dipole-velocity-computed rotational strength values.16 Boltzmann distributions were estimated from the ZPVE corrected B3LYP energies in the gas-phase calculations and from the B3LYP and B97D energies in the PCM model calculations. Torsional energy scans were performed at the B3LYP/6-31G(d) level in vacuo. The MOLEKEL software package was used for visualization of the results.

Figure 1. Structures of compounds 1−14. (#) Compounds were synthesized; (##) model compound for torsional energy scans and TS calculations. USA) was also used for column chromatography. All solvents for CC were of analytical grade. Plant Material. The alga L. okamurai was collected by hand along the coast of Nanji Island in the East China Sea, Zhejiang province, China, at a depth of 0.5−1 m, in December 2013, and the material was stored at −20 °C until processed. A voucher specimen (no. 13NJ-1) was deposited at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for inspection. Extraction and Isolation. The fresh alga material of L. okamurai (dry weight, 300 g) was exhaustively extracted with acetone at room temperature (1.0 L × 4). The acetone extract was then concentrated in vacuo, and the resulting residue was partitioned between Et2O and H2O and between n-BuOH and H2O, respectively. The Et2O-soluble extract (19.0 g) was then chromatographed on a silica gel column (200−300 mesh) using light petroleum ether with increasing amounts of CH2Cl2 as eluent to obtain nine major fractions (A−I). Fraction G (1.1 g) was then separated into seven subfractions (G1−G7) via a Sephadex LH-20 column, eluted with petroleum ether/CH2Cl2/ MeOH (2:1:1). Fraction G2 (37.9 mg) was then separated by silica gel CC (400−600 mesh) eluting with petroleum ether to yield compound 1 (2.0 mg), compound 2 (1.5 mg) and compound 11 (3.5 mg). Fraction G4 (20.7 mg) was further purified by silica gel CC (400−600 mesh) eluting with petroleum ether/CH2Cl2 (10:0 to 8:2) to obtain compound 3 (1.5 mg). Seven known compounds, namely, aplysin (4, 130.0 mg), debromo-aplysin (5, 40.0 mg), laurinterol (6, 150.0 mg), debromo-laurinterol (7, 200.0 mg), aplysinol (8, 2.5 mg), laureperoxide (9, 2.5 mg), and (2S,5R)-5,8,10,10-tetramethyl-2,3,4,5-tetrahydro-2,5-methanobenzo[b]oxepine (10, 2.0 mg), were afforded from fractions D−H by successively using silica gel CC, Sephadex LH-20 CC, and semipreparative HPLC. Dilaurokamurol A (1): white powder; [α]25D −14.3 (c 0.07, CHCl3); ECD {MeCN, λ [nm] (Δε)} 292 (0.77), 249 (0.75), 210 (−6.00), 198 (7.00); IR (KBr) νmax 3440, 2979, 2954, 2930, 2864, 1629, 1578, 1487, 1472, 1391, 1375, 1267, 1234, 1126, 1007, 881, 861 cm−1; for 1H and 13C NMR spectral data (CDCl3) see Table 1; HREIMS m/z 508.1977 [M]+ (calcd for C30H37O2Br, 508.1977). Dilaurokamurol B (2): white powder; [α]25D −20.5 (c 0.1, CHCl3); ECD {MeCN, λ [nm] (Δε)} 294sh (0.64), 285 (0.70), 246sh (1.22), 231 (1.43), 211 (−2.57), 200 (3.76); IR (KBr) νmax 3508, 3427, 2923, 2855, 1616, 1483, 1457,1396, 1224, 1120, 1011, 856, 802 cm−1; for 1H and 13C NMR spectral data (CDCl3) see Table 1; HREIMS m/z 430.2870 [M]+ (calcd for C30H38O2, 430.2872). Dilaurokamurol C (3): yellowish oil; [α]25D −10.3 (c 0.12, CHCl3); ECD {MeCN, λ [nm] (Δε)} 315 (−0.25), 249 (1.91), 233 (5.13), 216 (−4.39), 206 (5.88), 196 (−3.52); IR (KBr) νmax 3404, 3059, 2952, 2934, 2865, 1636, 1496, 1449, 1388, 1376, 1260, 1161, 1066,

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RESULTS AND DISCUSSION

The fresh alga materials were exhaustively extracted with acetone. The Et2O-soluble portion of the acetone extract was subjected to repeated column chromatography on silica gel, Sephadex LH-20, and RP-HPLC to yield pure compounds 1− 11, respectively (Figure 1 and the Supporting Information). The structures of the known compounds were readily identified as aplysin (4),17 debromo-aplysin (5),18 laurinterol (6),17 debromo-laurinterol (7),17 aplysinol (8),17 laureperoxide (9),8b aplysinal (10),19 and (2S,5R)-5,8,10,10-tetramethyl-2,3,4,5tetrahydro-2,5-methanobenzo[b]oxepine (11),18 respectively, by comparing their physical properties and spectroscopic data with those reported in the literature. Compounds 1−3 showed similar IR absorptions at νmax around 1600 and 1500 cm−1, indicative of the presence of aromatic rings. Their NMR spectra were reminiscent of those of co-occurring laurane sesquiterpenoids 5−7, respectively. In fact, compounds 1 and 3 exhibited the same partial structure B B

DOI: 10.1021/acs.jafc.6b05238 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. 1H (J in Hertz) and 13C NMR Data for Compounds 1−3 in CDCl3a 1 δH

position 1 2 3 4α 4β 5α 5β 6 7 8 9 10 11 12 13 14 15α 15β 1′ 2′ 3′ 4′α 4′β 5′α 5′β 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 7-OH 7′-OH

1.06−1.10 1.90−1.94 1.61−1.65 2.16−2.20 1.26−1.30

(m) (m) (m) (m) (m)

7.72 (s) 2.00 (s) 1.39 (s) 1.34 (s) 0.56−0.58 (t, 4.0) 0.53−0.56 (dd, 6.4, 4.0)

1.75−1.82 1.63−1.67 1.17−1.21 1.57−1.61 1.84−1.88

6.68 (s)

6.75 1.95 1.31 1.33 1.14 4.80

(s) (s) (s) (s) (s) (s)

(m) (m) (m) (m) (m)

2 δC 48.5 29.7 24.1 25.5 35.4 134.1 151 2 129.7 134.2 114.9 131.0 20.5 22.5 18.9 16.3 54.4 99.7 46.2 31.4 42.8 135.8 159.0 110.8 137.9 125.6 125.2 19.8 23.6 20.2 13.3

3

δH

1.06−1.10 1.90−1.94 1.61−1.65 2.20−2.24 1.30−1.34

δC 48.5 29.8 24.1 25.5

(m) (m) (m) (m) (m)

35.5

6.77 (d, 7.8) 7.46 (d, 7.8) 1.93 (s) 1.41 (s) 1.34 (s) 0.58 (t, 3.2) 0.49−0.51 (dd, 5.2, 3.2)

54.4 99.5 46.3 31.5

1.76−1.80 (m) 1.61−1.65 (m) 1.16−1.20 (m) 1.57−1.61(m) 1.84−1.88 (m)

42.7 135.5 159.2 110.7 137.9 125.7 125.5 19.8 23.7 20.2 13.3

6.68 (s)

6.82 1.97 1.31 1.33 1.14 4.80

131.9 151.9 128.3 134.9 120.5 127.2 19.9 22.8 19.2 16.4

(s) (s) (s) (s) (s) (s)

δH

1.06−1.10 1.61−1.65 1.88−1.92 1.28−1.32 2.17−2.22

(d, 4.0) (dd, 8.0, 4.0) (m) (m) (dd, 13.0, 8.0)

7.74 (s) 2.01(s) 1.39 (s) 1.34 (s) 0.59 (t, 4.0) 0.53−0.56 (dd, 8.0, 4.8)

1.10−1.14 1.66−1.68 1.95−1.98 1.36−1.38 2.10−2.15

(d, 4.0 Hz) (dd, 8.0, 4.0) (m) (m) (dd, 13.0, 8.0)

6.70 (s)

7.20 1.94 1.41 1.23 0.51 4.84 5.30

(s) (s) (s) (s) (t, 4.0); 0.46−0.49 (dd, 8.0, 4.8) (s) (s)

δC 48.5 29.7 24.1 25.4 35.3 134.2 151.4 129.6 134.1 114.9 131.0 20.6 22.6 19.0 16.3 48.2 29.7 24.6 25.5 36.5 133.3 154.5 119.0 136.7 125.9 131.6 19.0 23.9 18.9 16.3

a

Bruker-DRX-400 spectrometer (400 MHz for 1H and 100 MHz for 13C NMR) in CDCl3, chemical shifts (ppm) referred to CHCl3 (δH 7.26) and to CHCl3 (δC 77.0); assignments were deduced from analysis of 1D and 2D NMR spectra

as 6, whereas 1 and 2 shared the common partial structure A with 5. Laurokamurol A (1) was obtained as an optically active, white amorphous powder. Its molecular formula, C30H37O2Br, was determined by HREIMS (m/z 508.1977 [M]+, calcd 508.1977), suggesting 12 degrees of unsaturation. The obvious [M]+ peaks at m/z 508 and 510 with intensities of 0.99:1 in the HREIMS spectrum confirmed the presence of one bromine atom. The 1H and 13C NMR spectra (Table 1) of 1 showed two sets of resonances very similar to those of the co-occurring sesquiterpenes 5 and 6, consistent with the presence of the partial structures A and B. Furthermore, the 1H NMR spectrum (Table 1) of 1, in comparison with those of 5 and 6, exhibited only three aromatic protons, assignable to three olefinic protons on the benzene rings (H-8′, H-11′, and H-11, respectively), whereas the characteristic 1H NMR signals, δH 6.67 (H-10 in 5) and δH 6.61 (H-8 in 6), were absent, indicating the 8−10′ linkage position between the partial structures A and B. The HMBC cross peak from H-11′ (δH

6.75, s) to C-8 (δC 129.7) (Figure 2A) supported the 8−10′ connection of the two aromatic rings in compound 1. The relative configuration of 1 was biogenetically suggested to be the same as those of compounds 5 and 6, which has been further confirmed by the NOESY spectrum (Figure 2B). Particularly, the NOE correlations between CH3-13 (δH 1.39) and CH3-14(δH 1.34) and between CH3-14 and H-3 (δH 1.08) could support the relative configurations of the laurinterol moiety of 1, whereas the cross peaks between CH3-13′ (δH 1.31) and H-5′α (δH 1.59), H-5′α and H-4′α (δH 1.64), H-4′α and H-3′ (δH 1.79), H-3′, and CH3-14′ (δH 1.33) confirmed the debromo-aplysin moiety in compound 1 (Figure 2B). All 2D NMR experiments supported the planar structure and relative configuration of 1. Compound 2, named laurokamurol B, yielded an HREIMS peak at m/z 430.2870 of 78 mass units fewer than that of 1. The 1H and 13C NMR spectra of 2 revealed a close relationship with both 1 and 7. In particular, the 1H NMR spectrum (Table 1) of 2, in comparison with that of 1 and 7, clearly indicated the C

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compounds containing phenol groups, was conducted. However, once again, only both homodimers 12 (dimeric 7), which was previously discovered from the red alga L. nidif icain the Japanese Sea,9 and 13 were obtained. These results indicated that the heterodimers cannot be achieved by the simple oxidative couplings as the homodimers 12 and 13. Therefore, a plausible biosynthetic pathway of all the bislauranes was proposed as shown in Scheme 1. The homodimers Scheme 1. Plausible Biosynthetic Pathway of Dimeric Laurane-Type Sesquiterpenoids

Figure 2. (A) Key 1H−1H COSY and HMBC correlations for compounds 1 and 3. (B) Key NOESY correlations for 1 and 3.

bromine atom at C10 was replaced by an aromatic proton δH 6.67 (H-10, d, J = 7.8 Hz). All 1H and 13C NMR assignments according to structure 2 were confirmed by 1H−1H COSY, HMQC, and HMBC experiments. Compound 2 is the 10debromo-derivative of 1. The molecular formula of compound 3, named laurokamurol C, C30H37O2Br, was identical to that of 1, as deduced by HREIMS (m/z 508.1972 [M]+, calcd 508.1977). The 1H and 13 C NMR data (Table 1) of 3 showed two sets of signals assignable to those of the co-occurring 6 and 7, suggesting that 3, like 1 and 2, is also the heterodimer of the two abovementioned monomers. In particular, the HMBC cross peak from H-11′ (δH 7.20) to C-8 (δC 129.6) (Figure 2A) supported the 8−10′ linkage of compound 3. The NOESY correlations between CH3-13 (δH 1.39) and CH3-14, CH3-14 (δH 1.34), and H-3 (δH 1.08) could support the relative configurations of the laurinterol moiety of 3, whereas the cross peaks between CH313′(δH 1.41) and CH3-14′ (δH 1.23), CH3-14, and H-3′ (δH 1.12) confirmed those of the debromo-laurinterol moiety in compound 3 (Figure 2B). Thus, the planar structure and relative configurations of 3 were determined. To complete the structural characterization of 1−3, the remaining task is to determine their absolute configuration (AC), including both central chirality elements and axial chirality (or preferred biaryl helicity). As already indicated above, the precursors of bis-sesquiterpenoids 1−3 are 5−7, respectively, implying that the AC of chiral centers of all dimers should be logically the same as those of their corresponding monomers. Literature searching revealed that the ACs of monomers 5−7 were already determined by either X-ray diffraction analysis or chemical corelations.20 On the basis of the above evidence and the biogenetic consideration, the ACs of all the chiral centers of dimers 1−3 were suggested to be the same as those of the corresponding monomers 5−7. To confirm the correctness of the AC assignments, an effort was made to synthesize the dimers 1 and 3. For the dimer 1, the oxidative coupling of the two monomers 5 and 6 was carried out in the presence of MnO2.9 Unfortunately, instead of the expected product 1, we obtained only the self-coupling product 13 (dimeric 6), which was previously isolated from the red alga L. microcladia of the North Aegean Sea.10 The failure of this reaction should be attributed to the lack of free phenol group in 5, which prevented the oxidative coupling. Thus, the same procedure toward the dimer 3 by reaction of 6 and 7, two

12 and 13 could be obtained directly by homodimerization of the monomer 7 or 6 on C10−C10′ or C8−C8′, respectively, whereas for all of the new heterodimers, a first C8−C10′ homodimerization might be conducted, which either was followed by a C10 monobromination to afford 3 or underwent a nucleophilic reaction to open the cyclopropane toward 2. A C10 monobromination of 2 could finally proceed to give 1. However, this is not the only possible biosynthetic pathway. As reported by Kozlowski,21 oxidative cross-coupling of phenols could also be achieved by using different metal catalysts. Such a proposal could be adapted by compound 3, but might not be applicable for compounds 1 and 2 because their A moieties were not free phenols. Further biosynthesis or biomimetic synthesis of such compounds should be conducted to uncover their true biosynthetic pathways. Nevertheless, the last step to complete the structural elucidation of the new dimers 1−3 is to determine their axial chirality. It is worth pointing out that in the previous studies of the homodimeric bis-lauranes,9−11 the axial chirality between the two aromatic rings has not been discussed. In our case, intensive TDDFT-ECD calculations were applied to solve this puzzle. First, to explore if the ortho-trisubstituted biaryl moieties of 1−3 have hindered rotation along the biaryl axis and hence axial chirality, torsional energy scans and computation of the rotational transition state (TS) for the inversion of axial chirality were performed, whereas simultaneous rotation of substituents and overscanning made computations problematic. Therefore, the simplified truncated model 14 (Figure 1) comprising the same ortho substituents of the biaryl linkage was chosen to estimate rotational energy barrier for 1−3 (see the Supporting Information for detailed calculation of 14), and finally the rotational energy barrier of a similar trisubstituted biaryl 14 is expected in the range of 140 ± D

DOI: 10.1021/acs.jafc.6b05238 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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which was further confirmed by ECD calculations (see the Supportin Information for detailed calculations). The initial MMFF co nformational analysis of (1R,2S,3R,1′R,2′S,3′R)-3 resulted in 19 structures containing atropodiastereomers with both (aR) and (aS) axial chiralities, the DFT reoptimization of which afforded six (three aR and three aS) and seven (three aR and four aS) low-energy conformers above 2% at B3LYP/6-31G(d) in vacuo and B97D/ TZVP PCM/MeCN levels, respectively. In both cases, the lowest energy (aR) atropodiastereomer had slightly lower energy than the lowest energy (aS) one (ΔE = 0.5 kJ/mol). The ECD spectra computed for the B97D/TZVP PCM/ MeCN (aR,1R,2S,3R,1′R,2′S,3′R)-atropodiasteromer did not match well the experimental ECD curve, whereas that of the B3LYP/6-31G(d) in vacuo (aR,1R,2S,3R,1′R,2′S,3′R)-atropodiasteromer reproduced the measured ECD (Figure 4),

10 kJ/mol. This value is substantially higher than the one (∼93 kJ/mol) required for hindered rotation or axial chirality of biaryl systems.22 This result confirmed that the biaryl system has axial chirality in all three dimers. In light of this observation, detailed TDDFT-ECD calculations on 1−3 were performed. Because the AC of the central chirality of 1−3 has already been assigned and ECD spectra are mainly governed by the axial chirality, ECD calculations were run on the (aR,1R,2S,3R,1′S,2′S,3′S) and (aS,1R,2S,3R,1′S,2′S,3′S) atropodiastereomers of 2 to determine the axial chirality. The initial conformational search of (1R,2S,3R,1′S,2′S,3′S)-2 at MMFF level afforded 12 conformers including both (aR) and (aS) atropodiastereomers, because the MMFF analysis interconvert axial chirality. The reoptimization of these geometries at B3LYP/6-31G(d) in vacuo and B97D/TZVP (PCM/MeCN) levels resulted in two low-energy structures by both methods, one with (aR) and the other with (aS) axial chirality (Figure 3A). ECD calculations of these two atropodiastereomers were

Figure 4. Experimental ECD spectra of 3 in MeCN compared with the Boltzmann-averaged BH&HLYP/TZVP ECD spectrum of the lowenergy (aR,1R,2S,3R,1′R,2′S,3′R) and (aS,1R,2S,3R,1′R,2′S,3′R) diastereomers of 3 computed for the B3LYP/6-31G (d) in vacuo conformers. Bars represent the rotational strengths of the lowestenergy aR and aS conformers.

suggesting (aR) axial chirality of 3. This is also in line with the similarity of the experimental spectra of 3 to those of 1 and 2, and thus the absolute configuration of 3 was determined as (aR,1R,2S,3R,1′R,2′S,3′R). All of the isolated and synthesized compounds were evaluated for their PTP1B inhibitory activities. Among them, all of the dimers 1−3, 12, and 13 exhibited significant inhibitory effect with IC50 values of 8.1, 12.5, 6.1, 10.0, and 5.6 μM, respectively, comparable with that of the positive control oleanolic acid (IC50 = 3.3 μM), whereas all of the monomers were not active, except for 6 having weak inhibitory activity with IC50 values of 45.2 μM. In conclusion, three unusual heterodimeric laurane-type sesquiterpenoids 1−3 were isolated and fully characterized from the title alga, L. okamurai. The discovery of laurokamurols A−C (1−3) has added to an extremely diverse and complex array of laurane-type sesquiterpenoids, which is still expanding. Furthermore, the axial chirality of all the new bissesquiterpenoids was determined for the first time by using the TDDFT-ECD method. The potent PTP1B inhibitory activities of the dimeric bis-lauranes could provide a clue for the further function-oriented synthesis of such compounds toward new antiobesic or antidiabetic drug discovery. Such bioactive compounds also enable us to envisage the possible utilization of

Figure 3. (A) Structures of the lowest energy B97D/TZVP (PCM/ MeCN) (aR,1R,2S,3R,1′S,2′S,3′S) and (aS,1R,2S,3R,1′S,2′S, 3′S) atropodiastereomers of 2. (B) Experimental ECD spectrum of 2 in MeCN compared with the BH&HLYP/TZVP ECD spectra computed for the B97D/TZVP (PCM/MeCN) lowest-energy (aR) and (aS) atropodiastereomers of 2. Bars represent the rotational strengths of the atropodiastereomers.

performed with various functionals and TZVP basis set, and the resultant ECD spectra were compared with the experimental spectrum. Despite the rather similar geometry of the gas phase and solvent model atropodiastereomers, their calculated ECD spectra were markedly different (Figure 3B). Nevertheless, the ECD calculations of both gas-phase and solvent model atropodiastereomers suggested consistently (aR) axial chirality for the biaryl axis, even though the (aS)-2 atropodiastereomer has a slightly lower energy than (aR)-2 (ΔEB3LYP/6‑31G(d) in vacuo = 0.4 kJ/mol, ΔEB97D/TZVP PCM/MeCN = 0.5 kJ/mol). Thus, our TDDFT-ECD calculation approach on the two monomers and the biaryl derivative 2 allowed elucidation of the AC of 2 as (aR,1R,2S,3R,1′S,2′S,3′S). As the bromo-derivative of 2, the AC of 1 was immediately assigned to be the same (aR,1R,2S,3R,1′S,2′S,3′S) as that of 2, E

DOI: 10.1021/acs.jafc.6b05238 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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red algae of Laurencia as food additives and their functions for human healthcare, especially for antiobesity products.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05238. Detailed ECD calculations of compounds 1 and 14 and their corresponding figures, as well as the spectra of all of the new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(X.-W.L.) Phone: +86-21-50806600-3317. E-mail: xwli@ simm.ac.cn. *(Y.-W.G.) Phone/fax: +86-21-50805813. E-mail: ywguo@ simm.ac.cn. ORCID

Yue-Wei Guo: 0000-0003-0413-2070 Funding

This research work was financially supported by the Natural Science Foundation of China (No. 81520108028, 81273430, 41306130, 81302692, 41676073, 81603022), SCTSM Project (No. 14431901100, 15431901000), Institutes for Drug Discovery and Development, Chinese Academy of Sciences (No. CASIMM0120152039), and the SKLDR/SIMM Projects (SIMM 1501ZZ-03). X.-W.L. acknowledges the financial support of “Youth Innovation Promotion Association” (No. 2016258) from the Chinese Academy of Sciences, “Young Talent Supporting Project” from the China Association for Science and Technology (No. 2016QNRC001), and Shanghai “Pujiang Program” (No. 16PJ1410600). The research of the Hungarian authors was supported by the EU and cofinanced by the European Regional Development Fund under Project GINOP-2.3.2-15-2016-00008. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge The National Information Infrastructure Development Institute (NIIFI 10038) for providing CPU time. We thank Prof. Wen-Fei He from Wenzhou Medical University for the supply of the alga material.

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REFERENCES

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DOI: 10.1021/acs.jafc.6b05238 J. Agric. Food Chem. XXXX, XXX, XXX−XXX