Xylomolones AâD from the Thai Mangrove Xylocarpus moluccensis

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Xylomolones A–D from the Thai Mangrove Xylocarpus moluccensis: Assignment of Absolute Stereostructures and Unveiling a Convergent Strategy for Limonoid Biosynthesis Wan-Shan Li, Attila Mandi, Junjun Liu, Li Shen, Tibor Kurtán, and Jun Wu J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b03037 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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The Journal of Organic Chemistry

Xylomolones A–D from the Thai Mangrove Xylocarpus moluccensis: Assignment of Absolute Stereostructures and Unveiling a Convergent Strategy for Limonoid Biosynthesis Wan-Shan Li,†,‡ Attila Mándi,§ Jun-Jun Liu,¶ Li Shen,*† Tibor Kurtán,*§ and Jun Wu*‡ †Marine

Drugs Research Center, College of Pharmacy, Jinan University, 601 Huangpu Avenue

West, Guangzhou 510632, P. R. China ‡School

of Pharmaceutical Sciences, Southern Medical University, 1838 Guangzhou Avenue North,

Guangzhou 510515, P. R. China §Department ¶School

of Organic Chemistry, University of Debrecen, PO Box 400, 4002 Debrecen, Hungary

of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology,

Wuhan 430030, P. R. China

ABSTRACT GRAPHIC 11

O

Me

10 1 2 5 3 4

7 6

O

19

Me

O

6

Me Me

28

MeO

actinidiolide

2 3

4

6

O

MeO

Int I

1

Michael addition Cyclization Reduction

2 3

4

O

28

O

29

Me

A

13 9

OH

19

Me

10

H 6 7

MeO

5

1

Me

O 4

2 3 6

29

O 4

10 5

H

Me Me 28

O

19

7

MeO

1 4

Me

O 2 3

Me Me 28

O

7

29

Int II

MeO

5

O

1

10

O H 6

O

OH

19

2 3

4

OH

Me Me 28

O

1, 2

O

Me Me

7

29

10 5

H

AcO

Me Me

7

9

8

10 5

H

O

19

Me

1

H O 16

8

30

17

O B

Aldol reaction Reduction

29

A'

1 / 31

ACS Paragon Plus Environment

3

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ABSTRACT: Two new 9,10-seco limonoids with a central 3,4-dihydro-2H-pyran motif, named xylomolones A–B (1–2), possessing identical absolute configuration but reversed alignment of ring A, compared to the tricyclic core of rings B–D, were obtained from seeds of the Thai mangrove, Xylocarpus moluccensis, together with a highly modified 1,2-seco limonoid, named xylomolone C (3), containing a novel 3-oxabicyclo[3.2.1]octane-2,7-dione motif, and a new C11-terpenic acid methyl ester (4). The relative and absolute configurations of 1–4 were evidenced by extensive NMR investigations combined with density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD), specific optical rotation (OR), and 13C NMR data. The co-isolation of 1–4 allowed us to propose a novel convergent strategy for limonoid biosynthesis for the first time. This study demonstrates that mangroves of the genus Xylocarpus harbor new limonoid biosynthetic routes.

INTRODUCTION Limonoids occur mainly in the plant families Meliaceae, Rutaceae, and Simaroubaceae. The structural diversity of limonoids is deemed to be generated through oxidative ring cleavages and skeletal rearrangements. Ordinarily, limonoids are classified on the basis of seco styles and cyclization patterns of four main rings (A–D) in the triterpenoid backbone.1-10 9,10-seco limonoids are small group of natural products. To date, 103 ones, including classes of carapanolide, dukunolide, ecuadorin, entilin, thaixylomolin, xylogranatin, xylomexicanin, xylocarponoid, and xylogranatopyridine, have been reported.11-18 However, only six 9,10-seco limonoids, viz. xylogranatin A,11 9-epixylogranatin A,17 xylogranatumin A,17 kokosanolides A and C,19 and cipadonoid A,20 contain a central tetrahydropyrane motif with the oxygen bridge between C-1/C-9 or C-1/C-14. Previous phytochemical investigations on mangroves of the genus Xylocarpus 2 / 31


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afforded

thirty-seven

limonoids,11-18

9,10-seco

among

which

xylogranatins

F–H1

and

xylogranatopyridine B21 contain a unique central pyridine ring. To the best of our knowledge, xylogranatopyridine B is the only 9,10-seco limonoid with reversed alignment of ring A, compared to the tricyclic core of rings B–D.21 The total synthesis of (–)-xylogranatopyridine B has been newly achieved via a palladium-catalyzed oxidative stannylation of enones.22 In the course of our search for new skeletal limonoids from mangroves of the genus Xylocarpus,1-2,5-10,13-14 two new 9,10-seco limonoids, named xylomolones A–B (1–2), were isolated from seeds of the Thai X. moluccensis, together with a highly modified 1,2-seco limonoid, named xylomolone C (3), and a C11-terpenic acid methyl ester, named xylomolone D (4) (Figure 1). Xylomolone A contains an oxygen bridge between C-1/C-9; whereas xylomolone B consists of an oxygen bridge between C-3/C-9. Xylomolones A–B have identical absolute configuration, but reversed alignment of ring A, compared to the tricyclic core of rings B–D. Xylomolone C is the first 1,2-seco limonoid containing an unprecedented 3-oxabicyclo[3.2.1]octane-2,7-dione motif. Herein, we report the isolation and structural elucidation of 1–4, and the proposed convergent strategy for the biosynthesis of 1–3. O

23

E

22 18

Me

19

Me

6

9

12

C 14 8

B

7

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17

Me

O

axis 30

6

MeO

axis

31

O

31

28

4

Me

O

5

O

29

Me

o

180

5

6 7

3

A 10

6

O Me 28

F

C 14

8

B

13

H

17

21

H O

D 16

O

15

30

2 1

O

23

E O

O O 19 O 2 H OH Me 7

12

20

2 22

31

9

H OAc Me 19

1

MeO

11

H O

D 16

2

29

E

18

H O

15

H

Me Me 28

13

20

30

1 10 A 23 5 4

H

AcO

11

H O

O

23 22 21

A

3

12

1

5

11 10

9

H

H

4

H

30

8

17

13

C 14

15

D

O 16

O Me 18

OH

19

Me H

20 21

6 7

MeO 31

1 2 10 5 A 3 4

O

Me Me 29

28

O

Me 29

3

4

3 / 31



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Figure 1. Structures of xylomolones A–D (1–4).

RESULTS AND DISCUSSION Xylomolone A [1, [𝑎]25 D = + 110 (acetone)] was isolated as a white amorphous powder. HR-ESIMS measurements suggested a molecular formula of C29H34O9 (m/z 549.2093, calcd for [M + Na] 549.2095), indicating that 1 had 13 degrees of unsaturation. According to the 1H

and

13C

NMR data (Tables 1 and 2) of 1, eight degrees of unsaturation were due to a

ketone function, three ester groups, and four C=C bonds; thus, the molecule has to be pentacyclic. DEPT experiments revealed the presence of six methyl groups (a methoxy, an acetyl, a secondary methyl, and three tertiary methyl groups), three methylene groups, ten methine groups (four olefinic and three oxygenated), and ten quaternary carbons (four carbonyls and four olefinic carbons). Table 1. 1H (400 MHz) NMR Data for 1−3 in CDCl3 (δ in ppm, J in Hz) no. 5

1 2.72 (5.6)

2 2.69 br s

3 3.28 s

6 8 9 10 11α 11β 12β 12α 15 17 18 19 21 22 23 28 29

5.66 s 2.24 m 3.57 td (11.2, 4.4) 3.25 br s 1.79 m 2.16 m 1.40 m 1.68 m 6.02 br d (2.0) 5.05 s 1.17 s 1.34 d (6.8) 7.49 br s 6.45 br s 7.45 br s 1.26 s 1.22 s

5.47 s 2.36 t (11.2) 3.62 td (11.2, 4.4) 2.97 m 1.79 m 2.22 m 1.44 m 1.70 br d (14.0) 6.02 s 5.08 s 1.16 s 1.25 d (6.8) 7.50 br s 6.45 br s 7.45 br s 1.44 s 1.23 s

4.89 s

4 / 31


2.99 t (4.8) 1.76 m 2.11 m 1.97 m 1.38 m 6.03 s 5.24 s 1.04 s 1.24 s 7.55 br s 6.49 br s 7.45 br s 1.21 s 1.14 s

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30β 30α 31 33

2.04 m 2.71 dd (12.8, 3.2) 3.73 s 1.94 s

2.03 m 2.71 dd (11.2, 4.4) 3.74 s 2.01 s

5.56 s 5.35 s 3.89 s

Table 2. 13C (100 MHz) NMR Data for 1−3 in CDCl3 (δ in ppm) no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 7-OMe-31 32 33

1 166.4 qC 107.8 qC 200.7 qC 43.7 qC 50.5 CH 68.4 CH 171.2 qC 37.6 CH 77.9 CH 32.3 CH 25.8 CH2 31.7 CH2 39.1 qC 165.1 qC 115.2 CH 164.4 qC 81.1 CH 17.6 CH3 14.0 CH3 119.8 qC 141.1 CH 109.9 CH 143.2 CH 27.1 CH3 22.0 CH3 21.7 CH2 52.8 CH3 169.8 qC 20.3 CH3

2 197.5 qC 108.1 qC 172.2 qC 38.5 qC 51.4 CH 69.6 CH 171.0 qC 37.5 CH 78.3 CH 39.5 CH 26.2 CH2 31.8 CH2 39.1 qC 165.0 qC 115.2 CH 164.2 qC 81.1 CH 17.7 CH3 13.2 CH3 119.8 qC 141.1 CH 109.9 CH 143.2 CH 28.8 CH3 22.6 CH3 21.7 CH2 52.6 CH3 169.3 qC 20.6 CH3

3 210.1 qC 167.0 qC 88.0 CH 41.7 qC 49.4 CH 77.2 CH 169.4 qC 143.4 qC 48.8 CH 52.3 qC 22.0 CH2 30.2 CH2 39.8 qC 166.0 qC 113.5 CH 164.8 qC 80.6 CH 18.7 CH3 22.1 CH3 119.8 qC 141.3 CH 109.9 CH 143.2 CH 20.8 CH3 23.2 CH3 121.1 CH 53.4 CH3

The NMR data of 1 resembled those of thaixylomolin Q,23 being a 9,10-seco limonoid isolated from the same Thai mangrove species, except for the replacement of the seco-ring B in thaixylomolin Q by a 3,4-dihydro-2H-pyran moiety in 1. The connection of CH-9‒CH-8‒CH2-30 was evidenced by a proton spin system H-9‒H-8‒H2-30, which was deduced from 1H–1H COSY 5 / 31



Page 6 of 31

correlations (Figure 2). The presence of a Δ1,2 double bond was corroborated by HMBC cross-peaks between H2-30/C-1, H2-30/C-2, and H3-19/C-1; whereas the existence of the an oxygen bridge between C-1 and C-9 was established by the HMBC cross-peak from H-9 to C-1, being measured in the mixture of CD3OD:CDCl3 (1:1) (Figure 2 and Table S1). Thus, the constitution of 1 was identified as depicted.

22

Me

Me O

10

H

H

15

O

Me

O

O 29

1

6

MeO

7

O

31

Me 33

32

11

H O

Me

3

O

Me

29

28

21

18 20

H O

2

Me Me 7

8

22

16

14 9

4

28

MeO 31

17

1

5

6

O

13

30

33

32

11

H O

12

O

23

COSY HMBC

21

18 20

19

Me

O

23

COSY HMBC

12

13

8

5

H

3

10

H

H O 16

14 9

30 4

17

15

O

2 1

O

Me 19

O 2

Figure 2. Key 1H–1H COSY and HMBC correlations of 1‒2. The relative configuration of 1 was determined based on diagnostic NOE interactions (Figure 3). Those between H-5/H3-28, H-5/H-10, H-10/H3-28, H-9/H-12β, H-9/H-30β, and H-17/H-12β, revealed their cofacial relationship and were arbitrarily assigned as the β-oriented H-5, H-10, H3-28, H-9, and H-17, and the corresponding α-oriented H3-19 and H3-29; whereas NOE interactions between H-8/H-30α, H-8/H3-18, and H3-18/H-11α, led to the assignment of the α-oriented H-8 and H3-18. As shown in Figure 3, the β-oxygenated α,β-unsaturated ketone moiety (‒O‒C-1=C-2‒C-3=O) of 1 divides the whole molecule into two relatively isolated, NOE-independent substructures, viz. southwestern and northeastern parts (Figure 1), bearing no relationship of NOE interaction between each other. Moreover, the relative configuration of C-6 could not be determined from NOE interactions. Thus, the molecule of 1 consists of three uncorrelated chiral building blocks, viz. rings 6 / 31


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C‒E with four chiral centers, ring A with two chiral centers, and the C-6 chiral center of the conformationally flexible side-chain.

28 21 17

23

15

16 12

20

3

2

29

4 1

8

11

13

22

30

9

14 10

5 6

19

7

18

31 32 33

NOE

Figure 3. Diagnostic NOE interactions of 1. Based on NOE interactions shown on the lowest-energy computed conformer (Figure 3), the (8R*,9R*,13R*,17R*) relative configurations of rings C‒E and the cis relative configuration between H-5 and H-10 (3JH-5,10-H = 5.6 Hz) of ring A were assigned. The presence of three independently chiral building blocks in 1 implies eight possible stereoisomers or four pairs of enantiomers (Figure 4) for the absolute configuration of 1. Hence, stereoisomers 1A‒1D (Figure 4) were selected for TDDFT-OR/ECD, and DFT-NMR calculations. O

O Me H O

H

Me AcO

H O

Me

H O

AcO

1A

H O

H O

Me

O

H

Me AcO

H O

O

H O

Me

O

H O

O

H

Me AcO

Me Me MeO

1B

H O

O

Me Me MeO

O

H

Me

Me Me MeO

H O

O

O

H O O

H O Me Me

MeO 1C

O 1D

Figure 4. Four stereoisomers of 1 selected for DFT/TDDFT calculations. In order to elucidate the relative24-26 and absolute configurations25-30 of 1, conformational analysis and combined OR, ECD, and NMR theoretical studies were carried out on the respective stereoisomers 1A‒1D (Figure 4). The Merck Molecular Force Field (MMFF) conformational search 7 / 31



Page 8 of 31

resulted in 15‒17 low-energy conformers for 1A‒1D, which were reoptimized independently at five different levels producing five sets of conformers, viz. B3LYP/6-31G(d), B3LYP/6-31+G(d,p), B97D/TZVP28,31 PCM/MeCN, CAM-B3LYP/TZVP27,32 PCM/MeCN, and CAM-B3LYP/TZVP PCM/acetone. Optical rotation (OR) calculations33 performed at four different levels, viz. B3LYP, BH&HLYP, CAM-B3LYP, and PBE0 (all with the TZVP basis set and PCM model for acetone) for the CAM-B3LYP/TZVP PCM/acetone reoptimized conformers gave large positive values for all the four computed stereoisomers. Furthermore, all low-energy conformers of each stereoisomer had a positive computed OR value, which allowed the assignment of the absolute configuration of C-8, C-9, C-13 and C17, but afforded no information for the remaining three chiral centers, viz. C-5, C-6, and C-10. ECD calculations performed for the CAM-B3LYP/TZVP PCM/MeCN conformers gave rather similar results for the four selected stereoisomers and reproduced the main transitions of the experimental ECD spectrum. Similarly to the OR calculations, the ECD spectrum of 1 was governed by four chiral centers of rings C‒E, of which the absolute configuration could be unambiguously assigned.25 The ECD transitions of the ,-unsaturated lactone (ring-D) and the furan ring were determined by the C-13 and C-17 chirality centers; whereas the C-8 and C-9 ones influenced those of the ,-unsaturated ketone chromophore. Variations in the other three chiral centers, viz. C-5, C-6, and C-10, did not induce substantial changes that could be used to distinguish them. When the C-8, C-9, C-13, and C-17 chirality centers were all inverted, the Cotton effects (CEs) changed signs in the computed ECD spectra as well, regardless of the configurations of the other three chiral centers (C-5, C-6, and C-10). ECD spectra computed for the other sets of conformers were slightly different, but the major transitions were the same and the minor ones depended on the conformational distributions estimated at various levels (Figure 5). Based on the OR and ECD calculations, the 8 / 31


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(8R,9R,13R,17R) absolute configuration of rings C‒E could be determined.

Figure

5.

Experimental

ECD

spectrum

of

1

in

MeCN

compared

with

the

Boltzmann-weighted B3LYP/TZVP PCM/MeCN ECD spectrum of 1A computed for the CAM-B3LYP/TZVP PCM/MeCN conformers. Bars represent the rotational strength values of the lowest-energy conformer. To establish the absolute configurations of the remaining three chiral centers, viz. C-5, C-6, and C-10, DFT NMR calculations were performed. DFT

13C-NMR

calculations24-25,34-35 performed at

the mPW1PW91/6-311+G(2d,p) level36 for the B3LYP/6-31+G(d,p) conformers of the stereoisomers 1A‒1D gave quite similar overall deviations from the experimental

13C

chemical

shifts for the four stereoisomers. However, based on the different chemical shifts of ring A, C-6, and the connecting first carbons, the stereoisomers 1C and 1D could be excluded, particularly due to their bad agreement for the C-6 chiral center (Table 3). Based on the above NMR calculation results, the relative configuration (5R*,6R*,10S*) could be assigned.25 Overall, the agreement of the computed 13C-NMR chemical shifts was found to be slightly better for 1A, compared to that of 1B, suggesting the absolute configuration (5R,6R,8R,9R,10S,13R,17R) for 1, which was corroborated well by the (5R) configuration of limonoids containing an analogue scaffold of ring A as exemplified by hainangranatumins A‒E,14 prexylogranatopyridine,21 and thaixylomolin Q.23 DP4+ 9 / 31



Page 10 of 31

analysis performed for the relevant 11 carbons listed in Table 3 indicated 91.18% confidence for 1A, while the same analysis for all the carbons showed 88.28% confidence.37,38 Thus, the common biosynthetic origins of these limonoids further confirmed our configurational assignments. Table 3. Comparison of the computed

13C

NMR chemical shifts of 11 relevant carbons in the

vicinity of the investigated chiral centers of 1A‒1D with the experimental 13C NMR data. Carbon no. C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-10 C-19 C-28 C-29 Average

Exp.

1A

1B

1C

1D

Δδ1A

Δδ1B

Δδ1C

Δδ1D

166.4 107.8 200.7 43.7 50.5 68.4 171.2 32.3 14.0 27.1 22.0 N/A

167.27 109.16 199.04 46.27 52.25 68.68 173.58 34.60 13.77 25.74 20.89 N/A

168.14 109.40 199.22 46.56 53.06 68.82 173.63 35.21 13.78 25.48 21.15 N/A

170.14 109.14 199.01 47.83 49.95 72.45 173.00 36.06 12.81 25.07 22.69 N/A

171.25 108.88 199.10 47.56 47.40 73.89 172.64 36.40 13.82 23.19 22.92 N/A

0.87 1.36 1.66 2.57 1.75 0.28 2.38 2.30 0.23 1.36 1.11 1.44

1.74 1.60 1.48 2.86 2.56 0.42 2.43 2.91 0.22 1.62 0.85 1.70

3.74 1.34 1.69 4.13 0.55 4.05 1.80 3.76 1.19 2.03 0.69 2.27

4.85 1.08 1.60 3.86 3.10 5.49 1.44 4.10 0.18 3.91 0.92 2.78

Xylomolone B [2, [𝑎]25 D = + 52 (acetone)] provided the same molecular formula as that of 1 (m/z 549.2091, calcd for [M + Na] 549.2095). The 1H and

13C

NMR data (Tables 1 and 2) of 2 were

very similar to those of 1, except for the upshifted ketone group (δC 197.5 in 2 vs. 200.7 in 1) and the downshifted oxygenated olefinic quaternary carbon (δC 172.3 in 2 vs. 166.4 in 1). HMBC cross-peaks from protons of H3-19 [δH 1.25 (d, J = 6.8 Hz)] and H2-30 [δH 2.74 (dd, J = 11.2, 4.4 Hz), 2.03 (m)] to the carbon (δC 197.5) of the above ketone group assigned the connection of C-10‒C-1‒C-2‒C-30 (Figure 2). Owing to 13 indices of hydrogen deficiency of 2, a C-16‒C-17 ester linkage and a C-3‒O‒C-9 oxygen bridge have to be existed (Figure 2). The above deduction was corroborated in the positive-ion ESI-MS/MS of 2 by two groups of five diagnostic fragments, viz. m/z 485 and 389, and m/z 509, 467, and 449 (Figure S1); which originated from the subsequent loss of an ethenone unit and a furan-3-carbaldehyde unit,5 and from the subsequent loss of units of 10 / 31


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water and acetic acid or vice versa, respectively. Based on diagnostic NOE interactions shown on the lowest-energy computed conformer (Figure 6), the relative configuration of 2 was assigned. Those between H-9/H-12β, H-9/H-30β, H-17/H-12β, and H3-19/H3-29, assigned the β-oriented H-9, H-17, H3-19, and H3-29; whereas those between H-5/H-10, H-5/H3-28, H-8/H-30α, H-8/H3-18, and H3-18/H-11α, assigned the α-oriented H-5, H-10, H3-28, H-8, and H3-18. NOE

33

32 31 7

29

6 5

4

21 9

3 30

19

8

17 14

20

13

2

1

10

12

11

23

15

28

22

16 18

Figure 6. Diagnostic NOE interactions of 2. In order to determine the stereochemistry of 2, the same approaches of TDDFT-ECD/OR, and DFT 13C-NMR calculations were applied to 2. Four stereoisomers 2A‒2D (Figure 7) were selected for computation. As expected, TDDFT-ECD (Figure 8, Figure S4) and TDDFT-OR (Tables S6‒S9) calculations of 2 confirmed the (8R,9R,13R,17R) absolute configuration, since these chiroptical parameters are primarily governed by the north-east chiral block of 2. O Me H O

MeO

2A

H O

MeO

OAc

O Me

H O

MeO

OAc

2B

O Me

H O

Me OH

O Me

Me

H O

H

Me OH

O Me

Me

H O

H

Me OH

O Me OAc

Me O Me

H

Me Me OH

H O

O

O

O

MeO

OAc

2C

Figure 7. Four stereoisomers of 2 selected for DFT/TDDFT calculations. 11 / 31


H O

Me 2D

H O O


Page 12 of 31

Figure 8. Experimental ECD spectrum of 2 in MeCN compared with the Boltzmann-weighted B3LYP/TZVP PCM/MeCN ECD spectrum of 2A computed for the CAM-B3LYP/TZVP PCM/MeCN conformers. Bars represent the rotational strength values of the lowest-energy conformer. In contrast to the situation of 1, the DFT

13C-NMR

calculations performed on the four

stereoisomers of 2 gave very similar results (Table S11), which did not allow us to determine the absolute configurations of the other three chiral centers, viz. C-5, C-6, and C-10, independently. The DP4+ analysis of the computed 13C-NMR data gave only low confidence values (4.00-56.15%), on the basis of which the relative configuration could not be assigned, either.37,38 However, 2 can be considered as the close analogue of thaixylomolin Q (with X-ray crystal structure),23 obtained from the same batch of mangrove seeds, suggesting the (5R,6R,10S) absolute configuration, being in accordance with that of 1. Thus the absolute configuration of 2 was assigned as (5R,6R,8R,9R,10S,13R,17R). Xylomolone C [3, [𝑎]25 D = + 40 (acetone)] afforded a molecular formula of C27H30NaO9 (m/z 521.1781, calcd for [M + Na] 521.1782). The NMR data of 3 (Tables 1 and 2) showed the presence of a ketone group, three ester functions, and four C=C bonds; thus, the molecule was contemplated to be pentacyclic. DEPT experiments revealed the presence of five methyl groups (one methoxy 12 / 31


Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


group and four tertiary methyls), three methylene groups (one olefinic), eight methine groups (four olefinic and two oxygenated), and ten quaternary carbons (four carbonyls and three olefinic carbons). Comparison of the NMR data of 3 with those of swietmanin J,39 being an andirobin-type limonoid, suggested that both compounds had the same architecture of rings C, D, and E, whereas 3 possessed the totally different moiety of ring A from that of swietmanin J.39 The presence of a 3-hydroxy-19,28,29-trimethyl-cyclopentanone moiety (ring-A, C-1, C-3, C-4, C-5, C-10, C-19, C-28, and C-29, Figure 9) was established by HMBC cross-peaks between 3-OH/C-3, H-5/C-1, H-5/C-3, H-5/C-4, H-5/C-10, H3-19/C-1, H3-19/C-3, H3-19/C-5, H3-19/C-10, H3-28/C-3, H3-28/C-4, H3-28/C-5, H3-29/C-3, H3-29/C-4, and H3-29/C-5 (Figure 9). Further, the existence of a tetrahydro-2H-pyran-2-one moiety (ring-F, from C2 to C6, Figure 9) was evidenced by the 1H-1H COSY correlation between H-5/H-6 and HMBC cross-peaks between 3-OH/C-2, 3-OH/C-4, H-6/C-2,

and

H-6/C-4.

Taken

together,

the

bridged

bicyclic

substructure,

i.e.

3-oxabicyclo[3.2.1]octane-2,7-dione (from C1 to C6, and C10, Figure 9), was unequivocally elucidated. In addition, the location of a methoxycarbonyl group at C-6 was confirmed by HMBC cross-peaks from H3-31 and H-6 to C-7 (Figure 9). Thus, the constitution of 3 was identified as depicted.

O 2

O 31

MeO

21

HMBC

COSY

6

H

OH 1 3

7

O

O Me 28

23

H 19

Me

H

4

11 9

H

17

8

22

20

O

12

10 5

O

16

13 14 15

O Me 18

30

Me 29

13 / 31



Page 14 of 31

Figure 9. Key 1H-1H COSY and HMBC correlations of 3. The relative configuration of 3 was determined based on diagnostic NOE interactions (Figure 10). Those between H-5/H3-28, H-5/H3-29, H-5/H-9, H-11α/H3-18, and H-12α/H3-18 revealed their cofacial relationship and were arbitrarily assigned as α-oriented H-5, H-9, and H3-18. Similarly, NOE interactions between H-6/H3-19, H3-19/H-12β, and H3-19/H-17 assigned the β-orientation for H-6, H3-19, and H-17. The TDDFT-ECD protocol performed for (3S,5R,6R,9S,10R,13R,17R)-3 gave moderate to good agreement with the experimental curve at all the applied combinations of levels (Figure 11), allowing the elucidation of the absolute configuration as (3S,5R,6R,9S,10R,13R,17R) for 3.

NOE

21

19

16 17 20

15 2

31

6

7

28

1 3

5

14 30

10 9

8

23 22

13

12 11

18

4 29

Figure 10. Diagnostic NOE interactions of 3.

Figure 11. Experimental ECD spectrum of 3 in MeCN compared with the Boltzmann-weighted B3LYP/TZVP PCM/MeCN ECD spectrum of (3S,5R,6R,9S,10R,13R,17R)-3 computed for the 14 / 31


Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B97D/TZVP PCM/MeCN conformers. Bars represent the rotational strength values of the lowest-energy conformer. In order to unequivocally corroborate the structure of 3, quantum-chemical calculations of

13C

NMR chemical shifts were performed at the B3LYP/6-31G(d) level of theory for the structure 3 (Figure 12).40-41 The calculated

13C

NMR chemical shifts of 3 showed good agreement with the

experimental data with a correlation coefficient (R2) = 0.9986 (Figure 12). All the individual deviations, |Δδ|, between the predicted and experimental 13C NMR chemical shifts for 3, were less than 4.0 ppm (Figure 12, Table S12). Obviously, the calculated

13C

NMR chemical shifts of 3

nicely matched with the experimental 13C NMR data.

Figure 12. Calculated

13C

NMR chemical shifts for the structure of 3. (A) Linear correlations

between the calculated and experimental

13C

NMR chemical shifts of 3. (B) Individual deviations

between the calculated and experimental 13C chemical shifts.

Xylomolone D [4, [𝑎]25 D = + 25 (acetone)] gave a molecular formula of C12H18O4 as established by HR-ESIMS (m/z 227.1281, calcd for [M + H] 227.1278). It contains the same skeleton as that of the southwestern part (except for the 6-OAc group) of 1‒2 (Table 4). The relative configuration of 4 was determined based on diagnostic NOE interactions. Those between H-5/H3-28, H-5/H3-29, 15 / 31



Page 16 of 31

H-5/H3-19, H-3β/H3-28, and H-3α/H3-29 assigned the β-oriented H-5 and H3-28, and the α-oriented H3-29 (Figure 13). The absolute configuration of 4, containing the only C-5 chiral center, was determined by TDDFT-ECD calculations. The computed Boltzmann-weighted TDDFT-ECD spectra of (5S)-4 gave a moderate to good agreement with the experimental ECD curve at all applied combinations of levels (Figure 14). These results allowed us to assign the absolute configuration (5S) for 4, being in accordance with that of C-5 in 1 and 2. Interestingly, the n-π* transition of 4 was overestimated at all applied levels. Table 4. 1H (400 MHz) and 13C (100 MHz) NMR Data for 4 in CDCl3 (δ in ppm) no. 1 2 3α 3β 4 5 6a 6b 7 10 19 28 29 7-OMe 1-OH

δH, mult (J in Hz)

δC, mult 143.2 qC 193.1 qC

2.48 d (17.2) 2.22 d (17.2)

45.6 CH2 36.0 qC 47.2 CH

2.69 m 2.59 dd (16.0, 7.6) 2.33 dd (16.0, 4.8)

34.5 CH2 173.4 qC 130.6 qC 15.9 CH3 28.1 CH3 26.3 CH3 52.0 CH3

1.89 s 1.11 s 0.97 s 3.71 s 6.01, s

19

NOE

7

6

28

5 10 1

4 29

3

2

Figure 13. Diagnostic NOE interactions of 4. 16 / 31


Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 14. Experimental ECD spectrum of 4 in MeCN compared with the Boltzmann-weighted PBE0/TZVP ECD spectrum of (5S)-4 computed for the B3LYP/6-31G(d) conformers. Bars represent the rotational strength values of the lowest-energy conformer. The co-isolation of 1 and 2 enabled us to propose their biosynthetic origins from the same two building blocks, viz. A (Michael donor) and B (Michael acceptor), among which A could be originated from the precursor actinidiolide42 via the intermediate Int I by ring opening, reduction, oxidation, and esterification (Scheme 1). The Michael addition of A and B would generate the crucial intermediate C. Subsequent attack of the enolized oxygen atom, which could be derived from the C-1 carbonyl group of A, at the electrophilic C-9 of the intermediate C, then cyclization and reduction would give 1. Similarly, the tautomerization of the intermediate C would yield the intermediate D, in which the rotation of the building block A around the C-2‒C-30 sigma bond could produce the intermediate E. The attack of the enolized oxygen atom, which could be derived from the C-3 carbonyl group, at the electrophilic C-9 of the intermediate E, followed by cyclization and reduction would give 2. Thus, a convergent strategy was proposed for the biosynthesis of 1‒2. The same convergent strategy could be proposed for the biosynthetic origin of 3. The building block A' could be originated from the above-mentioned precursor, actinidiolide, via a series of intermediates, viz. Int I, Int II, Int IIIa/b, Int IVa/b, and Int V, by two possible subpathways: 17 / 31



Page 18 of 31

either through oxidation at C-2, followed by benzilic acid-like rearrangement, lactonization, and oxidation at C-3, or by oxidation at C-2 and C-3, subsequent benzilic acid-like rearrangement, and final lactonization. Then the aldol condensation of A' and B would generate the crucial intermediate C'. Finally, the reduction of the 9-OH group in the intermediate C' could give 3 (Scheme 1). The building blocks A, A', and B (Scheme 1) are too reactive to exist on their own. However, the presence of A and A' could be evidenced by the same carbon skeleton of a series of naturally occurring C11-terpenic lactones, such as actinidiolide, dihydroactinidiolide, and aeginetolide, obtained from plants of Actinidia polygama and Aeginetia indica;42-44 particularly xylomolone D (4) isolated from the same mangrove seeds as the enol form of Int II (Scheme 1); whereas the existence of B could be supported by the same scaffold of quite a number of previously assumed plant degraded limonoids, such as melazolides A‒B, azedaralide, and pyroangolensolide, isolated from the plant of Melia azedarach45-46 and dictamdiols A‒B, dictamdiol, and calodendrolide, obtained from Dictamnus dasycarpus.47 To the best our knowledge, the biosynthesis of various triterpenoids, including limonoid, is deemed to be originated from an acyclic triterpenoid precursor, i.e. squalene. However, the squalene biosynthetic route is rendered difficult to explain the formation of limonoid regioisomers 1 and 2. Obviously, the above-mentioned convergent strategy for the biosynthesis of 1‒3 is reasonable and supported by regioisomeric structures of 1 and 2. Furthermore, the co-isolation of 4 (the enol form of Int II), possessing the same skeleton as that of A and A', corroborates the presence of these building blocks.

18 / 31


Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O Me H O

9

Me H

O

30

3

H O

8

Me

O

2 1

9

O O Me

reduction O

8

180

30

MeO

E

3

O

30

9

O O

8

1

H

AcO

O 10 5

H

AcO 6

[O]

2 3

4

H

[+OAc]

O

28

MeO

10 5

6

O

2 3

4

28

MeO

O

O

1

3

O

Me

8

29

H

10 5

6

9

O

1 2 3 4

Me Me

7

28

MeO

actinidiolide

Int I

OH

19

Me

10 1 2 5 3 4

7 6

O

A

O

1

Me Me

Me Me

7

29

O

H 30

11

O

19

Me Me

7

MeO

C

Me

8

2

Me Me

Me Me O

1

H

AcO

9

O

Me

reduction

3

O

MeO

H O

Me H cyclization

30

2

O

O

H

Me

B

1

D

H O

Me

Michael addition

19

O

tautomerization

H O O

Me

2

O

8

O

O

H

Me Me

O

9

8

30

1

H

AcO

O

2

Me

Me

rotation

2 1

9

O O

o

Me OAc

MeO

H O

Me O

H

3

Me H

O

Me OAc

MeO

H O

Me O cyclization

H

O

29

O

4

[O] keto-enol tautomerization O

19

Me H 6

10 5

2 3

4

[O]

O

6

Me Me

7

28

MeO

O

19

Me H

O

1

O

2 3

4

Me

benzilic acid-like rearrangement

28

MeO

6

29

O 10 5

H

Int IIIa

O 6

4

3

Me Me

Me

lactonization

O

OH

5

28

7

2 3

4

Me

[O]

O

OH 29

10

H

6

5

O

1 2 3

4

Me Me

7

O

Int IVb

O

19

O

1

Me Me

MeO

MeO

10

H

6

28

7

O

19

O

29

lactonization

HO 2

28

MeO

29

O Int IVa

Int V enolization

O H Me O

10

H

5

6

O 2 3

4

28

O

A'

9

8

HO Aldol reaction

O

9

O

8

O 6

O

7

MeO

30

B

reduction

5

O

2 3

4

OH

6

Me Me 28

7

29

MeO

O

O

1

10

H

5

2 3

4

OH

Me Me 28

29

O 3

C'

H O O

O

19

Me

O

1

10

H

8

30

O

19

9

H

O

30

Me

H O

Me

H O

Me

29

Me

O

O OH

Me Me

7

MeO

1

O

O

Int II

1

2 3

4

28

MeO

benzilic acid-like rearrangement

19

HO

1

Me Me

7

O

Int IIIb

Me

10 5

H

O

Me Me

7

29

10 5

O

19

O

1

Scheme 1. Proposed biosynthetic routes for 1‒3. 19 / 31



Page 20 of 31

CONCLUSION In summary, three minor limonoids, named xylomolones A–C (1–3), among which the latter two are new skeletal limonoids, were isolated from the seeds of a Thai mangrove, X. moluccensis, together with a key biosynthetic precursor 4. The absolute configurations of these compounds were evidenced by extensive NMR investigations combined with the DFT and TDDFT calculations of ECD, OR, and

13C

NMR data. Most notably, xylomolone B (2)

is the first 9,10-seco limonoid that not only contains a 3,4-dihydro-2H-pyran motif, but also possesses the reversed alignment of ring A. Xylomolone C (3) is the first highly modified 1,2-seco limonoid with a bridged cyclic 3-oxabicyclo[3.2.1]octane-2,7-dione motif. The co-isolation of 1–4 allowed us to propose a novel convergent strategy for limonoid biosynthesis. This study demonstrates that mangroves of the genus Xylocarpus harbor new limonoid biosynthetic routes.

EXPERIMENTAL SECTION General Experimental Procedures. UV spectra were recorded on a GENESYS 10S UV-Vis spectrophotometer (Thermo Scientific) and HR-ESIMS obtained on a Bruker miXis ESI-QTOF MS in the positive-ion mode. NMR spectra were measured on a Bruker AV-400 spectrometer. Preparative HPLC was performed on a Waters 2535 pump equipped with a 2998 photodiode array detector and YMC C18 reversed-phase columns (250 × 10 mm i.d., 5 μm). For column chromatography, silica gel (100-200 mesh) (Qingdao Mar. Chem. Ind. Co. Ltd.) and C18 reversed-phase silica gel (ODS-A-HG 12 nm, 50 µm, YMC, Japan) were employed. Electronic circular dichroism (ECD) spectra were recorded on a Jasco 810

20 / 31


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spectropolarimeter in MeCN and optical rotations determined on an MCP500 modular circular polarimeter (Anton Paar GmbH).

Plant Material. From mangrove swamps of the Trang Province, Thailand, seeds of Xylocarpus moluccensis were collected in June 2013. The identification of the plant was performed by one of the authors (J.W.). Voucher sample (No. ThaiXM-03) is maintained in Marine Drugs Research Center, College of Pharmacy, Jinan University (Guangzhou).

Extraction and Isolation. The air-dried seeds (10.0 kg) were powdered, and extracted with 95% (v/v) EtOH (5  20 L) at room temperature to afford the resulting extract (680.0 g), which was partitioned between EtOAc and water to provide an EtOAc portion (296.0 g). The EtOAc portion was chromatographed on a silica gel column (120  10 cm i.d.), eluted with a gradient mixture of CHCl3/MeOH (100:0 to 5:1), to yield 160 fractions. Fractions 26−28 (31.6 g) were combined and further separated on an RP-18 column (100  5 cm i.d.), eluted with a gradient mixture of acetone/H2O (50:50 to 100:0), to yield 57 subfractions. The subfraction 23 (508 mg) was purified by semi-preparative HPLC (YMC-Pack 250  10 mm i. d., MeCN/MeOH/H2O, 30:20:50; MeOH/H2O, 53:47) to afford compounds 1 (1.5 mg, tR 67.8 min) and 2 (0.9 mg, tR 63.5 min). Fractions 29−40 (110.7g) were combined and further separated by an RP-18 column (100  5 cm i.d.), eluted with a gradient mixture of acetone/H2O (50:50 to 100:0), to yield 175 subfractions, among which subfractions 90 to 95 (3.20 g) were combined and purified by semi-preparative HPLC (YMC-Pack 250  10 mm i. d., MeOH/H2O, 60:40) to afford compound 3 (1.0 mg, tR 17.0 min); whereas the subfraction 5 was purified by semi-preparative HPLC (YMC-Pack ODS-5-A, 250 × 10 mm i.d., MeCN/H2O, 26:74) to afford compound 4 (5.4 mg, tR 21.6 min). 21 / 31



Page 22 of 31

Xylomolone A (1): White solid, [𝑎]25 D = + 110 (c = 0.02, acetone); UV (MeCN) λmax (log ε) 213.1 (4.3), 263.8 (4.1) nm; For 1H and 13C NMR spectroscopic data (see Tables 1–2); ECD (0.42 mM, MeCN) λmax (Δε) 190 (‒ 3.4), 222.4 (+ 5.9), 254.8 (+ 1.6) nm; HR-ESIMS m/z 549.2093 [M + Na]+ (calcd for C29H34NaO9, 549.2095) Xylomolone B (2): White solid, [𝑎]25 D = + 52 (c = 0.02, acetone); UV (MeCN) λmax (log ε) 199 (3.8), 259 (3.5) nm; For 1H and

13C

NMR spectroscopic data (see Tables 1–2); ECD (0.48 mM,

MeCN) λmax (Δε) 190 (‒ 1.1), 220.8 (+ 4.6), 262.4 (+ 1.9) nm; HR-ESIMS m/z 549.2091 [M + Na]+ (calcd for C29H34NaO9, 549.2095) Xylomolone C (3): white solid, [𝑎]25 D = + 40 (c = 0.02, acetone); UV (MeCN) λmax (log ε) 199 (3.9), 256 (3.7) nm; For 1H and

13C

NMR spectroscopic data (see Tables Tables 1–2); ECD (0.8

mM, MeCN) λmax (Δε) 190 (‒ 0.9), 246.1 (+ 7.3) nm; HR-ESIMS m/z 521.1781 [M + Na]+ (calcd for C27H30NaO9, 521.1782) Xylomolone D (4): white solid, [𝑎]25 D = + 25 (c = 0.05, acetone); UV (MeCN) λmax (log ε) 200 (3.7), 273.2 (3.4) nm; For 1H and 13C NMR spectroscopic data (see Table 4); ECD (1.1 mM, MeCN) λmax (Δε) 190 (‒ 1.2), 234 (+ 1.9), 267 (+ 0.2), 299 (+ 2.6) nm; HR-ESIMS m/z 227.1281 [M + H]+ (calcd for C12H19O4, 227.1278)

Computational Details By using Gaussian 09 program,41 all the configurations and conformations of 1‒3 were optimized at five different levels, viz. B3LYP/6-31G(d), B3LYP/6-31+G(d,p), B97D/TZVP PCM/MeCN, CAM-B3LYP/TZVP PCM/MeCN, and CAM-B3LYP/TZVP PCM/acetone. Optical rotation (OR) calculations performed at four different levels, viz. B3LYP, BH&HLYP, CAM-B3LYP, and PBE0 (all with the TZVP basis set and PCM model for 22 / 31


Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


acetone) for the CAM-B3LYP/TZVP PCM/acetone reoptimized conformers. DFT 13C-NMR calculations

were

performed

at

the

mPW1PW91/6-311+G(2d,p)

level

for

the

B3LYP/6-31+G(d,p) conformers of the stereoisomers 1A‒1D and 2A‒2D. For the calculations of

13C

NMR chemical shifts of 3, B3LYP/6-31G(d,p) method was used to

optimize the selected conformations. For all optimized structures, vibrational modes were calculated to ensure that no imaginary frequencies for energy minimum were obtained. NMR calculations were performed at the levels of mPW1PW91/6-31G(d,p) with the gauge-independent atomic orbital (GIAO) method.48-50 The solvent effect was considered by using chloroform for 3 in the calculations to resemble the experimental condition. The polarized continuum model (PCM) of Tomasiet al. was used.51-54 The calculated 13C NMR chemical shifts were analyzed by subtracting the isotopic shifts for TMS calculated with the same methods.48-50 Different conformers for the structure 3 were considered. The

13C

NMR chemical shifts in each compound were considered as

the average values of the same atoms in the different conformers. The average values were obtained by the Boltzmann distributions, using the relative Gibbs free energies as weighting factors.54 The differences Δδ were determined by subtracting the experimental chemical shifts δ exptl from the calculated chemical shifts δ calcd.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Figures S1‒S5, Tables S1‒S14, 1H- and 13C-NMR data of 1 in CDCl3:CD3OD (1:1) and Acetone-d6, ESI-MS/MS-fragmentation of 2‒3, Computational details; HR-ESIMS, 1D and 2D NMR spectra of 1–4 (PDF) 23 / 31



Page 24 of 31

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (L.S.) *E-mail: [email protected] (J.W.) *E-mail: [email protected] (K.T.). ORCID Attila Mándi: 0000-0002-7867-7084 Li Shen: 0000-0001-5770-1607 Tibor Kurtán: 0000-0002-8831-8499 Jun Wu: 0000-0003-0807-5229 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by grants from NSFC (U1501221, 31770377, and 81661148049), the Fundamental Research Funds for the Central Universities, China (21617474). The research of the Hungarian authors was supported by the EU and co-financed

by

the

European

Regional

Development

Fund

under

the

project

GINOP-2.3.2-15-2016-00008. T. K. thanks the National Research, Development and Innovation

Office

(NKFI

K120181)

for

financial

support.

The

Governmental

Information-Technology Development Agency (KIFÜ) is acknowledged for CPU time. We thank Dr. Patchara Pedpradab (Rajamangala University of Technology Srivijaya, Trang Province, Thailand) for providing the plant materials in this work. 24 / 31


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Xylomolones AâD from the Thai Mangrove Xylocarpus moluccensis

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