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약학석사학위논문

Structure Determination of Polybrominated Aromatics from

the Synoicum sp. Ascidian

군체멍게로부터 분리된 이차대사물질의 성분연구

February 2020

by Jongkyoon Bae

Natural Products Science Major, College of Pharmacy

Master Course in the Graduate School

Seoul National University

I

Abstract

Structure Determination of Polybrominated

Aromatics from the Synoicum sp. Ascidian

Jongkyoon Bae

Natural Products Science

College of Pharmacy

Master Course in the Graduate School

Seoul National University

Nine new polybrominated aromatic compounds, Isocadiolides A-H (1-8) and cadiolide N (9),

were isolated from Synoicum sp. Ascidian collected at offshore of Keomun-do (Island), Korea.

The planar- and stereo-structure were determined by extensive spectroscopic analyses. Notable

features of these compounds were that they all have tris-bromohydroxyphenyl moieties in

common, yet their core structures were diversified with different extents of rearrangements and

oxidation state (cyclopentenedione (1-5), dihydrofuran (6 and 7), pyranone (8), and furanone (9)).

No cytotoxicity against K562 and A549 cell lines (IC50 >10μM) were found of these compounds,

while, several of these exhibited weak antibacterial activities and moderate inhibition against

sortase A and isocitrate lyase.

II

Keywords : ascidian secondary metabolites, polybrominated aromatics, Sortase A inhibition.

Student number : 2018-24173

III

LIST OF CONTENTS

Abstract ...................................................................................... I

List of Contents ....................................................................... III

List of Scheme and Tables ....................................................... IV

List of Figures ........................................................................... V

Introduction ................................................................................ 1

Experimental Section ................................................................. 2

1. General Experimental Procedures .......................................... 3

2. Animal Material ..................................................................... 3

3. Extraction and Isolation ......................................................... 4

Results ........................................................................................ 7

1. Compound 1 ........................................................................... 7

2. Compound 2 ........................................................................... 8

3. Compound 3 ........................................................................... 9

4. Compound 4 ........................................................................... 9

5. Compound 5 ......................................................................... 10

6. Compound 6 ......................................................................... 11

7. Compound 7 ......................................................................... 12

8. Compound 8 ......................................................................... 13

9. Compound 9 ......................................................................... 14

Discussions ............................................................................... 26

References ................................................................................ 27

Abstract in Korean ................................................................... 71

IV

List of Scheme and Tables

Scheme 1. Isolation of compounds from ascidian

Synoicum sp. …………………………………………… 6

Table 1. 1H and 13C NMR assignment for compound 1............... 17

Table 2. 1H and 13C NMR assignment for compound 2………… 18

Table 3. 1H and 13C NMR assignment for compound 3………… 19

Table 4. 1H and 13C NMR assignment for compound 4…….…... 20

Table 5. 1H and 13C NMR assignment for compound 5…….…… 21





V

Lists of Figures

Figure 1. The 1H and 13C NMR spectrum of 1 ................................................... 29

Figure 2. The HSQC spectrum of 1 .................................................................. 30

Figure 3. The HMBC spectrum of 1 .................................................................. 31

Figure 4. The D-HMBC spectrum of 1 .............................................................. 32

Figure 5. The 1H and 13C NMR spectrum of 2 ................................................. 33

Figure 6. The HSQC spectrum of 2 .................................................................. 34

Figure 7. The HMBC spectrum of 2 ................................................................. 35

Figure 8. The D-HMBC spectrum of 2 ............................................................. 36

Figure 9. The 1H and 13C NMR spectrum of 3 .................................................. 37

Figure 10. The HSQC spectrum of 3 ................................................................ 38

Figure 11. The HMBC spectrum of 3 .............................................................. 39

Figure 12. The D-HMBC spectrum of 3 ........................................................... 40

Figure 13. The 1H and 13C NMR spectrum of 4 ................................................ 41


Figure 15. The HMBC spectrum of 4............................................................... 43

VI



Figure 18. The HSQC spectrum of 5 ................................................................. 46

Figure 19. The HMBC spectrum of 5 ................................................................. 47



Figure 22. The HSQC spectrum of 6................................................................. 50

Figure 23. The HMBC spectrum of 6 ............................................................... 51


Figure 25. The NOESY spectrum of 6 ............................................................. 53

Figure 26. The 1H and 13C NMR spectrum of 7 ............................................... 54



Figure 29. The NOESY spectrum of 7 ............................................................. 57

Figure 30. The 1H and 13C NMR spectrum of 8 ............................................... 58

VII



Figure 33. The D-HMBC spectrum of 8 ............................................................. 61

Figure 34. The NOESY spectrum of 8 ................................................................ 62


Figure 36. The HSQC spectrum of 9 ................................................................. 64

Figure 37. The HMBC spectrum of 9 ................................................................ 65


Figure 39. The 1H (DMSO-d6) NMR spectrum of 9 ......................................... 67

Figure 40. The NOESY spectrum of 9 .............................................................. 68

Figure 41. The 1-D Selective gradient NOESY (DMSO-d6) spectrum of 9 ... 69

Figure 42. Measured and calculated ECD profiles of

compounds 2, 4, and 6-9 ................................................................................. 70

1

Introduction

Ascidians are prolific sources of structurally unique and biologically active marine

natural products.1 Biosynthetically derived from various amino acids, a majority of ascidian

compounds naturally possess diverse nitrogen-containing functionalities.2 Among notable

exceptions are the halogenated polyaromatics such as the rubrolides3-6 and cadiolides.6-9

Despite the biosynthetic condensations of α-keto acids derived from phenylalanines and

tyrosines, these compounds contain nitrogen-free furanone cores with bis- and tris-

bromohydroxyphenyl substituents, respectively.3 The wide biological distribution of these

compounds in several ascidian genera, including Botryllus, Pseudodistoma, Ritterella, and

Synoicum, is quite remarkable. The significance of these compounds among ascidian

natural products is further highlighted by their diverse and often potent bioactivities such

as cytotoxicity and antibacterial (including anti-MRSA) and anti-inflammatory activities

as well as inhibitory activities against several enzymes.3-13 The recent finding of two new

rubrolides from a marine-derived Aspergillus terreus fungal strain is also notable.14

As a part of our search for bioactive marine natural products, we previously reported the

structures and bioactivities of cadiolides E and G-I and synoilides A and B from a Korean

Synoicum sp. ascidian.8 Among these compounds, cadiolide I and the synoilides possessed

novel carbon skeletons derived from the rearrangement of the furanone cores commonly

found in cadiolides and rubrolides, respectively, contributing to the structural diversity of

these halogenated aromatics. As a continuation of this study, we re-encountered this

Synoicum sp. ascidian offshore of Keomun-do (Island), Korea. The LC-ESIMS profile of

the organic extract revealed the presence of several brominated constituents in addition to

the previously identified cadiolides and synoilides, prompting an extensive chemical

investigation. Here, we report the structure determination of nine new brominated aromatic

2

compounds, isocadiolides A-H (1-8) and cadiolide N (9). All of these compounds possessed

tris-bromohydroxyphenyl moieties as a common structural motif, while their cores varied

(cyclopentenedione (1-5), dihydrofuran (6 and 7), pyranone (8) and furanone (9)),

reflecting different extents of rearrangement and oxidation. Several of the compounds

exhibited weak antibacterial activities against human pathogenic strains and moderate

abilities to inhibit the microbial enzymes sortase A (SrtA) and isocitrate lyase (ICL).

3

Experimental Section

1. General Experimental Procedures

Optical rotations were measured using a JASCO P-1020 polarimeter with a 1-cm cell. UV

spectra were acquired using a Hitachi U-3010 spectrophotometer. ECD spectra were

recorded on an Applied Photophysics Chirascan plus CD spectrometer. IR spectra were

recorded on a JASCO 4200 FT-IR spectrometer using a ZnSe cell. NMR spectra were

recorded in acetone-d6 (δH 2.05 and δC 29.84) and DMSO-d6 (δH 2.50 and δC 39.52) on a

Bruker Avance 400-, 600-, and 800- MHz spectrometer. The D-HMBC NMR experiments

were practiced in Bruker Avance 800- MHz instrument using the standard Bruker TopSpin

pulse sequence code (hmqcgpgf) with the data point of 2048 x 540 and optimized for the

measurement of JCH at 1 Hz. High-resolution FABMS spectrometric data were obtained at

the National Center for Inter-University Research Facilities (Seoul, Korea) and acquired

using a JEOL JMS 700 mass spectrometer with meta-nitrobenzyl alcohol (NBA) as a matrix.

HPLC was performed on a SpectraSYSTEM p2000 equipped with a refractive index

detector (SpectraSYSTEM RI-150) and a UV-Vis detector (Gilson UV-Vis-151). All

solvents used were of spectroscopic grade or were distilled prior to use.

2. Animal Material

Specimens of Synoicum sp. (sample number 16K-30) were collected by hand with scuba

equipment at a depth of 25 m off the coast of Keomun-do, Korea, on September 29-30,

4

2016. The colonial tunicates are dark-red, up to 27 mm high and up to 32 mm in maximum

dimension, rounded cushion-shaped, with smooth surface, sessile and fixed by a small basal

part. Six to eight zooids form a circular system around a central colonial cavity. Zooids are

red, nearly 14 mm long. The thorax of zooid is especially large (0.5 mm wide and 3.8 mm

long). The posterior abdomen of zooid is about three times the length of thorax. Each

branchial siphon has 6 lobes and the atrial siphon protruding. Zooid has 16 to 18 segmental

rows, moderately short gut-loop and almost spherical stomach with smooth surface. These

morphological features were very similar to the previous specimen of Synoicum collected

at the nearby location,8 indicated the specimen to belong to the same genus but the lack of

gonads prevented adequate species identification. The voucher specimens were deposited

at the Natural History Museum, Ehwa Womans University with the sample number of

EWNHMAS390, under the curatorship of Su-Yuan Seo.

3. Extraction and Isolation

Freshly collected specimens were immediately frozen and stored at -25 °C until use.

Lyophilized specimens were macerated and repeatedly extracted with MeOH (3 × 2 L) and

CH2Cl2 (3 × 2 L). The combined extracts (119.6 g) were successively partitioned between

H2O (65.4 g) and n-BuOH (48.9 g); the latter layer was repartitioned between H2O-MeOH

(15:85, 30.9 g) and n-hexane (16.1 g). Then, the H2O-MeOH layer was separated by C18

reversed-phase flash chromatography using sequential mixtures of MeOH and H2O as the

eluents (six fractions in a H2O-MeOH gradient, from 50:50 to 0:100) followed by acetone

and finally EtOAc.

Based on the results of the TLC and 1H NMR analyses, the fractions that eluted with

40:60 H2O-MeOH (1.72 g), 30:70 H2O-MeOH (1.68 g), and 20:80 H2O-MeOH (2.96 g)

5

were chosen for separation. The 40:60 H2O-MeOH fraction was separated by semi-

preparative reversed-phase HPLC (YMC-ODS column, 10 × 250 mm; H2O-MeOH, 30:70;

2.0 mL/min), yielding compounds 5 (tR = 34.0 min) and 3 (tR = 54.0 min). The former

compound was further purified by analytical HPLC (YMC-ODS column, 4.6 × 250 mm;

H2O-MeCN, 58:42; 0.7 mL/min; tR = 29.1 min). The 30:70 H2O-MeOH fraction was also

separated by semi-preparative reversed-phase HPLC (H2O-MeCN, 50:50; 2.0 mL/min), to

afford compounds 4 (tR = 64.0 min) and 1 (tR = 107.2 min). Then the former was further

purified by analytical HPLC (H2O-MeOH, 27:73; 0.7 mL/min; tR = 28.3 min).

The 20:80 H2O-MeOH fraction was separated by semi-preparative reversed-phase HPLC

(H2O-MeCN, 47:53; 2.0 mL/min), yielding, in order of elution, compounds 2 (tR = 30.2

min), 7 (tR = 56.4 min), 8 (tR = 59.2 min), 9 (tR = 60.8 min), and 6 (tR = 99.4 min). Then 2

was further purified by analytical HPLC (H2O-MeOH, 36:64; tR = 47.4 min). Compounds

7-9 were also purified by analytical HPLC (H2O-MeOH, 30:70; tR = 24.8, 28.2 and 32.6

min, respectively). The purified metabolites were obtained in the following amounts: 40.3,

4.3, 3.2, 6.5, 9.1, 9.8, 1.8, 5.4, and 3.1 mg of 1-9, respectively.

6

Scheme 1. Isolation of compounds from Ascidian Synoicum sp.

7

Results

1. Compound 1

The molecular formula of isocadiolide A (1) was deduced to be C25H12Br6O7,

corresponding to 16 degrees of unsaturation, based on HRFABMS analysis. The 1H NMR

spectrum of this compound showed only three signals (H 7.98 (2H, s), 7.45 (4H, s) and 3.94

(3H, s) while 20 carbon signals were observed in the C 155.1-111.5 region in 13C NMR

data. These indicate the highly aromatic nature of this compound. The 13C NMR data of

this compound showed signals for three carbonyl carbons at C 199.9, 196.3 and 163.6.

These were thought to be two keto groups and an ester carbon due to the strong absorption

bands at 1700 and 1731 cm-1 in the IR data.

The structure determination of 1 was aided by a combination of 2-D NMR analyses such

as HSQC, HMBC and D-HMBC experiments suitable for obtaining carbon-proton

correlations due to the lack of proton-proton correlations. That is, direct and 2- and 3-bond

carbon-proton correlations readily defined three identical and symmetric 3,5-dibromo-4-

hydroxyphenyl moieties (rings A-C). The olefinic carbon at C 151.5 (C-2) was placed at

the benzylic position based on its HMBC correlation with H-2’(H-6’). Subsequently, this

carbon was connected to adjacent carbons at C 199.9 (C-1) and 143.9 (C-3) by the D-

HMBC interactions of these carbons with H-2’ (H-6’). Similarly, the presence of a

carbomethoxy group with signals at C 163.6 (C-6) and 53.6 (6-OCH3) as well as its

attachment at C-3 were confirmed by the long-range correlations of the methoxy protons

with C-2, C-3 and C-4.

8

The carbon and proton chemical shifts of two of the rings A-C were almost identical,

suggesting these groups were in very similar chemical environments.

This was confirmed by HMBC experiments, which showed that both phenyl moieties were

directly linked to the nonprotonated aliphatic carbon at C 61.7 (C-5) based on the 3-bond

correlations of this carbon with the ortho-protons (H-2’’ and H-2’’’). Further extension of

the carbon-proton correlations was accomplished by D-HMBC experiments,15 which

showed the direct attachment of the C-1 ketone carbonyl and another carbon at C 196.3

(C-4) to C-5. Finally, C-3 and C-4 were linked to construct a 1,4-pentenedione moiety by

the key long-range correlation of 6-OMe/C-4. Thus, isocadiolide A (1) was determined to

be a new cadiolide analog bearing an unprecedented cyclopentene-1,4-dione core.

2. Compound 2

The 1H NMR spectrum indicated a flavonoid structure with aromatic signals at δH 7.25 (2H,

dd, J = 5.9, 1.7 Hz), 7.23 (1H, d, J = 8.5 Hz), 6.78 (2H, d, J = 8.3

The molecular formula of isocadiolide B (2) was deduced to be C25H14Br4O7 by HRFABMS

analysis. The spectroscopic data of this compound were very similar to those of 1. A

preliminary examination of its 13C and 1H NMR data readily revealed the presence of a

pentenedione and three bromohydroxyphenyl moieties analogous to those in 1. The most

noticeable differences in these data were the substitution modes of the phenyl rings that are

indicated by the difference in their molecular formulas. This interpretation was confirmed

by combined 2-D NMR experiments in which two of the rings A-C of 1 were found to be

replaced with 3-bromo-4-hydroxyphenyl groups (Table 2). According to the HMBC data,

these groups were attached at C-2 (ring A) and C-5 (ring C).

9

Indeed, the distinct phenyl substituents of 2 caused the observed differences in the NMR

data of these moieties, confirming the attachment of two phenyl groups at C-5 in 1. The

differentiation of these substituents resulted in stereogenicity at this position, and the

absolute configuration was investigated by ECD calculations. The measured ECD curve of

this compound displayed a positive Cotton effect (ε +2.54) at 374 nm with the same sign

as the calculated ECD data of the 5S model (Figure 42). It would be also noteworthy that

the wide variation of the specific optical rotation of 2 in the range of -12 and +15 is possibly

attributed from the significant conformational effects.16 Thus, the structure of isocadiolide

B (2) was defined to be analogous to isocadiolide A (1) with distinct phenyl substituents at

C-5.

3. Compound 3

Isocadiolide C (3) was isolated as a pale-yellow amorphous solid with a formula of

C23H10Br6O5 based on HRFABMS analysis. Although the spectroscopic data of this

compound were very similar to those of 1, an examination of the 13C and 1H NMR data

revealed the absence of the C-6 carbomethoxy group in this compound. In addition, an

olefinic methine (C 142.3, H 7.99) replaced an unprotonated carbon of 1 (Table 3). These

differences were readily confirmed by combined 2-D NMR experiments in which the new

methine (C-3) exhibited long-range correlations with adjacent atoms: H-3/C-1, C-5 and C-

1’ and H-2’(H-6’)/C-3. Thus, isocadiolide C (3) was defined as the decarbomethoxy

derivative of isocadiolide A (1).

4. Compound 4

The molecular formula of isocadiolide D (4) was established as C25H13Br5O7 based on

HRFABMS analysis. The 13C and 1H NMR data of this compound were similar to those of

10

1 and 2, implying the same polyaromatic pentenedione core of an isocadiolide. The most

conspicuous difference in these data was the replacement of a bromine with a hydrogen on

one of the phenyl substituents, as

indicated by the mass data. The 2-D NMR data confirmed this interpretation, and the

difference in substituents was found at C-5’’’ (Table 4). As for 2, the configuration at C-5

of 4 was assigned as 5S based on the comparison of the measured ECD curve, which

showed a positive Cotton effect (Δε +3.13) at 367 nm, with the calculated curve (Figure

42). As found for 2, the specific optical rotation of 4 varied in the wide range of -40 and

+15 possibly attributed from the significant conformational effect.16 Thus, isocadiolide D

(4) was defined as a debromo-derivative of isocadiolide A (1) at one of the phenyl moieties.

5. Compound 5

The molecular formula of isocadiolide E (5) was determined to be C24H11Br5O7 based on

HRFABMS analysis. The spectroscopic data of this compound were very similar to those

of other isocadiolides. However, detailed examination of its 13C and 1H NMR data revealed

two conspicuous structural features: the absence of the methyl group at the C-6 ester and

the presence of an asymmetric phenyl moiety (Table 5). A combination of 2-D NMR

experiments confirmed this interpretation, and the C-2 phenyl substituent was identified as

a 3-bromo-4-hydroxyphenyl group (ring A), while the C-5 phenyl groups were found to be

two identical 3,5-dibromo-4-hydroxyphenyl groups (rings B and C). Overall, the structure

of isocadiolide E (5) was defined as a 1,4-pentenedione-carboxylic acid bearing three

phenyl moieties.

11

6. Compound 6

The molecular formula of isocadiolide F (6) was established as C27H18Br6O9, with 16

degrees of unsaturation, based on HRFABMS analysis. Although the presence of three

brominated hydroxyphenyl groups was clearly indicated, a detailed examination of the 13C

NMR data revealed a significant structural difference between the core of this compound

and the cores of other isocadiolides. That is, two ketone carbonyls (C-1 and C-4) and an

ester/acid carbon (C-6) that are commonly found in other isocadiolides were replaced by

two esters and an acetal/ketal carbon with signals at C 169.8, 163.3 and 114.9, respectively

(Table 6). The signals of the nonprotonated carbon (C-4) was also remarkably shifted to C

92.9. In addition, three methoxy groups were indicated by the signals as C 53.7, 52.7 and

51.7, and the corresponding singlet methyl protons were observed at H 3.96, 3.62 and 3.26

in the 1H NMR data (Table 6).

As for the other compounds, the structure of 6 was determined by a combination of 2-D

NMR experiments. The HSQC and HMBC data readily defined three identical 3,5-

dibromo-4-hydroxyphenyl moieties. A three-bond correlation to an aromatic proton at H

7.31 (H-2’/H-6’) placed the nonprotonated olefinic carbon at C 148.1 (C-2) at the benzylic

position. Then, aided by the D-HMBC data, the neighboring carbons were identified as

those at C 114.9 (C-1) and 133.0 (C-3) (Table 6). A carbomethoxy group (C-6, C 169.8;

6-OCH3, C 53.7, H 3.96) was also identified and attached to the latter carbon.

The carbon chemical shift of C-1 (C 114.9) was indicative of a ketal group. The

substituents on this carbon were a brominated hydroxyphenyl group (ring C) and a methoxy

(1-OCH3) group based on the HMBC data, and the D-HMBC data confirmed the

interactions of the methoxy and aromatic protons with the C-2 and C-3 carbons: H-2” (H-

6”)/C-2, 6-OCH3/C-2 and C-3. The attachments of the remaining phenyl group (ring B)

and carbomethoxy (C-5 and 5-OCH3) group to an oxygenate nonprotonated carbon (C 92.9

12

(C-4)) were confirmed based on the H-2’’ (H-6’’)/C-1’’ and C-5, 5-OCH3/C-4 and C-5

correlations. Although it was not directly indicated by the carbon-proton correlations, the

carbon chemical shifts at C 114.9 and 92.9 constructed an ether bridge between C-1 and

C-4. As a result, the nonprotonated C-3 and C-4 carbons must be directly linked to construct

a dihydrofuran moiety and form the final ring required by the mass data.

The dihydrofuran moiety of 6 possessed two stereogenic centers (at C-1 and C-4). The

NOESY data of this compound showed cross-peaks for 1-OCH3/H-2’’ (H-6’’) and 5-

OCH3/H-2’’’ (H-6’’’) assigning 1S* and 4S* relative configurations. ECD could not be

used to assign the absolute configuration as the measured and calculated curves showed

significant discrepancies (Figure 42). Overall, the structure of isocadiolide F (6) was

defined as a dihydrofuran-bearing tris-dibromo-hydroxyphenyl compound.

7. Compound 7

A structural analogue, isocadiolide G (7), was also isolated as a yellow, amorphous solid,

and it was found to have a formula of C27H19Br5O9 based on HRFABMS analysis. The

spectroscopic data of this compound were very similar to those of 6, with the replacement

of a bromine with a hydrogen as the most noticeable difference in the 13C and 1H NMR data

(Table 7). The resulting 3-bromo-4-hydroxyphenyl moiety was assigned to ring C based on

a combination of 2-D NMR data. The NOESY data also revealed the same 1-OCH3/H-2’’

(H-6’’) and 5-OCH3/H-2’’’ cross peaks as those seen for 6, defining 7 as a debrominated

derivative of 6. The ECD profile of this compound showed a noticeable positive Cotton

effect (Δε +3.14) at 280 nm, enabling the assignment of the 1S,4S absolute configuration

(Figure 42). Although they were not directly assigned, their structural similarity and

biogenetic considerations suggested dihydrofuran-bearing isocadiolides F (6) and G (7)

would have the same absolute configuration.

13

8. Compound 8

The molecular formula of isocadiolide H (8) was established as C27H19Br5O9 based on

HRFABMS analysis. The 13C and 1H NMR data of this compound were highly reminiscent

of those of other isocadiolides, in particular, the presence of three phenyl groups, two 3,5-

dibromo-4-hydroxyphenyls and one 3-bromo-4-hydroxyphenyl group, was suggested.

Similar to 6 and 7, the 13C NMR data of 8 showed two olefinic and three methoxy carbons

at C 146.9, 133.9, 53.0, 52.9, and 51.5, respectively (Table 8). However, the remaining

carbons were a ketone carbonyl, an ester carbonyl and two ketals/acetals at C 189.4, 165.1,

100.6, and 98.6, respectively, indicating this compound has unique cyclic moiety.

The connectivity of these nonprotonated carbons was determined by 2-D NMR

experiments. The HMBC cross-peaks of the aromatic protons at H 7.19 (H-2’/H-6’) placed

the olefinic carbon at C 133.9 (C-2) in the benzylic position. The connection of this carbon

to neighboring carbons at C 189.4 (C-1) and 146.9 (C-3), establishing an ,-unsaturated

ketone, was aided by the D-HMBC data. The presence of a carbomethoxy group (C-6 and

OCH3) as well as its attachment to C-3 was determined similarly. The nonprotonated carbon

at C 100.6 (C-5) was directly attached to both a methoxy group (1-OMe) and the 3-bromo-

4-hydroxyphenyl group (ring C) according to the HMBC data, and C-5 was attached to a

ketone carbonyl (C-1) based on the D-HMBC data.

14

Based on a similar interpretation, the substituents on the nonprotonated carbon at C 98.6

(C-4) and its linkage to C-3 of an unsaturated keto group were determined. Although it was

not directly indicated by 2-D NMR correlations, the remaining oxygen linked the C-4 and

C-5 carbons, making both of these carbons ketals. This interpretation was consistent with

their characteristic chemical shifts at C 98.6 and 100.6, respectively. Thus, a pyranone

moiety (C-1-C-5) was constructed to account for the additional ring required by the mass

data. Overall, isocadiolide H (8) was defined as a polybrominated tris-phenolic compound

bearing a pyranone core. The relative configurations at the C-4 and C-5 stereogenic centers

of 8 were assigned as 4R* and 5S* by the NOESY cross-peaks of 4-OCH3/5-OCH3 and H-

2’’ (H-6’’)/H-6’’’. However, the ECD-based assignment of the absolute configuration was

not possible due to the severe discrepancy between the measured and calculated ECD

profiles, similar to what was seen for 6.

9. Compound 9

The molecular formula of cadiolide N (9) was deduced to be C26H19Br5O7, corresponding

to 15 degrees of unsaturation, based on HRFABMS analysis. However, the 13C NMR data

of this compound showed far more signals than anticipated, mostly paired carbons. Similar

trends were also observed in the 1H NMR data, suggesting that 9 was isolated as a mixture

of either two structurally similar compounds or conformers: 9a and 9b (1:0.69 by

integration of the 1H NMR signals acquired in acetone-d6). Because further separation into

individual constituents was unsuccessful, the structure of 9 was analyzed as a mixture. The

13C NMR data of 9 showed signals of a carbonyl and an oxymethine carbon at C

170.1/170.0 and 66.0/65.1, respectively (Table 9).

15

Based on the chemical shifts and signal intensities, the carbon signals in the region of C

157.8-107.0 were tentatively attributed to three benzene rings, one double bond and one

ketal/acetal, accounting for 13 degrees of unsaturation. Therefore, compound 9 must have

an additional ring to satisfy the mass data. The remaining signals in the 13C NMR data were

from one methylene and two methyl carbons at C 42.7/42.5, 61.0/60.8 and 56.5/56.5,

respectively.

Compound 9 was determined to be a cadiolide by a combination of 2-D NMR analyses.

First, the 1H-1H and direct and long-range 13C-1H correlations readily defined three

aromatic moieties as one 3-bromo-4-methoxyphenyl group (ring C), one 3,5-dibromo-4-

hydroxyphenyl group (ring B) and one 3,5-dibromo-4-methoxyphenyl group (ring A)

(Table 9). The attachment of an oxymethine group (C-6: C 65.1, H 5.62 for 9a) to ring A

was indicated by the HMBC cross-peaks of H-2’(H-6’)/C-6 and H-6/C-1’. The further

connection of the oxymethine to an ,-unsaturated carbonyl group (C-1-C-3) was also

determined based on HMBC cross-peaks (H-6/C-1, C-2, and C-3) in conjunction with the

D-HMBC cross-peak of H-2’(H-6’)/C-2. The ring B was also attached to C-3 by the HMBC

data (H-2”(H-6”)/C-3).

A methylene group (C-5, C 42.7, H 3.23 and 3.11 for 9a) was attached at ring C based on

the HMBC cross-peaks of H2-5/C-1’’’ and C-2’’’(C-6’’’) and H-2’’’(H-6’’’)/C-5. Then,

the connectivity was extended to a deshielded oxygenated nonprotonated carbon (C-4, C

107.0) by the long-range correlations of H2-5/C-4 and H-2’’’(H-6’’’)/C-4. Subsequently,

this substructure was connected to the previously described unsaturated carbonyl group by

the crucial H2-5/C-3 correlation in the HMBC data.

The HRFABMS data of 9 required the presence of another ring. Because all of the 1H-13C

correlations in this compound were assigned based on the 2-D NMR data, the remaining

ring must contain an oxygen bridge. That is, among the oxygen-bearing carbons (C-1, C-4

(two) and C-6 carbons), two were connected by this bridge, while others bore hydroxy

groups. This problem was readily solved by acquiring the 1H NMR spectrum in DMSO-d6

as both H-6 and OH-6 protons showed substantial vicinal coupling constants: H H-6, 5.46;

16

OH-6, 6.40; JH-6,OH-6 = 5.5 Hz. Thus, a butenolide (C-1-C-4) was constructed, and hydroxy

groups were placed at C-4 and C-6. The resulting hemiketal functionality at C-4 was also

supported by the deshielded chemical shift of this carbon at C 107.0.

The combined 2-D NMR analyses also provided the structure of minor constituent 9b and

allowed confident assignment of all of its carbons and protons (Table 9). The very similar

chemical shifts throughout the entire molecule suggested these compounds were

conformers. This interpretation was confirmed by 1-D selective gradient NOE experiments

in which irradiation of the H-2’(H-6’) and 6-OH protons of 9a resulted in the simultaneous

irradiations of the same protons in 9b and vice versa (Figure 41).17 Furthermore, the ratios

of 9a/9b were remarkably changed by different NMR solvents. However, NOESY

experiments could not be used to elucidate the relative configurations at the C-4 and C-6

stereogenic centers because reliable cross-peaks were not present possibly due to the

significant distance between these groups. The MTPA method was also unsuccessful due

to the severe crowding around the 6-OH group.8 Consequently, this problem was

approached by calculating the ECD curves of all four possible absolute configurations. The

results indicated that regardless of the configuration at C-6, the calculated ECD profiles

possessing the 4R configuration were in good accordance with the measured curve (Figure

42). Then, the 6S configuration was assigned based on biogenic considerations and a

comparison of the NMR data with those of the congeners cadiolide G8 and cadiolide H9 in

the literature, which possess the same partial structure. Thus, cadiolide N (9) was

determined to be a new member of the cadiolide class bearing a -hydroxyfuranone moiety.

17

1

Table 1. 1H and 13C NMR assignment for compound 1 in acetone-d6

Position 1H (ppm, mult) 13C (ppm, type)

1 199.9, C 2 151.5, C 3 143.9, C 4 196.3, C 5 61.7, C 6 163.6, C 1’ 122.2, C

2’/6’ 7.98, s 135.3, CH 3’/5’ 111.5, C

4’ 155.1, C 1’ ’ 131.4, C

2’ ’/6’ ’ 7.45, s 133.2, CH 3’ ’/5’ ’ 111.8, C

4’ ’ 152.0, C 1’ ’ ’ 131.4, C

2’ ’ ’/6’ ’ ’ 7.45, s 133.2, CH 3’ ’ ’/5’ ’ ’ 111.8, C

4’ ’ ’ 152.0, C 6-OMe 3.94, s 53.6, CH3

18

2



1 200.9, C 2 151.8, C 3 143.1, C 4 196.8, C 5 62.5, C 6 164.2, C 1’ 120.8, C 2’ 8.06, d (2.2) 136.3, CH 3’ 111.7, C 4’ 159.0, C 5’ 7.22, d (8.6) 117.6, CH 6’ 7.64, dd (8.6, 2.2) 132.2, CH 1’ ’ 132.0, C

2’ ’/6’ ’ 7.42, s 133.3, CH 3’ ’/5’ ’ 111.7, C

4’ ’ 151.9, C 1’ ’ ’ 129.9, C 2’ ’ ’ 7.39, d (2.3) 133.6, CH 3’ ’ ’ 110.9, C 4’ ’ ’ 155.4, C 5’ ’ ’ 7.06, d (8.6) 117.5, CH 6’ ’ ’ 7.12, dd (8.6, 2.3) 129.6, CH

6-OMe 3.93, s 53.5, CH3

19

3



1 201.7, C 2 154.2, C 3 7.99, s 142.3, CH 4 199.9, C 5 62.6, C 1’ 124.0, C

2’/6’ 8.42, s 134.8, CH 3’/5’ 111.8, C

4’ 155.0, C 1’ ’ 132.4, C

2’ ’/6’ ’ 7.43, s 133.2, CH 3’ ’/5’ ’ 111.7, C

4’ ’ 151.7, C 1’ ’ ’ 132.4, C

2’ ’ ’/6’ ’ ’ 7.43, s 133.2, CH 3’ ’ ’/5’ ’ ’ 111.7, C

4’ ’ ’ 151.7, C

20

4



1 200.5, C 2 151.1, C 3 143.5, C 4 196.6 ,C 5 62.4, C 6 163.8, C 1’ 121.8, C

2’/6’ 7.99, s 135.3, CH 3’/5’ 111.7, C

4’ 155.5, C 1’ ’ 132.0, C

2’ ’/6’ ’ 7.42, s 133.4, CH 3’ ’/5’ ’ 111.5, C

4’ ’ 151.6, C 1’ ’ ’ 129.9, C 2’ ’ ’ 7.40, d (2.3) 133.6, CH 3’ ’ ’ 110.7, C 4’ ’ ’ 155.3, C 5’ ’ ’ 7.07, d (8.6) 117.5, CH 6’ ’ ’ 7.13, dd (8.6, 2.3) 129.7, CH

6-OMe 3.93, s 53.6, CH3

21

5



1 200.6, C 2 151.0, C 3 144.7, C 4 196.8, C 5 61.7, C 6 164.3, C 1’ 121.4, C 2’ 8.14, d (2.1) 136.3, CH 3’ 110.7, C 4’ 158.4, C 5’ 7.18, d (8.6) 117.4, CH 6’ 7.77, dd (8.6, 2.1) 132.4, CH 1’ ’ 131.7, C

2’ ’ /6’ ’ 7.46, s 133.2, CH 3’ ’ /5’ ’ 111.7, C

4’ ’ 151.9, C 1’ ’ ’ 131.7, C

2’ ’ ’/6’ ’ ’ 7.46, s 133.2, CH 3’ ’ ’/5’ ’ ’ 111.7, C

4’ ’ ’ 151.9, C

22

6



1 114.9, C 2 148.1, C 3 133.0, C 4 92.9, C 5 163.3, C 6 169.8, C 1’ 125.6, C

2’/6’ 7.31, s 133.6, CH 3’/5’ 110.8, C

4’ 152.4, C 1’ ’ 133.6, C

2’ ’/6’ ’ 7.78, s 132.7, CH 3’ ’/5’ ’ 110.8, C

4’ ’ 151.8, C 1’ ’ ’ 131.9, C

2’ ’ ’/6’ ’ ’ 7.54, s 132.8, CH 3’ ’ ’/5’ ’ ’ 111.2, C

4’ ’ ’ 152.3, C 1-OMe 3.26, s 51.7, CH3 5-OMe 3.62, s 52.7, CH3 6-OMe 3.96, s 53.7, CH3

23

7



1 115.6, C 2 148.7, C 3 133.6, C 4 92.8, C 5 163.4, C 6 169.9, C 1’ 126.0, C

2’/6’ 7.28, s 133.6, CH 3’/5’ 110.6, C

4’ 152.2, C 1’ ’ 133.9, C

2’ ’/6’ ’ 7.80, s 132.8, CH 3’ ’/5’ ’ 110.7, C

4’ ’ 151.7, C 1’ ’ ’ 130.0, C 2’ ’ ’ 7.58, d (2.3) 133.7, CH 3’ ’ ’ 110.1, C 4’ ’ ’ 155.8, C 5’ ’ ’ 7.00, d (8.5) 116.8, CH 6’ ’ ’ 7.10, dd (8.5, 2.3) 129.1, CH

1-OMe 3.25, s 51.4, CH3 5-OMe 3.59, s 52.5, CH3 6-OMe 3.95, s 53.6, CH3

24

8



1 189.4, C 2 133.9, C 3 146.9, C 4 98.6, C 5 100.6, C 6 165.1, C 1’ 127.2, C

2’/6’ 7.19, s 133.6, CH 3’/5’ 111.3, C

4’ 152.7, C 1’ ’ 132.3, C

2’ ’/6’ ’ 7.80, s 132.6, CH 3’ ’/5’ ’ 111.5, C

4’ ’ 152.6, C 1’ ’ ’ 128.4, C 2’ ’ ’ 7.76, d (2.2) 133.9, CH 3’ ’ ’ 110.6, C 4’ ’ ’ 156.3, C 5’ ’ ’ 7.16, d (8.5) 117.4, CH 6’ ’ ’ 7.48, d (8.5, 2.2) 129.9, CH

4-OMe 3.51, s 53.0, CH3 5-OMe 3.35, s 51.5, CH3 6-OMe 3.48, s 52.9, CH3

25

9

Table 9. 1H and 13C NMR assignment for compound 9 (9aa/9ba) in acetone-d6


1 170.0 / 170.1 , C 2 140.9 / 142.0, C 3 157.8 / 156.9, C 4 107.0 / 107.4, C 5 3.11, d (14.3) / 3.06, d (14.3) 42.7 / 42.5, CH2 3.23, d (14.3) / 3.22, d (14.3)

6 5.62, brs / 5.65, brs 65.1 / 66.0, CH 1’ 132.0 / 132.2, C

2’/6’ 7.45, s / 7.47, s 131.5 / 130.7, CH 3’/5’ 118.1 / 118.3, C

4’ 153.6 / 153.7, C 1’ ’ 125.3 / 125.8, C

2’ ’/6’ ’ 7.71, s / 7.72, s 134.2 / 134.4, CH 3’ ’/5’ ’ 111.0 / 111.1, C

4’ ’ 153.0 / 153.0, C 1’ ’ ’ 128.3 / 128.2, C 2’ ’ ’ 7.14, d (2.1) / 7.03, d (2.1) 135.9 / 135.7, CH 3’ ’ ’ 110.0 / 110.0, C 4’ ’ ’ 153.6 / 153.7, C 5’ ’ ’ 6.90, d (8.4) / 6.88, d (8.2) 112.5 / 112.4, C 6’ ’ ’ 7.06, dd (8.4, 2.1) / 7.03, dd (8.2, 2.1) 131.7 / 131.5, CH

4’-OMe 3.76, s / 3.87, s 60.8 / 61.0, CH3 4’ ’ ’-OMe 3.85, s / 3.86, s 56.5 / 56.5, CH3

a9a and 9b are conformers.

26

Discussion

The previous chemical investigation of Korean Synoicum sp. Ascidian has afforded

diverse secondary metabolites possess tris-brominated aromatic moieties in common,

however, the core carbon skeletons were highly diverse and unprecedented

(cyclopentenedione (1-5), dihydrofuran (6 and 7), pyranone (8), and furanone (9)).

Cadiolides are well-known family of ascidian metabolite possessing brominated aromatic

moieties. Cadiolides have been reported about their potent antibacterial activity against

several Gram (+), Gram (-) and MRSA strains. Nine new compounds were isolated by

chromatographic methods. The planar and stereochemical structures were determined

based on extensive spectroscopic analyses.

Especially, diverse NMR techniques were attempted to determine the planar and

stereochemical structures. Epimers and conformers issue was readily solved by 1D

Selective Gradient NOESY experiment and 2D decoupled-HMBC experiment made it

possible to clarify

Finally, the absolute configurations of compounds were determined by computational

methods (comparison of measured ECD graphs and calculated ECD plots).

Antibacterial activity of these compounds was generally weaker than reported cadiolides,

however, inhibition against SrtA and isocitrate lyase enzymes were comparable to positive

control.

27

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16. K. B. Wiberg, P. H. Vaccaro, J. R. Cheeseman, J. Am. Chem. Soc. 2003, 125, 1888-1896.

17. D. X. Hu, P. Grice, S. V. Ley, J. Org. Chem. 2012, 77, 5198-5202.

28

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R. Acharya, FEBS J. 2015, 282, 2097-2114. (e) A. W. Jacobitz, M. D. Kattke, J.

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29

Figure 1. [ The 1H (800 MHz) and 13C NMR (200 MHz) acetone-d6 ] NMR spectrum of 1

30

Figure 2. The HSQC (800 MHz, acetone-d6) spectrum of 1

31

Figure 3. The HMBC (800 MHz, acetone-d6) spectrum of 1

32

Figure 4. The D-HMBC (800 MHz, acetone-d6, JCH = 1 Hz) spectrum of 1

33

Figure 5. [The 1H (800 MHz) and 13C NMR (200 MHz) acetone-d6] NMR spectrum of 2

34


35


36


37


38


39


40


41


42


43


44


45


46


47


48


49


50


51


52


53

Figure 25. The NOESY (800 MHz, acetone-d6) spectrum of 6

54


55


56


57

Figure 29. The NOESY spectrum (800 MHz, acetone-d6) spectrum of 7

58


59


60


61


62

Figure 34. The NOESY spectrum (800 MHz, acetone-d6) spectrum of 8

63


64


65


66


67

Figure 39. The 1H NMR (800 MHz, DMSO-d6) spectrum of 9

68

Figure 40. The NOESY (800 MHz, DMSO-d6) spectrum of 9

69

Figure 41. The 1-D Selective gradient NOESY (800 MHz, DMSO-d6) spectrum of 9

(Irradiation at δH a) 7.55 (H-2'/6' of 9a), b) 7.64 (H-2'/6' of 9b),

c) 6.10 (6-OH of 9b), and d) 6.32 (6-OH of 9a)

a)

b)

c)

d)

70

Figure 42. Measured and calculated ECD profiles of compounds 2, 4, and 6-9

Wavelength (nm)

300 400 500 600

M-1cm

-1)

-6

-4

-2

0

2

4

6

expt. for 4

calc. for 4 (5R)

calc. for 4 (5S)

Wavelength (nm)

300 400 500 600

M-1cm

-1)

-6

-4

-2

0

2

4

6

expt. for 2

calc. for 2 (5R)

calc. for 2 (5S)

Wavelength (nm)

240 260 280 300 320 340 360

(M-1cm

-1)

-6

-4

-2

0

2

4

6

expt. for 7

calc. for 7 (1R, 4R)

calc. for 7 (1S, 4S)

Wavelength (nm)

240 260 280 300 320 340 360

(M-1cm

-1)

-6

-4

-2

0

2

4

6

expt. for 6



Wavelength (nm)

250 300 350 400 450

M-1cm

-1)

-6

-4

-2

0

2

4

6

expt. for 8

calc. for 8 (4R, 5S)

calc. for 8 (4S, 5R)

Wavelength (nm)

250 300 350 400

M-1cm

-1)

-6

-4

-2

0

2

4

6

expt. for 9



calc. for 9 (4R, 6S)

calc. for 9 (4S, 6R)

71

국문초록

시노이컴 속 군체멍게에서 분리한

다브롬화 방향족 화합물의 구조결정

서울대학교 대학원

약학과 천연물과학전공

배 종 균

한국 거문도 연안에서 채집한 시노이컴 속 군체멍게로부터 9 종의 신규 다브롬화

방향족 화합물인 isocadiolide A-H (1-8), cadiolide N (9)을 분리하였다.

고해상도 질량분석, 핵자기공명분석, 적외선 분광법, 자외선-가시광선 분광법 등

각종 분광학적인 분석을 통해 이 화합물들은 공통적으로 tris-

bromohydroxyphenyl 부분구조를 가지고 있음을 확인하였다.

이는 선행 연구되었던 cadiolide 계열의 구조적 특징과 유사하였으나, 그 중심 탄소

골격은 cyclopentenedione (1-5), dihydrofuran (6 and 7), pyranone (8),

furanone (9)으로 다양한 산화상태와 재배열을 하고 있는 것으로 확인하였다.

72

이 화합물들 중 일부는 약한 항박테리아 생리활성을 나타내었고 sortase A 와

isocitrate lyase 효소에 중간 정도의 저해능력을 보였다.

주요어 : 멍게 대사물질, 다브롬화 방향족 화합물, sortase A 저해

학 번 : 2018 – 24173

Introduction Experimental Section 1. General Experimental Procedures 2. Animal Material 3. Extraction and Isolation

Results 1. Compound 1 2. Compound 2 3. Compound 3 4. Compound 4 5. Compound 5 6. Compound 6 7. Compound 7 8. Compound 8 9. Compound 9

Discussions References Abstract in Korean

10Introduction 1Experimental Section 3 1. General Experimental Procedures 3 2. Animal Material 3 3. Extraction and Isolation 4Results 7 1. Compound 1 7 2. Compound 2 8 3. Compound 3 9 4. Compound 4 9 5. Compound 5 10 6. Compound 6 11 7. Compound 7 12 8. Compound 8 13 9. Compound 9 14Discussions 26References 27Abstract in Korean 71

disclaimer - seoul national university...despite the biosynthetic condensations of α-keto acids...

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