Integrating Molecular Networking and 1H NMR To Target the Isolation

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Article 1

Integrating Molecular Networking and H NMR to Target the Isolation of Chrysogeamides from a Library of Marine-Derived Penicillium Fungi Xue-Mei Hou, Yue-Ying Li, Yun-Wei Shi, Yao-Wei Fang, Rong Chao, Yu-Cheng Gu, Chang-Yun Wang, and Chang-Lun Shao J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02614 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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The Journal of Organic Chemistry

Integrating Molecular Networking and

1H

NMR to Target the Isolation of

Chrysogeamides from a Library of Marine-Derived Penicillium Fungi

Xue-Mei Hou,†,‡ Yue-Ying Li,† Yun-Wei Shi,§ Yao-Wei Fang,† Rong Chao,†,‡ Yu-Cheng Gu,┴ Chang-Yun Wang, †,‡* Chang-Lun Shao †,‡* †

Key Laboratory of Marine Drugs, The Ministry of Education of China, School of

Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Laboratory

for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine

Science and Technology, Qingdao 266200, People’s Republic of China §

Co-innovation Center of Neuroregeneration, Key Laboratory of Neuroregeneration of

Jiangsu and Ministry of Education, Nantong University, Nantong 226001, People’s Republic of China ┴

Syngenta Jealott’s Hill International Research Centre, Bracknell, Berkshire, RG42 6EY,

United Kingdom

* Corresponding Author: Chang-Lun Shao, e-mail: [email protected], tel.: 86-532-82031381; Chang-Yun Wang, e-mail: [email protected], tel.: 86-532-82031536.

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The Journal of Organic Chemistry 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

Abstract A challenging problem in natural product discovery is to rapidly dereplicate known compounds and expose novel ones from complicated components. Herein, integrating the LC-MS/MS-dependent molecular networking and 1H NMR techniques efficiently and successfully enabled the targeted identification of seven new cyclohexadepsipeptides, chrysogeamides A–G (1–7), from the coral-derived fungus Penicillium chrysogenum (CHNSCLM-0003) which was targeted from a library of marine-derived Penicillium fungi. Compound 4 features a rare 3-hydroxy-4-methylhexanoic acid (HMHA) moiety which was firstly discovered from marine-derived organisms. Interestingly, isotope labelling feeding experiments confirmed that 13C1-L-Leu was transformed into 13C1-D-Leu moiety indicating that D-Leu could be isomerized from L-Leu. Compounds 1 and 2 obviously promoted angiogenesis in zebrafish at 1.0 μg/mL, with non-toxic to embryonic zebrafish at 100 μg/mL. Combining molecular networking with 1H NMR as a discovery tool will be implemented as a systematic strategy, not only for known compounds dereplication but also for untapped reservoir discovery.


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Introduction Marine-derived fungi are increasingly major focus of natural products (NPs) research efforts and rich sources of novel and bioactive compounds.1 Among the diversity microbial phyla in marine ecosystems, the genus Penicillium is represented by more than two hundred species in the marine environments. It should be emphasized that there are still many unidentified Penicillium species, namely Penicillium sp. Over the last 40 years, more than 660 new compounds have been isolated from marine-derived Penicillium.2 These compounds accounted for 14% of the natural products from marine-derived fungi. Particularly, the natural product ligerin displayed tremendous value of anticancer drug development.3 Interestingly, its semisynthetic products CKD-732 and PPI-2458 have been entered Phase I clinical trials for the treatment of solid tumors.4,5 Obviously, the genus Penicillium represents huge potential for new and interesting bioactive compounds. Natural product researchers face the challenge of maximizing the discovery of new compounds while minimizing the reevaluation of known compounds. Therefore, dereplication becomes critical to avoid re-isolation of known molecules. A recent advance in the field of NP research with broad implications to metabolite dereplication, based on the greatly increased availability of LC-MS/MS instrumentation in NP research laboratories, has been the MS/MS-based Molecular Networks algorithm.6-11 This platform associates MS/MS spectra based on similar mass fragmentation patterns with the underlying concept that structurally related molecules will fragment in similar ways to give analogous patterns. In addition, new molecules that are related to known substances in the database can rapidly be assigned to specific structural families, thereby accelerating the discovery and characterization process.12 The nuclear magnetic resonance (NMR) spectroscopy is valued for its ability to shed light on molecular structure. In recent 20 years, this powerful method has been widely used in drug discovery.13,14 Especially, proton-detected NMR experiments on submicrogram amounts from complex mixtures are indubitable in identifying structural features. The combination of the cheminformatics strategy of MS/MS-dependent molecular networking with highly sensitive 1H NMR could shed light on structural relationships and expedite dereplication of compounds in crude extracts to improve the efficiency of discovering new 3 ACS Paragon Plus Environment


compounds. To explore the chemical diversity of bioactive metabolites in the marine derived Penicillium species, 270 Penicillium strains using HPLC-MS/MS, molecular networking, and the GNPS platform were screened (the 270 Penicillium species were selected from our microbial library, Table S1). A comprehensive network was generated according to MS/MS spectra and visualized in Cytoscape. Structurally related molecules (nodes) connected by edges is termed a ‘molecular family’. Nearly 341 unannotated molecular families from these 270 samples suggest that the Penicillium contain a significant number of natural products that remain explored. Investigating the molecular families with dense nodes, several known compounds were identified, such as the blennolides,15,16 quinolinone alkaloids,17,18 cyclopeptides,19 and cyclodepsipeptides20 (Figure S1). Analyzing the cluster with the densest nodes, many putative new cyclodepsipeptides were identified. After searching for the networks of each Penicillium strains, the cyclodepsipeptides-cluster was specially targeted as metabolites by Penicillium chrysogenum CHNSCLM-0003. Combining visual molecular networking and 1H NMR, a series of novel cyclohexadepsipeptides, chrysogeamides A–G (1–7), nodupetide (8)21 and scopularides A and B (9, 10),20 were isolated (Figure 1). Herein, we report the discovery, isolation, structure elucidation, and biological evaluation of these chrysogeamides. R

O (1)

O O

O

O O

H N

HN

NH O

NH H N

O

R

O NH

HN

HN

Chrysogeamide C (3) R = (CH2)4CH3 Chrysogeamide D (4) R = CH3 Chrysogeamide E (5) R = (CH2)2CH3

O O

O

(2)

O

Chrysogeamide A (1) R = (CH2)2CH3 Chrysogeamide B (2) R = (CH2)4CH3 Nodupetide (8) R = CH3

O

H N

O NH

NH

O

O

R

R

O

NH

NH NH

O

O O

O

O

NH O

O Chrysogeamide F (6) R = (CH2)2CH3 Chrysogeamide G (7) R = (CH2)4CH3

NH

NH

N

H N

O

HN

O

O Scopularide A (9) R = (CH2)4CH3 Scopularide B (10) R = (CH2)2CH3


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Figure 1. Chemical structures of the isolated cyclohexadepsipeptides. (1) chemical structures of 1–10, (2) X-ray crystallographic analysis of 9.

Results and discussion Identification of chrysogeamides by molecular networking and 1H NMR. The fungus P. chrysogenum (CHNSCLM-0003) was cultured on rice-potato solid medium and extracted with EA, EA/CH2Cl2, and acetone successively to afford three organic extracts. These three organic extracts were subjected to untargeted HPLC-MS/MS analysis, respectively. Then a visualized molecular networking was constructed with the converted MS/MS data. Several families of molecules were easily visualized from the full network (Figure 2). Clearly, a concentrated cluster with more than 80 nodes revealed regular MS/MS patterns which showed neutral loss of amino acid fragments indicating a peptide family (Figure S2). The ions at m/z 596.2 (1), and m/z 624.2 (2), m/z 568.2 (8) indicated three putative analogues for their molecular weights differ by 28Da. Additionally, the MS/MS spectra analysis of 1, 2, and 8 yielded the same [Val–Ala–Leu] amino acid sequence tag. In addition, ions at m/z 686.3 (3), m/z 630.2 (4), and m/z 658.3 (5) revealed the homologs of 3–5, and the ions at m/z 642.2 (6) and m/z 670.3 (7) indicated plausible analogues, so as the ions at m/z 672.3 (9) and m/z 644.6 (10). Careful analysis the MS/MS spectra of 3–7, 9 and 10 revealed the same [Phe–Ala–Leu] tag. The cluster, containing these 10 nodes, was interesting because of the presence of these analogues, and formed a peptide subfamily (Figure 2).



Figure 2. Cyclohexadepsipeptide-cluster from the molecular networking in P. chrysogenum CHNSCLM0003. (1) Molecular networking of all organic extracts from 270 Penicillium fungi, (2) molecular networking of the organic extract from P. chrysogenum CHNSCLM-0003, (3) cluster corresponding to compounds of the chrysogeamide family observed in the molecular networking, (4) MS/MS spectrum of the node (9), (5) ESI MS/MS fragment ions of 9. 1H

NMR analysis of the total organic extracts showed the characteristic signals of

peptides, including exchangeable protons (NH) at δH 9.00−7.50, and α-protons belonging to amino acids at δH 4.50−3.50 (Figure S3). Further separation and purification of the organic extracts under the guidance of 1H NMR led to the obtaining of three sub-fractions containing peptides (Fr. M-2-3, Fr. M-2-4-1, and Fr. M-2-4-2). UPLC-MS analysis of these three sub-fractions showed the peaks with ten molecular weights at: Fr. M-2-3) m/z 568.2, m/z 596.2, m/z 624.2, Fr. M-2-4-1) m/z 644.6, m/z 672.3, Fr. M-2-4-2) m/z 642.2, m/z 670.3, m/z 630.2, m/z 658.3, and m/z 686.3. The molecular weights detected from the UPLC-MS were in accordance with that of molecular networking, indicating that the cyclohexadepsipeptides existed in these three sub-fractions. Combined molecular networking and MS/MS fragmentation pattern strategies with NMR were used to discover and identify these molecules. Purification of these sub-fractions led to the isolation of ten cyclohexadepsipeptides (1–10). Scopularides A (9) and B (10),20 were elucidated as cyclo-[(CO)L-Phe–L-Ala–DLeu–L-Val–Gly–HMDA/HMOA(O)] on the basis of NMR, ESI-MS/MS and Marfey’s methods. The MS/MS fragmentation pathway of 9 and 10 was characteristic as the first breakage at the ester bond and then sequentially neutral loss of phenylalanine (Phe), alanine (Ala), and leucine (Leu) (Figure 2, Figures S33 and S34). Additional putative analogues with mass differences attributed to varying fatty acid chains with the MS shifts of 28 or 42 Da. Structure elucidation of chrysogeamides A−G (1−7). Chrysogeamide A (1) was isolated as a white microcrystalline powder. HR-ESI-MS of 1 gave a [M + H]+ at m/z 596.4031 indicating a molecular formula of C30H53N5O7 with 7 degrees of unsaturation. The 1H NMR (C5D5N) spectrum (Table 1) showed the characteristic of a peptidic structure, particularly with the signals of α-protons at δH 4.94–3.95, and amide (NH) proton signals at δH 9.72–8.37. The 13C NMR spectrum gave 30 carbons including six ester or amide-type 6 ACS Paragon Plus Environment

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carbonyls (δC 173.3−170.3), one oxygenated sp3-hybridized carbon (δC 77.3), five nitrogenated sp3-type carbons (δC 59.8−43.8), and other sp3-type carbon signals. However, six α-protons at δH 4.94–3.95 and 21 methyl protons at δH 1.00–0.50 were significantly overlapped which greatly impeded the assembly of the planar structure of 1. Nevertheless, different deuterated reagents, including CD3OD, DMSO-d6, and pridine-d5 were used for improving the dispersion between these NMR signals (Table 1, Table S2). Careful comparison the 1H NMR data of 1 with those of nodupetide (8) showed close structure between these two compounds. The most obvious differences were the presence of two more methylene groups (δH 1.28, δC 29.5, 27-CH2; δH 1.23, δC 23.1, 28-CH2) in 1 than 8. Detailed analysis of the 1D and 2D NMR spectra revealed that 1 showed same amino acid residues as 8, including one glycine (Gly), one alanine (Ala), one leucine (Leu), and two valines (Val) (Figure 3). The 2D NMR data revealed those unaccounted 9 carbon signals forming a 3-hydroxy-4-methyloctanoic acid (HMOA) group. These signals accounted for six out of seven degrees of unsaturation, indicating the final degree of unsaturation arising from the cyclic nature of 1. HMBC correlations from the amide (NH) protons to their adjacent carbonyls were used to connect the residues in 1 as [(CO)Val2–Ala–Leu–Val1– Gly–HMOA(O)]. Additionally, the chemical shift of the proton in the β-position of the HMOA residue (δH 5.43) suggested that the oxygen bound at C-24 should be a part of an ester group between HMOA and Val2 residues. However, in the HMBC experiment, no obvious correlation was observed between H-24 to C-17 due to the present of oxygen atom. Finally, a ROESY correlation between H-24 (δH 5.43) and H-18 (δH 4.84) was detected to conclude the cyclic structure of 1. Additionally, the key ESI-MS/MS fragmentation revealed the peptide fragment CO-Val2−Ala−Leu-NH. Finally, the cyclic planar structure of 1 was established as cyclo-[(CO)Val2–Ala–Leu–Val1–Gly–HMOA(O)].



(1) 30

O

Val2 20

O Ala

27 24

17

19

O

22

NH

14

(2)

HMOA O

NH 8

2

O Leu

H N

HN

29

O 497.3 [M + H]+ O 479.3 [M + H - H2O]+

Gly O

1

NH

NH NH

6

3

O O

4

O

10

Initial Fragmentation

NH

15

16

(3)

25

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O

Val1

H N

HN

O

O

13 1

H-1H COSY

HMBC

+

408.3 [M + H - H2O]

ROESY

295.2 [M + H - H2O]+

Figure 3. 2D NMR correlations and ESI-MS/MS fragment ions of 1. (1) and (2): the 1H-1H COSY, key HMBC and ROESY correlations for 1; (3): ESI-MS/MS fragment ions of 1.

The absolute configurations of the amino acid residues were established by HPLCDAD and UPLC-MS analysis of the acid hydrolysate derivatized with Marfey’s reagent (Nα-(2,4-dinitro-5-fluorophenyl)-L-alalinamide, L-FDAA).22 The retention times of the acid hydrolysate of 1 derivatized with Marfey’s reagent on HPLC were 20.6, 26.5, 51.5, and 95.7 min, respectively (Figure S40). Additionally, UPLC-MS analysis of the acid hydrolysate of 1 derivatized with Marfey’s reagent displayed retention times and negative ions at 5.16 min (m/z 326 [M − H]−, 653 [2M − H]−), 6.21 min (m/z 340 [M − H]−), 9.26 min (m/z 368 [M − H]−), and 14.22 min (m/z 765 [2M − H]−) (Figure S41). Comparing these retention times with that of standards indicated Gly, L-Ala, L-Val, and D-Leu residues in 1. Thus, the chemical structure of chrysogeamide A (1) was fully elucidated as cyclo[(CO)L-Val2–L-Ala–D-Leu–L-Val1–Gly–HMOA(O)] (Figure 1). Chrysogeamide B (2) was also a white powder, with a molecular formula of C32H57N5O7 from HR-ESI-MS at m/z 624.4340 [M + H]+ (calcd for C32H58N5O7, 624.4331). Compound 2 displayed similar NMR spectra with 1 (Table S2), showing two more methylene groups than 1. Careful analysis of the NMR data suggested a 3-hydroxy4-methyldecanoic acid (HMDA) unit in place of the HMOA residue in 1. The HMBC correlations, ESI-MS/MS and Marfey’s methods elucidated 2 as cyclo-[(CO)L-Val–L-Ala– D-Leu–L-Val–Gly–HMDA(O)]

(Figures 1, S4, S5, S40, and S41).

Table 1. NMR data (in pridine-d5) for chrysogeamide A (1) Residue Gly

No. 1

δC, type 170.3, C

δH (J in Hz)

1H-1H

COSY


HMBC

ROESY

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2a 2b Val1

Leu

Ala

Val2

43.8, CH2

Gly-NH

9.09, t (4.6)

2a/b

1 22 3, 6, 7

NH 3 4

173.3, C 59.8, CH

4.79, m

Val1-NH, 5

5

30.4, CH

2.48, m

4, 6, 7

6 7

19.4, CH3 19.7, CH3

1.13, d (7.0) 1.14, d (7.0)

5 5

9.02, d (8.6)

4 Leu-NH, 10

1

4, 23a

2a, 6, 7 4 4

NH 8 9 10a 10b 11

172.8, C 53.3, CH 39.8, CH2 25.2,CH

4.82, m 1.94, m 1.78, m 1.90, m

12

22.9, CH3

0.87, d (6.1)

11

9

13

21.7, CH3

0.84, d (6.1)

11

9

9.67, d (6.4)

9

9

1 12, 13 8, 12, 13

12, 13

NH 14 15

173.2, C 49.6, CH

4.92, m

Ala-NH, 16

16

18.3, CH3

1.67, d (7.1)

15

9.31, d (7.7)

15

3 18 14

NH 17

172.5, C

18

58.6, CH

4.84, d (6.8)

Val2-NH, 19

19

31.3, CH

2.19, dq (13.5, 6.8)

18, 20, 21

20

18.3, CH3

1.09, d (6.8)

19

18

21

19.3, CH3

1.05, d (6.8)

19

18

8.44, d (7.9)

18

14

24

22

NH HMOA

4.77, dd (16.9, 6.6) 4.00, dd (16.9, 4.2)

22 23a 23b 24

171.7, C 39.9, CH2

25

37.8, CH 32.4, CH2

1.82, m

24, 30

1.52, m 1.08, m

27

27

29.5, CH2

1.28, m

26

28 29 30

23.1, CH2 14.2, CH3 14.6, CH3

1.23, m 0.83, t (6.7) 0.90, d (6.8)

29 28 25

26a 26b

77.7, CH

2.72, m 2.63, m 5.43, m

15, 20, 21, 24 17

23, 25

2a 18, 30

27 24, 26

24

Chrysogeamide C (3) was isolated as a white powder with a molecular formula of C37H59N5O7. The 1H NMR (pridine-d5) (Figure S22) spectrum showed five NH at 9.72, 9.19, 9.05, 8.87, and 8.82 ppm, a mono-substituted benzene at δH 7.38 (2H), 7.27 (2H), and 9 ACS Paragon Plus Environment


7.25 (1H), and an ester bond between amino acid and hydroxy acid at δH 5.51 (1H). The similar 1H NMR data of 3 with 9 indicated that 3 was also a cyclohexadepsipeptide. The delicate difference of the 1H NMR spectrum between 3 and 9 was additional methylene proton signals [δH 1.98 (1H) and 1.79 (1H)] in 3. It is indicated that a Val residue in 9 was replaced by a Leu in 3. Detailed analyzing the 1H NMR data revealed a Gly, two Leu, an Ala, a Phe, and a HMDA. These component amino acid residues were also confirmed by Marfey’s method followed by UPLC-MS analysis, with the retention times of the acid hydrolysate of 3 derivatized with Marfey’s reagent were 5.25 min (m/z 326 [M − H]−, Gly), 6.02 min (m/z 340 [M − H]−, L-Ala), 11.52 min (m/z 416 [M − H]−, L-Phe), and 14.30 min (m/z 765 [2M − H]−,

D-Leu)

(Figure S42). Additional ESI-MS/MS fragment ions

corresponding to the neutral losses of [Phe], [Phe−Ala], and [Phe−Ala−Leu] were also observed (Figure S27). So, the chemical structure of 3 was identified as cyclo-[(CO)L-Phe– L-Ala–D-Leu–D-Leu–Gly–HMDA(O)]

(Figure 1).

Chrysogeamides D (4) and F (6) were isolated as a mixture with a trace quantity indicating the molecular formulas of C33H51N5O7 and C34H51N5O7 by HR-ESI-MS, respectively.

1H

NMR (DMSO-d6) (Figure S23) analysis of 4/6 suggested the

characteristics of cyclohexadepsipeptides, with a contain ratio of 1:0.4. Detailed analysis the 1H NMR data of 4/6 and comparing with that of 8−10 revealed ten amino acid residues, including two Gly, three Leu, two Ala, two Phe, and one proline (Pro), together with two fatty acid units, including HMHA and HMOA. The retention times of the acid hydrolysate of 3 derivatized with Marfey’s reagent via UPLC-MS were 5.16 min (m/z 326 [M − H]−, 653 [2M − H]−, Gly), 6.02 min (m/z 340 [M − H]−, L-Ala), 6.68 min (m/z 366 [M − H]−, LPro), 11.60 min (m/z 416 [M − H]−, 833 [2M − H]−, L-Phe), and 14.44 min (m/z 765 [2M − H]−, D-Leu) (Figure S43). Based on the molecular networking analysis, the same [Phe– Ala–Leu] tag was deduced in the structures of 4 and 6 (Figures S28 and S30). The molecular weight of 4 was 56 Da less than compound 3, indicating four methylene groups less than 3. It is suggested that the HMDA unit in 3 was replaced by a HMHA unit in 4. Additionally, the similar node of 4 with 3 indicated the same amino acids fragments between the two compounds. Combining the molecular weight and the connected sequence of the units confirmed 4 as cyclo-[(CO)L-Phe–L-Ala–D-Leu–D-Leu–Gly–HMHA(O)], and 6 as cyclo-[(CO)L-Phe–L-Ala–D-Leu–L-Pro–Gly–HMOA(O)] (Figure 1). 10 ACS Paragon Plus Environment

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Chrysogeamides E (5) and G (7) were also isolated as a mixture with a contain ratio of 1:0.5. Their molecular formulas were elucidated as C35H55N5O7 and C36H55N5O7 by HRESI-MS, respectively. The similar 1H NMR (DMSO-d6) (Figure S24) spectrum of 5/7 with that of 4/6 indicated that 5/7 were also cyclohexadepsipeptides. Careful analysis the 1H NMR data of 5/7 indicated the presence of ten amino acid residues, including two Gly, three Leu, two Ala, two Phe, and one Pro. These amino acids were also verified by UPLCMS analysis of the acid hydrolysate derivatized with Marfey’s reagent (Figure S43). The molecular weight of 5 was 28 Da less than compound 3, indicating two methylene groups less than 3. It is suggested that the HMDA unit in 3 was replaced by a HMOA unit in 5. The same [Phe–Ala–Leu] tag as well as the similar node between 5 and 3 suggested the chemical structure of 5 as cyclo-[(CO)L-Phe–L-Ala–D-Leu–D-Leu–Gly–HMOA(O)]. The structure of 7 was confirmed to be cyclo-[(CO)L-Phe–L-Ala–D-Leu–L-Pro–Gly– HMDA(O)] in the same way as that of 5 (Figure 1). It was reported that the relative configuration of HMA unit could be determined by the coupling constant and NOE correlations.20,23 However, the conformational flexibility would preclude the use of this method. In theory, hydrolysis of the lactone and subsequent esterification using the Mosher’s method would determine the absolute configuration of C24. However, attempts to isolate the linear peptide by acid hydrolysis or methanolysis of 1 and 2 were unsuccessful. Fortunately, scopularide A (9) formed good crystals in the solvent of acetone/cyclohexane/H2O by slow evaporation at room temperature. The X-ray crystallographic analysis of 9 by using Cu Kα radiation gave a (3S,4S)-3-hydroxy-4methyldecanoic acid (HMDA) moiety with Flack parameter as 0.10 (7) (Figure 1). Owing to the same biosynthetic pathway of 1–7 as that of 9, the absolute configuration of HMA unit in 1–7 was established as 24S,25S. Carefully analysis of the ESI-MS/MS spectra of 1– 10 revealed the cleavage features of these cyclohexadepsipeptides, including a primary breakage between the ester bond then neutral losses of three amino acid fragments, successively (Figure 4). Chemical investigation of these cyclohexadepsipeptides indicated that they showed characteristic features of [Gly(CO)–HMA(O)] and [Ala(CO)–Leu(NH)] tags, which provided clues to the structure elucidation of the minor 3–7.



constant tag

initial fragmentation neutral losses of three amino acid fragments, successively

variable tag R3 O

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NH

HMA O

R1 O

NH

Ala NH Leu O

O

Gly HN

H N

O R2

O

variable tag

constant tag

Figure 4. MS/MS fragmentation pattern and structural features of 1–10.

There are a few cyclodepsipeptides containing an HMA residue that differ from the number of the constituent residues and the sequence of residues, defining the cyclotetradepsipeptides (CTDPs) and cyclohexadepsipeptides (CHDPs) (Table 2). The CTDPs include the HMOA-containing beauveriolides I, III, IV–VII, and IX, 23,24 and the HMDA-containing beauveriolides II and VIII. 23,24 Until now, these CTDPs were terrestrial origin. The CHDPs with HMOA (scopularide B20 and arenamide B25), HMDA (emericellamide D,26 scopularide A,20 arenamides A and C,25 and oryzamides A–C27), or HMLA (emericellamide F26) unit were isolated from marine-derived organisms. Previously, HMHA unit has been found in terrestrial natural products, including the peptides nodupetide from insect-derived fungus Nodulisporium sp.21 and polypeptin C from terrestrial bacterium Peanibacillus ehimensis,28 and triterpenoid saponin from terrestrial plant Quillaja saponaria Molina29. Interestingly, 4 features a rare HMHA motif firstly isolated from marine resources. Ultimately, trends can be deduced from constituent residues and the residue sequence in CHDPs. For example, the second and fifth modules are assembled by unaltered Gly and L-Ala, respectively, while the fourth module is assembled by Leu unit only differs from L or D configuration. Additional trend arises from the regular sequence of the six residues, HMA(O)−Gly−amino acida−Leu−Ala−amino acid(CO)b. Table 2. Cyclodepsipeptides containing HMA unit and their residue sequences Compd.

Source

Residue


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1st Cyclotetradepsipeptides (CTDPs) terrestrial beauveriolide I 23 beauveriolide II 23 beauveriolide III 24 beauverioide IV 24 beauveriolide V 24 beauveriolide VI 24 beauveriolide VII 24 beauveriolide VIII 24 beauveriolide IX 24 Cyclohexadepsipeptides (CHDPs) marine emericellamide D 26 emericellamide F 26 scopularide A (9) 20 scopularide B (10) 20 arenamide A 25 arenamide B 25 arenamide C 25 oryzamide A 27 oryzamide B 27 oryzamide C 27 chrysogeamide A (1) chrysogeamide B (2) chrysogeamide C (3) chrysogeamide D (4) chrysogeamide E (5) chrysogeamide F (6) chrysogeamide G (7) marine and nodupetide (8)21 terrestrial a HMLA: 3-hydroxyl-4-methyllauric acid

2nd

3rd

3S,4S-HMOA 3S,4S-HMDA 3S,4S-HMOA HMOA HMOA HMOA HMOA HMDA HMOA

4th

5th

6th

L-Phe

L-Ala

D-Leu

L-Phe

L-Ala

D-Leu

L-Phe

L-Ala

D-allo-Ile

L-Val

L-Ala

D-Val

L-Val

L-Ala

D-allo-Ile

L-Val

L-Ala

D-Leu

L-Phe

L-Ala

D-Val

L-Val

L-Ala

D-allo-Ile

L-Phe

L-Phe

D-allo-Ile

3S,4S-HMDA 3S,4S-HMLAa 3S,4S-HMDA 3S,4S-HMOA 3S,4S-HMDA 3S,4S-HMOA 3S,4S-HMDA 3S,4S-HMDA 3S,4S-HMDA 3S,4S-HMDA 3S,4S-HMOA 3S,4S-HMDA 3S,4S-HMDA 3S,4S-HMHA 3S,4S-HMOA 3S,4S-HMOA 3S,4S-HMDA

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

L-Val

L-Leu

L-Ala

L-Ala

L-Val

L-Leu

L-Ala

L-Ala

L-Val

D-Leu

L-Ala

L-Phe

L-Val

D-Leu

L-Ala

L-Phe

L-Val

L-Leu

L-Ala

L-Phe

L-Val

L-Leu

L-Ala

L-Phe

L-Val

L-Leu

L-Ala

L-Met

L-Val

D-Leu

L-Ala

L-Leu

L-Val

D-Leu

L-Ala

L-Tyr

L-Val

D-Leu

L-Ala

L-Met

L-Val

D-Leu

L-Ala

L-Val

L-Val

D-Leu

L-Ala

L-Val

D-Leu

D-Leu

L-Ala

L-Phe

D-Leu

D-Leu

L-Ala

L-Phe

D-Leu

D-Leu

L-Ala

L-Phe

L-Pro

D-Leu

L-Ala

L-Phe

L-Pro

D-Leu

L-Ala

L-Phe

3S,4S-HMHA

Gly

L-Val

D-Leu

L-Ala

L-Val

To study the effect of addition of Val, Phe, Ala, Leu, or Gly on cyclohexadepsipeptides production, L-Val, D-Val, L-Phe, D-Phe, L-Ala, D-Ala, L-Leu, D-Leu or Gly was added to fermentation broth. Interestingly, only the addition of L-Leu enhanced the yield of 9 (2.7→5.6 mg/L) and 10 (2.2→6.0 mg/L), while addition of D-Leu or other amino acids showed almost no influence on the production (Table S3). This result indicated that L-Leu might be ingested selectively by P. chrysogenum and isomerized to D-Leu to form 9 and 10. To verify the metabolic pathway of the L-Leu precursor, isotope labelling experiments were conducted by feeding 13C1-L-Leu. HPLC analysis showed that 9 and 10 still existed in the fungal metabolites. The

13C

NMR spectrum of the isolated 9 (or 10) showed the

overflow signal at δC 175.0 ppm, which is the chemical shift of the carbonyl carbon of Leu 13 ACS Paragon Plus Environment


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(Figure 5). This Leu residue was also elucidated as D-Leu by acid analysis (Figures S44), demonstrating that 13C1-L-Leu was isomerized to 13C1-D-Leu and installed into the peptide scaffold by a non-ribosomal peptide synthetase (NRPS). It is assumed that

D-Leu

is

generated from L-Leu by the racemase. To probe the existence possibility of racemase, a hypothetic gene cluster responsible for the biosynthesis of chrysogeamides was calculated by antiSMASH through mining four reported Penicillium chrysogenum genome sequence (strain No.s NCPC10086, HKF42, KF25, and Wisconsin 54-1255) (Figure S45).30 An epimerization (E) domain was identified existing in a core gene cluster which controlled the biosynthesis of the [Val-Ala-Leu] tag. Genome sequencing and bioinformatic analysis, genomic library screening, and gene inactivation experiments will be done for validating the presence of racemase in the E domain. 23

21/25

21

O

20 18

O

17

natural abundance

1 20

NH

1

NH C8

11

8

9 15

18

23

28

4

2

14

O

O

26

NH 15

26 14 17 3

5

O 28

22/24

H N 9

HN

O

13

8*

C1-L-Leu feed

4 3

O 9

Figure 5. 13C NMR spectrum of natural abundance of 9 compared through 13C1-L-Leu feeding experiment. The enhanced peak is marked with star*. The spectra were measured in CD3OD at 125 MHz.

Angiogenesis is an essential process during organ growth and repair, while imbalance in this process results in serious disease.31 Insufficient angiogenesis leads to poor circulation and tissue death, and always underlies conditions such as coronary heart disease, stroke and delayed wound healing.32 Intensive efforts should be undertaken to develop therapeutic agents to promote angiogenesis in ischaemic tissues. Thus, 1, 2, and 8–10 were investigated their pro-angiogenic activity using Tg(kdrl:EGFP) transgenic zebrafish line. These compounds promoted angiogenesis at 1.0 μg/mL toward zebrafish embryo, with 1, 2, and 8 obviously promoting the cavity of blood vessels (Figure S47). More importantly, 1, 2, and 8 didn’t exhibit obvious toxicity in embryonic zebrafish at 100 μg/mL (Figure S48). Additionally, 1, 2, and 8 were inactive against human breast cancer 14 ACS Paragon Plus Environment

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(MCF-7) and liver cancer (BEL-7402, HepG2, and SMMC7721) cell lines, while 9 and 10 showed strong and selective cytotoxic activity against HepG2 and SMMC7721 with IC50 values of 26.81 and 33.75 μM, respevtively, which were equivalent to that of the positive controls 5-FU (IC50, >100 and 15.79 μM) and cisplatin (IC50, 28.27 and 21.81 μM). Compounds 1, 2, and 8–10 were also evaluated for their antibacterial, anti-tubercular, antiviral, enzyme inhibitory, and antifouling activities, only 8 displayed weak antibacterial activity against Pseudomonas aeruginosa with an MIC value of 50 μM (Table S4–S7). In conclusion, we generated a comprehensive network reflecting the molecular diversity from 270 Penicillium strains isolating different marine environment. The abundant network and diverse clusters allowed us to observe the chemical diversity of Penicillium metabolites. Combining molecular networking with the 1H NMR tracking technique enabled efficiently the isolation of a class of new cyclodepsipeptides, chrysogeamides A–G (1–7). Interestingly, the isotope labelling experiments validated the D-Leu

in these compounds was isomerized from L-Leu. Compounds 1, 2 and 8 possess

obvious pro-angiogenic activity and safety in zebrafish and represent a new class of proangiogenic agents. Further investigations should be focused on the biosynthetic gene cluster of these cyclohexadepsipeptides especially verifying the presence of leucine racemase in P. chrysogenum (CHNSCLM-0003). This study also encourages us for further research to inform the chemical and biological diversity of these 270 Penicillium species. Experimental section General Experimental Procedures. Optical rotations were measured on a JASCO P1020 digital polarimeter (JASCO Ltd., Tokyo, Japan). UV spectra were obtained on a Beckman DU 640 spectrophotometer (Beckman Instruments Ltd., California, America). IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets (Perkin Elmer Ltd., Boston, America). NMR spectra were recorded on a JEOL JEM-ECP NMR spectrometer (JEOL Ltd., Tokyo, Japan; 500 MHz for 1H and 125 MHz for

13C),

using

TMS as an internal standard. The ESI-MS spectra were obtained from a Micromass Q-TOF spectrometer (Waters Ltd., Massachusetts, America). Single-crystal data were obtained on an Agilent Gemini Ultra diffractometer (Cu Kα radiation) (Agilent Technologies Inc., California, America). HPLC-MS was performed on an Agilent series 1290 Infinity HPLC 15 ACS Paragon Plus Environment


instrument (Agilent, Technologies, Santa Clara, CA, USA), coupled with a Q-TOF Mass spectrometer (Thermo Scientific, Bremen, Germany), with a YMC C18 column [(YMC Co., Ltd., Tokyo, Japan) YMC-Park, ODS-A, 250 × 2.1 mm, S-5 μm, 12 nm, 0.5 mL/min]. UPLC-MS was performed on Waters UPLC® system (Waters Ltd., Massachusetts, America) using a C18 column [(Waters Ltd., Massachusetts, America) ACQUITY UPLC® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min] and ACQUITY QDa ESIMS scan from 100 to 1200 Da. HPLC analysis was was performed on a Hitachi L-2000 system (Hitachi Ltd., Tokyo, Japan) using a C18 column [(YMC Co., Ltd., Tokyo, Japan) YMC-Park, ODS-A, 250 × 4.6 mm, S-5 μm, 12 nm, 1.0 mL/min]. Semi-preparative HPLC was performed on a Hitachi L-2000 system (Hitachi Ltd., Tokyo, Japan) using a C18 column [(Eka Ltd., Bohus, Sweden) Kromasil 250 × 10 mm, 5 μm, 2.0 mL/min]. Silica gel (Qingdao Haiyang Chemical Group Co., Qingdao, China; 200–300 mesh), octadecylsilyl silica gel (YMC Co., Ltd., Tokyo, Japan; 45−60 μm), and Sephadex LH-20 (GE Ltd., Connecticut, America) were used for column chromatography. Precoated silica gel plates (Yantai Zhifu Chemical Group Co., Qingdao, China; G60, F-254) were used for thin layer chromatography. Fungal Material. The fungal strain CHNSCLM-0003 was isolated from the gorgonian coral Carijoa sp. (GX-WZ-2010001) collected from Weizhou coral reefs, South China Sea in April, 2010. The fungus was identified as Penicillium chrysogenum according to its morphological traits and a molecular biological protocol by amplification and sequencing of the DNA sequences of the ITS region of the rRNA gene. The 532 base pair ITS sequence had 100% sequence identity to that of P. chrysogenum (KC009773). The strain was deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, PR China, with the Genbank (NCBI) accession number KP759287. Other 269 marine-derived Penicillium fungal strains were summarized in Table S1. Fermentation and Extraction. The fungus CHNSCLM-0003 was grown stationary on rice-potato solid medium (fifty 1000 mL Erlenmeyer flasks, each containing 70 g of rice and 70 mL of potato-salt liquid) at room temperature. After 55 days’ cultivation, the fermented substrate was extracted with ethyl acetate (EA), EA/CH2Cl2, and acetone successively to give three organic samples [Fr.1 (12.1 g), Fr.2 (5.7 g), and Fr.3 (2.3 g)]. 16 ACS Paragon Plus Environment

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The fermentation and extraction of other fungi were the same as that of fungal strain CHNSCLM-0003. LC-MS/MS and Molecular Networking Analysis. LC-MS-MS was performed on an Agilent series 1290 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA, USA), coupled with a Q-TOF Mass spectrometer (Thermo Scientific, Bremen, Germany), with a YMC C18 column [(YMC Co., Ltd., Tokyo, Japan) YMC-Park, ODS-A, 250 × 2.1 mm, S-5 μm, 12 nm, 0.5 mL/min]. Fr.1 (Fr.2 and Fr.3) (0.5 mg/mL, 10 μL) was analyzed by LC-MS with a gradient program of MeCN-H2O (0.1% formic acid) [0–2 min 10%, 2– 17 min 10–100%, 17–19 min 100%; 0.5 mL/min; MS scan 150-2000 Da], and then with an automated full dependent MS-MS scan. Differentiation of the molecular ions, adducts, and fragment ions was done by identification of the [M + H]+ ion. All MS/MS data were converted to .mzXML format files using MSConver software. The molecular networking was performed using the GNPS data analysis workflow using spectral clustering algorithm.33 The spectral networks were imported into Cytoscape 3.6.1 and visualized using the force-directed layout (Figure S1 and S2). Isolation. The mixed Fr.1, Fr.2, and Fr.3 (CHNSCLM-0003) was subjected to normal silica gel column chromatography (CC) and eluted by a gradient of petroleum ether (PE)/EA to EA, and then EA/CH3OH to afford four sub-fractions (Fr.M-1–Fr.M-4) on the basis of TLC analysis. Fr.M-2 was re-chromatographed on normal CC with CH2Cl2/CH3OH to provide four sub-fractions (Fr.M-2-1–Fr.M-2-4). Fr.M-2-3 was separated repeatedly by Sephadex LH-20 eluting with CH2Cl2–CH3OH (v:v, 1:1), normal CC with CH2Cl2/CH3OH, then purified by semi-preparative HPLC (MeCN (A), H2O (B); 60% A; 2 mL/min) to afford compounds 1 (15.3 mg), 2 (8.5 mg), and 8 (10.7 mg). Fr.M2-4 was separated repeatedly by Sephadex LH-20 eluting with CH3OH, and then by ODS (CH3OH/H2O, 40–80%) to afford Fr.M-2-4-1 and Fr.M-2-4-2. Fr.M-2-4-1 was purified by semi-preparative HPLC (MeCN (A), H2O (B); 65% A; 2 mL/min) to afford compounds 9 (20.5 mg) and 10 (23.4 mg). Fr.M-2-4-2 was purified by semi-preparative HPLC (MeCN (A), H2O (B); 60% A; 2 mL/min) to afford compounds 3 (0.6 mg), 4/6 (0.7 mg), and 5/7 (0.8 mg).



Chrysogeamide A (1): white powder; [α]20 D −47 (c 0.09, CH3OH); UV (MeOH) λmax (log ε) 201 (4.65) nm; IR (KBr) νmax 3403, 2960, 1735, 1668, 1646, 1539 cm–1; 1H NMR (C5D5N, 500 MHz, DMSO-d6, 500 MHz; CD3OD, 500 MHz) and 13C NMR (C5D5N, 125 MHz; CD3OD, 125 MHz), see Table 1 and Table S2; ESI-MS/MS m/z 497.3 [M + H – Val]+, m/z 479.3 [M + H – H2O – Val]+, m/z 408.3 [M + H – H2O – Val – Ala]+, m/z 295.2 [M + H – H2O – Val – Ala – Leu]+; HR-ESI-MS m/z 596.4031 [M + H]+ (calcd for C30H54N5O7, 596.4018). Chrysogeamide B (2): white powder; [α]20 D −48 (c 0.07, CH3OH); UV (MeOH) λmax (log ε) 201 (4.66) nm; IR (KBr) νmax 3433, 2959, 1748, 1640, 1539 cm–1; 1H NMR (CD3OD, 500 MHz; DMSO-d6, 500 MHz) and 13C NMR (CD3OD, 125 MHz; DMSO-d6, 125 MHz), see Table S2; ESI-MS/MS m/z 525.4 [M + H – Val]+, m/z 507.4 [M + H – H2O – Val]+, m/z 436.3 [M + H – H2O – Val – Ala]+, m/z 323.2 [M + H – H2O – Val – Ala – Leu]+; HR-ESI-MS m/z 624.4340 [M + H]+ (calcd for C32H58N5O7, 624.4331). Chrysogeamide C (3): white powder; 1H NMR (C5D5N, 500 MHz, J in Hz) δH 9.05 (1H, d, J = 8.8 Hz, Gly-NH), 4.76 (1H, dd, J = 16.7, 6.1 Hz, Gly-Ha-2), 4.05 (1H, dd, J = 16.7, 4.5 Hz, Gly-Hb-2), 8.82 (1H, overlapped, Leu-NH), 4.88 (1H, m, Leu-H-2), 1.96 (1H, m, Leu-Ha-3), 1.79 (1H, m, Leu-Hb-3), 1.96 (1H, m, Leu-H-4), 1.14 (3H, overlapped, LeuH-6), 0.88 (3H, overlapped, Leu-H-5), 9.72 (1H, d, J = 6.3 Hz, Leu-NH), 4.87 (1H, m, Leu-H-2), 1.98 (1H, m, Leu-Ha-3), 1.78 (1H, m, Leu-Hb-3), 1.98 (1H, m, Leu-H-4), 0.88 (3H, overlapped, Leu-H-5), 0.89 (3H, overlapped, Leu-H-6), 9.19 (1H, d, J = 7.3 Hz, AlaNH), 4.93 (1H, m, Ala-H-2), 1.12 (3H, m, Ala-H-3), 8.87 (1H, overlapped, Phe-NH), 5.22 (1H, m, Phe-H-2), 3.45 (1H, dd, J = 14.0, 7.3 Hz, Phe-Ha-3), 3.32 (1H, dd, J = 14.0, 8.2 Hz, Phe-Hb-3), 7.38 (2H, d, J = 7.3 Hz, Phe-H-5/9), 7.27 (2H, m, Phe-H-6/8), 7.25 (1H, m, Phe-H-7), 5.51 (1H, m, HMDA-H-3), 2.76 (1H, dd, J = 14.5, 8.4 Hz, HMDA-Ha-2), 2.64 (1H, d, J = 14.5 Hz, HMDA-Hb-2), 1.67 (1H, m, HMDA-H-4), 1.34 (1H, m, HMDAHa-5), 1.02 (1H, m, HMDA-Hb-5), 1.24 (2H, m, HMDA-H-6), 1.18 (2H, m, HMDA-H7), 1.17 (2H, m, HMDA-H-8), 1.25 (2H, m, HMDA-H-9), 0.88 (3H, m, HMDA-H-10), 0.78 (3H, m, HMDA-4-CH3); ESI-MS/MS (initial fragmentation at ions of m/z 686.5 [M + H]+, m/z 668.4 [M + H – H2O]+) m/z 539.4 [M + H – Phe]+, m/z 521.4 [M + H – H2O – Phe]+, m/z 468.3 [M + H – Phe – Ala]+, m/z 450.3 [M + H – H2O – Phe – Ala]+, m/z 337.3


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[M + H – H2O – Phe – Ala – Leu]+; HR-ESI-MS 686.4491 [M + H]+ (calcd for C37H60N5O7, 686.4487). Chrysogeamide D (4): white powder; ESI-MS/MS (initial fragmentation at ions of m/z 630.4 [M + H]+, m/z 612.4 [M + H – H2O]+) m/z 483.3 [M+H–Phe]+, m/z 465.3 [M + H – H2O – Phe]+, m/z 412.3 [M + H – Phe – Ala]+, m/z 394.3 [M + H – H2O – Phe – Ala]+, m/z 299.2 [M + H – Phe – Ala – Leu]+, m/z 281.3 [M + H – H2O – Phe – Ala – Leu]+; HRESI-MS m/z 630.3865 [M + H]+ (calcd for C33H52N5O7, 630.3861). Chrysogeamide E (5): white powder; ESI-MS/MS (initial fragmentation at ions of m/z 658.4 [M + H]+, m/z 640.4 [M + H – H2O]+) m/z 511.4 [M + H – Phe]+, m/z 493.3 [M + H – H2O – Phe]+, m/z 440.3 [M + H – Phe – Ala]+, m/z 422.3 [M + H – H2O – Phe – Ala]+, m/z 327.2 [M + H – Phe – Ala – Leu]+, m/z 309.2 [M + H – H2O – Phe – Ala – Leu]+; HR-ESI-MS 658.4155 [M + H]+ (calcd for C35H56N5O7, 658.4174). Chrysogeamide F (6): white powder; ESI-MS/MS (initial fragmentation at ions of m/z 642.4 [M + H]+, m/z 624.4 [M + H – H2O]+) m/z 495.3 [M + H – Phe]+, m/z 477.3 [M + H – H2O – Phe]+, m/z 424.3 [M + H – Phe – Ala]+, 406.3 [M + H – H2O – Phe – Ala]+, m/z 311.2 [M + H – Phe – Ala – Leu]+, m/z 293.2 [M + H – H2O – Phe – Ala – Leu]+; HRESI-MS m/z 642.3865 [M + H]+ (calcd for C34H52N5O7, 642.3861). Chrysogeamide G (7): white powder; ESI-MS/MS (initial fragmentation at ions of m/z 670.4 [M + H]+, m/z 652.4 [M + H – H2O]+) m/z 523.4 [M + H – Phe]+, 505.3 [M + H – H2O – Phe]+, m/z 452.3 [M + H – Phe – Ala]+, m/z 434.3 [M + H – H2O – Phe – Ala]+, m/z 321.2 [M + H – H2O – Phe – Ala – Leu]+; HR-ESI-MS m/z 670.4155 [M + H]+ (calcd for C36H56N5O7, 670.4174). Marfey’s Analysis of 1–10. A solution of 1 (2–10, 0.1 mg) was hydrolyzed by heating HCl (6M, 1 mL) for 10 h at 100 ºC. The solution was evaporated to dryness and redissolved in H2O (250 μL). The acid hydrolysate solution (50 μL) was treated with a 1% solution of L-FDAA (20 μL) in acetone followed by NaHCO3 (1M, 10 μL). The mixture was heated at 55 ºC for 1 h, then stopped by HCl (2M, 5 μL). The amino acid standards (Gly, L-Ala, D/L-Ala, L-Val, D/L-Val, L-Pro, D/L-Pro, L-Leu, and D/L-Leu) were derivatized with LFDAA in the same manner as that of 1. All derivatives were analyzed by HPLC-DAD (MeCN (A), H2O (0.1% HCOOH) (B); linear gradient: 0–30 min 25% A, 30–40 min 25%– 30% A, 40–120 min 30%–40% A; monitor: 340 nm; 1.0 mL/min). The retention times of 19 ACS Paragon Plus Environment


the L-FDAA-derivatized amino acid standards were as follows: Gly (20.5 min), L-Ala (25.3 min), D-Ala (39.3 min), L-Val (51.0 min), D-Val (70.6 min), L-Leu (73.0 min), D-Leu (95.7 min). The retention times of the acid hydrolysate derivatives of 1, 2, and 8 were as follows: 20.6 (Gly), 25.5 (L-Ala), 51.5 (L-Val), and 95.7 (D-Leu) min. All derivatives were also analyzed and detected by UPLC-MS (MeCN (A), H2O (0.1% HCOOH) (B); linear gradient: 0–15 min 25%–50% A, 15–17 min 50%–100% A, 17–19 min 100% A, 19–20 min 100%–25% A; UV: 190–400 nm; 0.5 mL/min; ESI-MS scan: 100–1200 Da). Retention times (min) and negative ESI-MS of the L-FDAA-derivatized amino acid standards were as follows: Gly (5.12 min; m/z 326 [M − H]−, 653 [2M − H]−); L-Ala

(6.17 min), D-Ala (7.43 min; m/z 340 [M − H]−, 681 [2M − H]−); L-Pro (6.45 min),

D-Pro

(7.14 min, m/z 366 [M − H]−, 733 [2M − H]−); L-Val (9.16 min), D-Val (11.58 min,

m/z 368 [M − H]−, 737 [2M − H]−); L-Phe (11.43 min), D-Phe (13.51 min, m/z 416 [M − H]−, 833 [2M − H]−); L-Leu (11.76 min), D-Leu (14.18 min, m/z 382 [M − H]−, 765 [2M − H]−); L-FDAA (7.30 min, m/z 271 [M − H]−). The retention times of the acid hydrolysate derivatives of 1–10 on UPLC-MS were as follows: 1 (2 or 8): 5.16 min (Gly), 6.21 min (LAla), 9.26 min (L-Val), and 14.22 min (D-Leu); 3: 5.25 min (Gly), 6.02 min (L-Ala), 11.52 min (L-Phe), and 14.30 min (D-Leu); 4/6 (or 5/7): 5.16 min (Gly), 6.02 min (L-Ala), 6.68 min (L-Pro), 11.60 min (L-Phe), and 14.44 min (D-Leu); 9 (or 10): 5.25 min (Gly), 6.02 min (L-Ala), 9.18 min (L-Val), 11.60 min (L-Phe), and 14.42 min (D-Leu). X-ray Crystallographic Analysis of Scopularide A (9). Upon crystallization from CH3OH-acetone-H2O (10:1:1), the regular needle crystals of 9 were obtained. The singlecrystal X-ray diffraction data were collected at 150(10) K for 9 on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.54184 Å). Crystallographic data for 9 has been deposited in the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]). Crystal data for 9: C36H57N5O7 Mr = 671.87, monoclinic, space group P2(1) with a= 26.18193 (18) Å, b = 9.22358 (7) Å, c = 16.79917 (11) Å, α = 90°, β = 107.1512 (7)°, γ = 90°, V = 3876.44 (5) Å3, Z = 4, Dx = 1.151 mg/m3, μ (Cu Kα) = 0.647 mm−1, and F(000) = 1456. Crystal dimensions: 0.120 × 0.120 × 0.110 mm3. Independent reflections: 26811, the 20 ACS Paragon Plus Environment

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final R1 value was 0.0425 (6665), wR2 = 0.1518 (6809) (I > 2σ(I)), Flack parameter = 0.10 (7). CCDC number: 1830395. Gene Mining of the Chrysogeamides Biosynthetic gene cluster. The genomes of four Penicillium chrysogenum strains, namely P. chrysogenum NCPC10086, P. chrysogenum HKF42, P. chrysogenum KF25, and Penicillium chrysogenum Wisconsin 541255 were sequenced and deposited in GenBank. The non-ribosomal peptide synthetases (NRPS) gene clusters of the four strains were mined with antiSmash.30 NRPS domains were identified in the clusters using the NRPSpredictor3 server. If the predicted core structure of the NRPS gene cluster has the same amino acid sequence with that in the chrysogeamides, it was preliminary determined as the chrysogeamides biosynthetic gene cluster. Biological Assay. Pro-angiogenic activity in zebrafish embryos was conducted conforming to the Local Institutional Laws and the Chinese Law for the Protection of Animals. The Tg(kdrl:EGFP) transgenic zebrafish embryos and adults were raised and maintained under standard conditions as described by Wang et al.34 The bioassay was evaluated in duplicate, testing at 100, 10, and 1.0 μg/mL. Toxicity in the embryonic zebrafish was tested at 100 μg/mL. Confocal images of trunk vessels were captured at 55 hpf. Antimicrobial, antiviral, cytotoxic, and enzyme inhibitory activities were shown in the Supporting Information. ASSOCIATED CONTENT Supporting Information General experimental procedures, biological assays, 1D and 2D NMR spectra for 1 and 2, 1H NMR spectra for 3−7, HR-ESI-MS spectra of 1−7, MS/MS spectra for 1−10, UPLCMS analysis of Marfey’s analysis of standard amino acids as well as derivative products of 1−10, a comprehensive network of 270 Penicillium species, and an expanded view of chrysogeamide-cluster of P. chrysogenum CHNSCLM-0003 are presented. The Supporting Information is available free of charge on the ACS Publications website.



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AUTHOR INFORMATION Corresponding Author * Chang-Lun Shao, e-mail: [email protected], tel.: 86-532-82031381; Chang-Yun Wang, e-mail: [email protected], tel.: 86-532-82031536. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Syngenta for the fellowship to Xue-Mei Hou. We also thank Prof. W.H. G. (UCSD) for assistance with molecular networking analysis. This work was supported by the Program of National Natural Science Foundation of China (Nos. 81874300, U1706210, 41776141, and U1606403), AoShan Talents Program Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASTP-ES11), the Program of Natural Science Foundation of Shandong Province of China (No. JQ201510), the Fundamental Research Funds for the Central Universities (No. 201841004), the Taishan Scholars

Program,

China,

the

China

Postdoctoral

Science

Foundation

(No.

2017M622285), and the Qingdao Postdoctoral Researcher Applied Research Project, China. REFERENCES (1) Rateb, M. E.; Ebel, R. Secondary metabolites of fungi from marine habitats. Nat. Prod. Rep. 2011, 28, 290–344. (2) Blunt, J. W.; Carroll, A. R.; Copp, B. R.; Davis, R. A.; Keyzers, R. A.; Prinsep, M. R. Marine natural products. Nat. Prod. Rep. 2018, 35, 8–53. (3) Vansteelandt, M.; Blanchet, E.; Egorov, M.; Petit, F.; Toupet, L.; Bondon, A.; Monteau, F.; Bizec, B. L.; Thomas, O. P.; Pouchus, Y. F.; Bot, R. L.; Grovel, O. Ligerin, an antiproliferative chlorinated sesquiterpenoid from a marine-derived Penicillium strain. J. Nat. Prod. 2013, 76, 297–301. (4) Shin, S. J.; Ahn, J. B.; Park, K. S.; Lee, Y. J.; Hong, Y. S.; Kim, T. W.; Kim, H. R.; Rha, S. Y.; Roh, J. K.; Kim, D. H.; Kim, C.; Chung, H. C. A phase Ib pharmacokinetic 22 ACS Paragon Plus Environment

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Graphical abstract R1 O R3 O 13 C

O 13

C1-L-Leu OH

S,S-HMA O

O

NH

NH

Gly

L-Ala 13

NH2 D-Leu

O

NH C

H N

HN

O R2

O Chrysogeamides


Integrating Molecular Networking and 1H NMR To Target the Isolation

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