Neuroprotective Glycosylated Cyclic Lipodepsipeptides

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Article Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX

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Neuroprotective Glycosylated Cyclic Lipodepsipeptides, Colletotrichamides A−E, from a Halophyte-Associated Fungus, Colletotrichum gloeosporioides JS419 Sunghee Bang,† Changyeol Lee,† Soonok Kim,‡ Ji Hoon Song,§ Ki Sung Kang,§ Stephen T. Deyrup,∥ Sang-Jip Nam,⊥ Xuekui Xia,*,# and Sang Hee Shim*,† †

College of Pharmacy, Duksung Women’s University, Seoul 01369, South Korea Biological Resources Assessment Division, National Institute of Biological Resources, Incheon 22689, South Korea § College of Korean Medicine, Gachon University, Seongnam 13120, South Korea ∥ Department of Chemistry and Biochemistry, Siena College, Londonville, New York12211, United States ⊥ Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea # Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, Shandong Province, China

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‡

S Supporting Information *

ABSTRACT: Glutamate neurotoxicity has been implicated in neuronal death in both acute CNS injury and in chronic neurodegenerative diseases. Five unique cyclic depsipeptides with neuroprotective activity, colletotrichamides A−E (1−5), were isolated from cultures of a halophyte Suaeda japonicaassociated fungus, Colletotrichum gloeosporioides JS419. Spectroscopic analysis revealed that they were glycosylated cyclic lipodepsipeptides. Their relative configurations were determined by ROESY and J-based configuration analysis, and absolute configurations were established by chemical reactions including modified Mosher’s method, advanced Marfey’s method, and sugar derivatization. This is the first report of a glycosylated dimethyl-trioxygenated dodecanoyl moiety, and the relative as well as absolute stereochemistry was elucidated herein for the first time. Colletotrichamide C exhibited strong neuroprotective activity against glutamate in hippocampal HT22 cells.

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stress, a notable mutualistic symbiosis.4 Since glutamate toxicity is due, at least in part, to an influx of ions, fungi that increase plant survival in the environment of high salinity, i.e., high ion concentration, are likely to produce compounds that can reduce glutamate toxicity. Colletotrichum sp., a filamentous fungus distributed worldwide, has been reported as an endophyte as well as a major plant pathogen causing anthracnose disease.5 Colletotrichum gloeosporioides JS419 was isolated from the inner tissue of Suaeda japonica, a halophyte known for having leaves that change color from green to red.6 Extracts of C. gloeosporioides were investigated for neuroprotective agents, leading to the isolation of five new compounds, named colletotrichamides A−E (1−5) (Figure 1).

INTRODUCTION Glutamate toxicity has been implicated in neuronal cell death in both acute CNS injury and in chronic diseases. The presence of excess extracellular glutamate, sometimes due to a decrease in the expression and/or activity of glutamate transporters, causes an influx of Ca2+ ions, ultimately leading to cell death.1 This mechanism is deemed to be responsible for much of the severe symptoms associated with acute CNS trauma and hemorrhagic stroke, as well as in diseases of chronic neuronal degeneration, which include amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and some forms of epilepsy.2 Although a few compounds have been found to help mitigate glutamate neurotoxicity, most notably β-lactam antibiotics, estrogen, and atorvastatin, they require pretreatment of the neurons for full effect. Further drug candidates for these critical conditions are urgently needed, especially ones that can rescue cells without pretreatment.1,3 In our search for molecules that can reduce glutamate toxicity, we targeted fungi associated with halophytes. Halophytes live in high salinity, and associated fungi have been shown to help the halophytes survive against this abiotic © XXXX American Chemical Society

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RESULTS AND DISCUSSION Colletotrichamide A (1) was isolated as a white amorphous solid with a molecular formula of C36H58N2O11 (nine degrees of unsaturation), obtained by NMR including 13C NMR and Received: June 14, 2019

A

DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

Article

The Journal of Organic Chemistry

Figure 1. Structures of compounds 1−5 isolated from C. gloeosporioides JS419.

carbonyl carbon (C-9) of Phe allowed for the linkage of N-MeIle with Phe (Figure 3). Strong HMBCs of the α-amino proton of Phe (H-10) with the carbonyl carbon (C-19) and of a secondary methyl H3-31 of the long chain with C-19 suggested that Phe was linked to the long chain through an amide bond. In addition, N-Me-Ile was found to be connected to C-23 of the long chain through an ester linkage by HMBCs of the αamino proton of N-Me-Ile (H-2) with the ester carbonyl carbon (C-1) and of the oxymethine proton (H-23) of the long chain with C-1, which was supported by the deshielded chemical shift of H-23 (δH 4.63). The mannose unit was found to be attached to C-29 based on the HMBC of the anomeric proton H-1′ (δH 4.43) with C-29 of the long chain. Thus the planar structure of 1 was established to be a 12-membered cyclic lipodepsipeptide. Colletotrichamide B (2) was isolated as an amorphous powder and had a molecular formula of C36H56N2O11 by (+)HRESIMS data (observed [M + Na]+ at m/z 715.3779). The 1H and 13C NMR spectra of 2 were similar to those of 1, suggesting that 2 was also composed of two amino acids, mannose, and the long lipid chain with fourteen carbons (Table 1). The NMR data of 2 suggested the presence of unsaturation in the long polyketide chain, which was evident by the olefinic proton signals at δH 5.42 and 5.39 (each 1H, ddd). The presence of unsaturation was also supported by the 2 amu deficient of molecular weight when compared with that of compound 1. The 1H−1H COSY and HMBC data (Table S2) indicated that the olefinic group was between C-26 and C27 with the E geometry determined by the 1H NMR coupling constant (3JH‑26, H‑27=16 Hz). The molecular formula for colletotrichamide C (3) was determined to be C35H56N2O11 by (+)HRESIMS. Based on the HRMS, the deficient was 14 amu in this compound compared with compound 1. The NMR data for 3 were also very similar to those for 1 except that one secondary methyl group was missing in 3 compared with 1, which was supported by the reduced molecular weight (Tables 1 and S3). By interpretation of 1H−1H COSY and HMBCs, 3 was found to be de-methylated at C-20 when compared with 1.

HSQC (Tables 1 and S1), which was verified by highresolution ESIMS (observed [M + Na]+ at m/z 717.3936). The 1H and 13C NMR spectra showed a characteristic amide NH signal (δH 8.61) and two α-amino group signals (δH 5.06 and 4.77; δC 59.7 and 49.6) for a peptide, indicating that it was a dipeptide. Two amino acids were found to be N-methyl isoleucine and phenylalanine (Phe) by interpretation of oneand two-dimensional (2D) NMR. The isoleucine moiety was determined to be methylated by observing the heteronuclear multiple bond correlations (HMBCs) between the N-CH3 group (H3-8, d, δH 2.66) and the α-carbon (C-2, d, δC 59.7) of the isoleucine. Since N-methyl isoleucine contains two consecutive chiral centers at C-2 and C-3, it has four possible stereoisomers: N-Me-Ile with (2R, 3R) or (2S, 3S) or N-Meallo-Ile with (2R, 3S) or (2S, 3R) configuration. The large vicinal coupling constant (3JH‑2,H‑3 = 12 Hz) and a ROESY correlation between H3-8 and H2-4 provided the support that 1 contained an N-Me-Ile residue (Figure 2a). In addition, the presence of a hexose unit was suggested by the 1H and 13C NMR resonances for five oxygenated methine and one oxygenated methylene groups. The hexose was determined to be mannose by direct comparison of its acid hydrolysate with an authentic sample by thin-layer chromatography (TLC), which was further supported by the 13C NMR data.7 At this point, one carbonyl carbon and thirteen sp3 carbons, three of which were oxygenated, remained unexplained. Careful interpretation of 1H−1H correlation spectroscopy (COSY) afforded a partial structure for a long aliphatic chain, CH 3 −CH−CH(O)−CH 2 −C(O)−CH(CH3)−CH2−CH2−CH2−CH2−CH(O)−CH3 corresponding to C-31/C-20/C-21/C-22/C-23/C-24(C-32)/C-25/C26/C-27/C-28/C-29/C-30 unit. Chemical shift values suggested that C-20 of this chain was linked to the carbonyl carbon (C-19) to form a dimethyl, trioxygenated dodecanoyl moiety, which was confirmed by HMBCs. Thus, this compound was composed of two amino acids, a dimethyl, trioxygenated dodecanoyl moiety, and a mannose. An HMBC of the α-amino proton (H-2) of N-Me-Ile with the amide B

DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

(d, 12) (m) (m) (m) (m) (m) (d, 6.5)

C

1.51 (m) 1.34 (dt, 14, 6.5) 3.77 (sextet, 6.0)

36.9

72.3

25.3

26.1

(m) (dd, 11, 3.0) (m) (m) (m)

(d, 5.5) (m) (dd, 15, 3.5) (dt, 11, 3.0) (m)

1.55 0.99 1.23 1.17 1.26

4.51 1.51 1.44 4.63 1.86

31.7

76.0 33.5

31.4

3.87 (ddd, 8.5, 5.5, 3.5)

67.0

(d, 7.0) (d, 7.0) (m) (d, 7.0) (d, 7.0) (d, 9.5)

2.57 (dq, 6.5, 3.5)

7.22 7.24 7.19 7.24 7.22 8.61

3.08 (dd, 13, 9.5) 2.84 (dd, 13, 6.0)

5.06 (dt, 9.5, 6.0)

2.66 (s)

4.77 1.83 2.00 0.95 0.68 0.68 0.79

δH, multi (J in Hz)

137.7 129.1 128.2 126.4 128.2 129.1 NH 173.5 46.3

35.8

9.8 15.3 N 29.8 172.9 49.6

δC

168.6 59.7 29.8 24.3 23.3

no

1 2 3a 3b 4a 4b 5 6 7 8 9 10a 10b 11a 11b 12 13 14 15 16 17 18 19 20a 20b 21a 21b 21-OH 22a 22b 23 24a 24b 25a 25b 26a 26b 27a 27b 28a 28b 29

1 δC

72.6

40.1

128.3

129.5

35.0

75.5 33.5

31.4

67.0

137.6 129.1 128.1 126.3 128.1 129.1 NH 173.4 46.2

35.7

9.7 15.2 N 29.7 172.9 49.6

23.2

168.5 59.7 29.8 (m) (m) (m) (d, 6.5)

(m) (m) (m) (m) (m) (d, 10)

(d, 6.0) (dd, 15, 8.5) (dd, 15, 3.5) (br d, 11) (m)

2.31 (m) 2.11 (m) 3.81 (q, 6.0)

5.42 (ddd, 16, 8.5, 6.5)

2.24 (m) 1.85 (m) 5.39 (ddd, 16, 9.0. 6.5)

4.58 1.54 1.44 4.66 1.87

3.88 (br t, 8.0)

2.59 (dq, 6.5, 3.5)

7.22 7.25 7.19 7.25 7.22 8.63

3.08 (dd, 13, 9.5) 2.84 (dd, 13, 6.0)

5.06 (dt, 9.5, 6.5)

2.66 (s)

0.95 0.68 0.68 0.78

4.77 (d, 12) 1.83 (m)

δH, multi (J in Hz)

2

72.3

36.8

25.3

28.6

31.7

76.1 33.5

22.1

65.0

137.7 129.1 128.1 126.3 128.1 129.1 NH 170.7 35.8

35.7

9.8 15.3 N 29.8 172.9 49.6

23.3

168.7 59.8 29.9

δC

Table 1. 1H and 13C NMR Data of Colletotrichamides A−E (1−5) in DMSO-d6a

(m) (m) (d, 3.5) (d, 6.5)

(m) (m) (m) (m) (m) (d, 9.5)

(d, 7.0) (m) (m) (dt, 11, 3.0) (m)

1.51 (m) 1.34 (m) 3.76 (q, 6.5)

1.27 (m)

1.58 (m) 1.01 (m) 1.23 (m)

4.71 1.51 1.27 4.64 1.86

2.49 (m) 1.80 (d, 11) 3.82 (m)

7.23 7.23 7.18 7.23 7.23 8.62

3.05 (dd, 13, 9.0) 2.85 (dd, 13, 6.5)

5.03 (dt, 9.0, 7.0)

2.68 (s)

0.96 0.70 0.70 0.79

4.76 (d, 12) 1.86 (m)

δH, multi (J in Hz)

3 δC

72.2

36.9

25.3

26.2

31.7

76.4 33.9

32.1

67.4

137.8 129.1 128.1 126.3 128.1 129.1 NH 173.1 46.2

35.9

23.0 21.5 N 29.6 172.4 49.3

23.9

169.8 52.9 34.7 (dd, 11, 5.5) (m) (m) (m)

(m) (m) (m) (m) (m) (d, 9.5)

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

(m) (m) (dt, 10, 3.0) (m)

1.50 (m) 1.33 (m) 3.77 (sextet, 6.0)

1.55 1.02 1.24 1.17 1.26

1.55 1.39 4.62 1.89

3.92 (dd, 8.5, 3.5)

2.58 (dq, 6.5, 3.5)

7.23 7.23 7.18 7.23 7.23 8.61

3.08 (dd, 13, 9.0) 2.86 (dd, 13, 6.5)

5.10 (dt, 9.0, 7.0)

2.68 (s)

0.78 (d, 6.5) 0.76 (d, 6.5)

5.24 1.55 1.35 1.10

δH, multi (J in Hz)

4 δC

19.2

72.3

36.9

25.3

26.0

33.5 31.7

75.9

31.5

129.1 128.1 126.3 128.1 129.1 NH 173.3 46.2 67.0

137.7

19.2 N 29.6 172.9 49.5 35.7

17.4

168.5 61.2

(m) (m) (m) (m) (m) (d, 9.5)

(m) (m) (br d, 11) (dd, 15, 4.0) (m) (m)

1.23 (d, 6.5)

1.54 (m) 1.34 (m) 3.77 (sextet, 6.0)

1.87 1.54 0.99 1.43 1.19 1.26

4.64 (dt, 11, 3.0)

1.54 (m) 1.43 (dd, 15, 4.0)

2.57 (dq, 6.5, 3.5) 3.88 (m)

7.23 7.24 7.18 7.24 7.23 8.63

5.07 (dt, 9.0, 7.0) 3.09 (dd, 13, 9.0) 2.85 (dd, 13, 6.5)

2.68 (s)

0.84 (d, 6.5)

0.49 (d, 6.5)

4.69 (d, 13)

δH, multi (J in Hz)

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DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

Article

(s) (br s) (br s) (m) (m) (m) (m) (m) (br d, 12) (dd, 12, 6.0) (m)

The molecular formula of colletotrichamide D (4) was found to be C36H58N2O11 by high-resolution ESIMS as well as 13C and 2D NMR data (Tables 1 and S4). Even though the molecular formula of 4 was exactly similar to that of 1, the NMR data for 4 were different from those for 1. The difference between 4 and 1 was the replacement of N-Me-Ile in 1 with NMe-Leu, which was evident by changes in the 1H NMR data and was further confirmed by 2D NMR. Colletotrichamide E (5) was also a mannosyl cyclic lipodepsipeptide with the molecular formula of C35H56N2O11. Based on the highresolution mass spectrometry (HRMS), one unit of CH2 was presumed to be missing in this compound compared with compound 1 as shown in compound 3. Unlike compound 3, two of the secondary methyl groups in the lipid chain were not missing in this compound. Instead, the presence of N-Me-Val was evident based on two germinal methyl protons at δH 0.49 and 0.84, together with an absence of one methylene group when compared with 1. The relative configuration of the long lipid chain was determined by J-based configuration analysis (JBCA) and ROESY correlations.8 2JC,H and 3JC,H obtained from HETLOC experiments together with vicinal coupling constants obtained from proton-decoupling experiments afforded the relative configuration as shown. The small 3JH‑20, H‑21 (3.5 Hz), large 3 JC‑31, H‑21 (5.9 Hz), and large 2JC‑21, H‑20 (6.5 Hz) indicated that the methyl group at C-20 and the hydroxyl group at C-21 were in a syn orientation (Figure 4a). The strong ROESY correlation between the methyl group at C-20 and the hydroxyl proton also supported the syn configuration. JBCA was not useful for the C-23 and C-24 methines since the vicinal coupling constant 3JH‑23, H‑24 was quite large (10.5 Hz) indicative of anti orientation. Instead, the strong ROESY correlation between H2-22 and H2-25 indicated that they were in threo configuration (Figures 2b and 4b). Stereochemical relationship between C-21 and C-23 was elucidated by JBCA as well as ROESY correlations.8 A strong ROESY correlation between H-21 and H-24, which is an opposite orientation to H-23, indicated the relative stereochemistry as shown (Figure 2b). In addition, the methylene protons at C-22 were distinguishable in the 1H NMR, which allowed for consecutive JBCA for the elucidation of relative configurations from C-21 to C-23 through C-22. Large 3JC‑23, H‑21 (8.2 Hz), large 2 JC‑21, H‑22a (7.2 Hz), and small 2JC‑21, H‑22b (1.3 Hz) values indicated that H-22a was in the same orientation as the hydroxyl group at C-21 (Figure 4c), whereas large 3JC‑21, H‑23 (8.2 Hz), small 2JC‑23, H‑22a (0.2 Hz), and large 2JC‑23, H‑22b (6.9 Hz) values indicated that H-22a was in the opposite orientation to the oxygenation at C-23 (Figure 4d). Thus, the oxygenation substituents at C-21 and C-23 were deduced to be in the opposite orientation. The results obtained from JBCA agreed with those from ROESY. The relative configuration of mannose was also confirmed by 1JCH values and ROESY correlations. Since the H-2′ of mannose was in equatorial configuration, the coupling constant of the anomeric proton was not meaningful for relative stereochemistry of mannose. The anomers in carbohydrates have different 1JCH values depending on the equatorial/axial configurations. Since the 1JC‑1′, H‑1′ of mannose in 1 had 158.3 Hz, mannose was found to be in β configuration.9 Strong ROESY correlations between H-1′ with H-3′ and H-5′ axial orientations also supported the β configuration of the mannose moiety (Figure 2c).

77.4 61.4 3.01 (ddd, 8.5, 5.5, 1.5) 3.67 (m) 3.44 (dd, 12, 6.5) 77.4 61.4

67.2 3.29 (m) 67.2

a1

77.4 61.4

67.2

73.8

77.4 61.4

67.3

73.9

H and 13C NMR were measured at 500 and 125 MHz, respectively.

77.4 61.4

67.3

73.8

4.43 3.53 4.19 3.26 4.53 3.28 4.72 3.01 3.67 3.44 4.37 97.4 71.2

(s) (dd, 5.0, 1.5) (d, 5.0) (m) (d, 5.5) (m) (m) (ddd, 8.5, 5.5, 2.0) (ddd, 12, 5.5, 2.0) (dt, 12, 5.5) (t, 5.5)

73.9

3.26 (m)

73.8

4.43 3.53 4.19 3.26 4.78 3.27 4.60 3.00 3.67 3.44 4.43 97.5 71.2

0.84 (d, 6.0) 0.71 (d, 7.0)

δH, multi (J in Hz) δC

7.5 15.6

(d, 6.0) (d, 6.5) (d, 6.5) (s) (br d, 1.0)

δH, multi (J in Hz)

1.06 0.83 0.71 4.43 3.53 19.2 7.5 15.7 97.5 71.2

δC δH, multi (J in Hz)

1.05 (d, 6.5) 0.71 (d, 7.0)

δC

19.2 15.6

(d, 6.0) (d, 7.0) (d, 7.0) (s) (m) (d, 5.5) (m) (m) (m) (m) (br t, 6.0) (br dd, 12, 5.5) (dt, 12, 6.0) (t, 6.0) 1.04 0.84 0.71 4.46 3.55 4.20 3.27 4.55 3.26 4.73 3.01 3.67 3.43 4.39

δH, multi (J in Hz) δC

18.7 7.4 15.7 97.8 71.1

δC

19.2 7.5 15.6 97.5 71.2

no

30 31 32 1’ 2’ 2’-OH 3’ 3’-OH 4’ 4’-OH 5’ 6’a 6’b 6’-OH

δH, multi (J in Hz)

(d, 6.0) (d, 6.5) (d, 6.5) (s) (dd, 5.0, 2.0) (d, 5.0) (m) (d, 5.0) (m) (d, 4.5) (ddd, 8.5, 5.5, 2.0) (ddd, 12, 5.5, 2.0) (dt, 12, 5.5) (t, 5.5)

4 3 2 1

Table 1. continued

1.05 0.84 0.71 4.43 3.54 4.18 3.26 4.53 3.28 4.71 3.01 3.67 3.44 4.37

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D

DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry

Figure 2. ROESY correlations of N-Me-Ile (a), trioxygenated, dimethyl dodecanoyl moiety (b), and mannose (c) in 1.

Table 2. LC−MS Analysis of the FDLA Derivatives of Colletotrichamides A, D, and E (1, 4, and 5)

Figure 3. Key COSY and HMBCs for 1.

colletotrichamide A (1)

Phe (D)

N-Me-Ile (L)

tRL (min) tRD (min) elution order colletotrichamide D (4) tRL (min) tRD (min) elution order colletotrichamide E (5) tRL (min) tRD (min) elution order

38.86 17.60 D → L Phe (D) 36.98 25.28 D → L Phe (D) 36.85 25.08 D → L

28.94 39.03 L → D N-Me-Leu (L) 30.16 37.19 L → D N-Me-Val (L) 23.35 31.82 L → D

Chromatographic comparison of the derivatives of the hydrolysate with those of authentic mannose allowed the assignment of mannose to be D configuration (tR: D-mannose derivative 22.07 min, L-mannose derivative 5.87 min). The absolute configuration of the two oxymethine centers at C-21 and C-29 was determined by the modified Mosher’s method.12 Acid hydrolysis of 1 and 4 generated compounds with a free hydroxyl group at C-29, which were subsequently converted into S- and R-α-methoxy-α-(trifluoromethyl) phenylacetic acid (MTPA) esters (1a/4a and 1b/4b) by treatment with R- and S-MTPA-Cl, respectively. The proton chemical shifts for these derivatives (1a/4a and 1b/4b) were assigned by the analysis of 1 H NMR and 1H−1H COSY spectral data. Calculation of ΔδH values (ΔδH = δS − δR) established the absolute configurations of both C-21 and C-29 as R and subsequently defined 20R, 23S, and 24S configuration on the basis of their relative configuration (Figure 5). Colletotrichamides A−E (1−5) have an uncommon 12membered ring system, which was found in taumycins, tausalarins, and acremolides.13,14 They have an interesting

Absolute configurations of the compounds were elucidated using both chemical reactions and chromatographic methods as described below. The absolute configurations of amino acids were determined by the advanced Marfey’s method.10 Acid hydrolysis of 1 afforded free amino acids, which were then derivatized with D/L-FDLA (α-2,4-dinitro-5-fluorophenylleucinamide) for the analysis by liquid chromatography− mass spectrometry (LC−MS). Based on the LC−MS elution sequence, N-Me-Ile and Phe were found to possess L and D configurations, respectively (Table 2). Similarly to 1, the absolute configurations of amino acids in 4 and 5 were also determined by the application of advanced Marfey’s methods. Consequently, Phe in 4 and 5 had D configurations while NMe-Leu in 4 and N-Me-Val in 5 had L configurations (Table 2). The absolute configuration of mannose was also assigned by application of chemical reaction followed by chromatographic analysis. The free mannose moiety was obtained by acid hydrolysis and reacted with L-cysteine methyl ester hydrochloride and o-tolyl isothiocyanate, which was analyzed by high-performance liquid chromatography (HPLC).11

Figure 4. J-based configuration analysis of C-20 and C-21 (a), C-23 and C-24 (b), C-21 and C-22 (c), and C-22 and C-23 (d) in 1. E

DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

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from halophytes could be invaluable sources for the discovery of new neuroprotective compounds.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was measured at room temperature on a JASCO P-2000 polarimeter (JASCO, Easton, PA) using a 1 cm cell. IR spectra were recorded on an Agilent Cary 630 FTIR spectrometer (Agilent Technologies, Santa Clara, CA). High- resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired on a UHR ESI Q-TOF mass spectrometer (Bruker, Billerica, MA). NMR spectra were obtained using Varian NMR systems 500 MHz (1H: 500 MHz, 13C: 125 MHz) (Varian, Palo Alto, CA) and Bruker magnet system 800/45 ASCEND (1H: 800 MHz, 13C: 200 MHz) (Bruker, Billerica, MA) with DMSOd6 (Cambridge Isotope Laboratories, Inc., Tewksbury, MA). Fungal Materials. The fungal strain (JS419) was isolated from S. japonica Makino, which was collected from a swamp in Suncheon, South Korea, in September 2011. Tissues of this plant were cut into small pieces (0.5 × 0.5 cm) and rinsed sequentially for 1 min with 2% sodium hypochlorite, 70% ethanol, and sterilized distilled water to remove external microorganisms. After being air-dried, the sterilized plant tissues were incubated on malt extract agar medium (4 g of yeast extract, 10 g of malt extract, 4 g of potato dextrose broth, and 18 g of agar per 1 L of sterilized distilled water) with 50 ppm kanamycin, 50 ppm chloramphenicol, and 50 ppm Rose Bengal at 22°C for 7 days. The actively growing fungus was transferred onto potato dextrose agar (PDA) medium (24 g of potato dextrose broth and 18 g of agar per 1 L of sterilized distilled water). The fungal strain was identified as C. gloeosporioides on the basis of the internal transcribed spacer sequences by one of the authors (S.K.). It was deposited as a 20% glycerol stock in a liquid nitrogen tank at the Wildlife Genetic Resources Bank of the National Institute of Biological Resources (Incheon, Korea). Mass Cultivation of a Fungus. The JS419 strain was cultured on a solid PDA medium for 7 days at room temperature. Agar plugs were cut into small pieces (0.5 × 0.5 cm) under aseptic conditions and these pieces were inoculated on a solid rice medium (60 g of rice, 90 mL distilled water per 500 mL Erlenmeyer flasks). After incubation at room temperature for 30 days, each 200 mL of ethyl acetate was poured to each culture flasks (50 × 500 mL Erlenmeyer flasks) and placed one day for extraction. They were then filtered to separate supernatants from the solid mycelia. The supernatants were evaporated under reduced pressure at 35 °C to yield the extract (110 g). Cell Culture. Murine hippocampal HT22 cells were grown in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and antibiotics. The cells were kept in an incubator at 37°C with humidified condition supplemented with 5% carbon dioxide. Extraction and Isolation. The crude extract (110 g) was fractionated by vacuum liquid column chromatography over silica gel by a stepwise gradient of n-hexane/acetone/MeOH (from 1:0:0, 60:1:0, 25:1:0, 7:1:0, 5:1:0, 4:1:0, 3:1:0, 2:1:0, 0:1:0, to 0:0:1, each 5 L) to obtain RM01-RM10 fractions. The RM10 fraction (4.25 g) was subjected to silica gel vacuum liquid column chromatography and separated into RM10A and RM10J fractions, which were eluted by a gradient of CHCl3/MeOH (from 70:1, 30:1, 13:1, 10:1, 7.5:1, 5:1, 3.5:1, 2:1, 1:1, to 0:1, each 4 L). Fraction RM10D (180 mg) was separated with C18 vacuum liquid column chromatography using a gradient of MeOH in H2O (40, 50, 60, 70, and 100%, v/v, each 200 mL). The 100% MeOH fraction (102 mg) was further purified by reversed-phase HPLC (Phenomenex, Luna C18 (2), 5 μm, 250 × 10.0 mm, room temperature, 2.5 mL/min, isocratic 45% aqueous ACN, UV 210 nm) to yield 1 (70.0 mg, tR = 29.0 min), 4 (13.0 mg, tR = 26.5 min), and 5 (2.5 mg, tR = 19.5 min). Fraction RM10E (262 mg) was separated by C18 vacuum liquid column chromatography using a stepwise gradient of MeOH in H2O (50, 60, 70, 80, 90, and 100%, v/v, each 300 mL). The 80% aqueous MeOH fraction (13 mg) was further purified by reversed-phase HPLC (Phenomenex, Luna C18 (2), 5 μm, 250 × 10.0 mm, room temperature, 2.1 mL/min,

Figure 5. ΔδS‑R values obtained for the bis-S-(1a and 4a) and bis-RMTPA ester (1b and 4b) in DMSO-d6.

biosynthetic origin. They are polyketide synthase−nonribosomal peptide synthetase (PKS−NRPS) hybrid metabolites with structurally unique moieties in several aspects. They are composed of a 3,5,11-trihydroxy-2,6-dimethyl dodecanoic acid moiety, a mannose, and two amino acids, one of which is N-methylated. The polyketide chain, 3,5,11-trihydroxy-2,6dimethyl dodecanoic acid moiety, was linked to the two amino acids through an amide bond and an ester bond, which allowed the formation of a 12-membered ring system. To form a 12membered ring system, the NRPS-PKS hybrid has infrequently been encountered, with only two examples, HA23 from Fusarium sp., and acremolides A−D from Acremonium sp.14 Even though this polyketide chain, incorporated into cyclic depsipeptides, has precedent in two cases, the previous reports did not elucidate relative or absolute configuration. Furthermore, the mannose moiety attached to C-11 of the polyketide chain through O-glycosidic linkage represents a late-stage structural modification in the biosynthetic process. Colletotrichamides A−E are the first example of cyclic lipodepsipeptide with a glycosylated aliphatic chain. All of the isolated compounds were evaluated for their protective activities on HT22 hippocampal cell death induced by glutamate, and compounds 2, 3, and 5 were found to be active. Compound 3 exhibited almost 100% cell viability at a concentration of 100 μM (Figure 6). Additionally, the activity

Figure 6. Neuroprotective activities of compounds 3 and 5 against glutamate-induced neurotoxicity in HT22 cells. **P < 0.001, *P < 0.05 compared with glutamate-treated value.

was observed when the compounds were added simultaneously with glutamate. This indicates that 3, or related compounds, could have significant advantages over known molecules that reduce glutamate toxicity, since those required pretreatment of the cells,1,3 especially for the treatment of traumatic CNS injury or hemorrhagic stroke. The discovery of colletotrichamides A−E is a good example to show that microorganisms F

DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

Article

The Journal of Organic Chemistry isocratic 45% aqueous ACN, UV 210 nm) to afford 2 (3.0 mg, tR = 19.0 min) and 3 (2.0 mg, tR = 20.5 min). Colletotrichamide A. White amorphous solid (1, 70.0 mg); [α]D25 = +70.4 (c 0.0625, MeOH); IR νmax 3303, 2930, 1733, 1636, 1023 cm−1; for 1H and 13C NMR data (500 MHz in DMSO-d6), see Table 1; HRMS (ESI-TOF) m/z: [M + Na]+, Calcd for C36H58N2O11Na 717.3933; found 717.3936. Colletotrichamide B. White amorphous solid (2, 3.0 mg); [α]D25 = −109.5 (c 0.1, MeOH); IR νmax 3405, 3366, 2929, 1731, 1636, 1071 cm−1; for 1H and 13C NMR data (500 MHz in DMSO-d6), see Table 1; HRMS (ESI-TOF) m/z: 715.3776 [M + Na]+, calcd for C36H56N2O11Na; found 715.3779. Colletotrichamide C. White amorphous solid (3, 2.0 mg); [α]D25 = −113.8 (c 0.1, MeOH); IR νmax 3390, 3340, 3299, 3252, 2966, 1734, 1637, 1058 cm−1; for 1H and 13C NMR data (500 MHz in DMSOd6), see Table 1; HRMS (ESI-TOF) m/z: [M + Na]+, calcd for C35H56N2O11Na 703.3776; found 703.3778. Colletotrichamide D. White amorphous solid (4, 13.0 mg); [α]D25 = −114.6 (c 0.1, MeOH); IR νmax 3565, 3432, 2932, 1721, 1638, 1070 cm−1; for 1H and 13C NMR data (500 MHz in DMSO-d6), see Table 1; HRMS (ESI-TOF) m/z: [M + Na]+, Calcd for C36H58N2O11Na 717.3933; found 717.3939. Colletotrichamide E. White amorphous solid (5, 2.5 mg); [α]D25 = −100.5 (c 0.1, MeOH); IR νmax 3354, 3298, 2962, 1732, 1643, 1065 cm−1; for 1H and 13C NMR data (500 MHz in DMSO-d6), see Table 1; HRMS (ESI-TOF) m/z: [M + Na]+, calcd for C35H56N2O11Na 703.3776; found 703.3780. Derivatization of the Sugar Moieties. Compound 1 (3.0 mg) was hydrolyzed with 3.0 mL of 1 N HCl at 80 °C and extracted with EtOAc three times, dividing the organic and aqueous layers. The aqueous fraction of hydrolysate, which included the sugar moiety, was evaporated and then compared with authentic mannose (SigmaAldrich, St. Louis, MO) by LC analysis on silica gel plates with CHCl3/MeOH/acetone/H2O (14:6:2:1). The aqueous fraction of hydrolysate was dissolved in 0.1 mL of pyridine, with 0.5 mg of Lcysteine methyl ester hydrochloride, and then heated at 60 °C for 1 h. Additionally, 10 μL of o-tolyl isothiocyanate was added into the reaction mixture, followed by incubation at 60 °C for 1 h. The reaction mixture was evaporated in vacuo and then dissolved with HPLC-grade MeOH to a concentration of 1.0 mg/mL. A 10 μL aliquot of the resulting mixture was analyzed by HPLC (Agilent Technologies, ZORBAX SB-C18, 5 μm, 250 × 4.6 mm, 35 °C, 0.8 mL/min, UV 250 nm) with an isocratic elution of 25% ACN in H2O for 40 min. Both authentic D-mannose and L-mannose were reacted in the same manner as described above. Retention time (tR, min) of the derivative of the sugar in colletotrichamide A (1): 5.87. Retention time (tR, min) of the derivative of the authentic Dmannose: 5.88. Retention time (tR, min) of the derivative of the authentic Lmannose: 22.07. FDLA Derivatization of the Amino Acids. Compound 1 was hydrolyzed with 0.7 mL of 6 N HCl in an oil bath at 115 °C for 1 h. The reaction vial was cooled in an ice-water bath for 3 min. The reaction mixture was evaporated under reduced pressure, followed by the addition of H2O three times for the removal of HCl completely. The hydrolysate, including the free amino acids, was divided into two vials and dissolved in 50 μL of 1 N NaHCO3. Each vial was treated with 150 μL of either 1% N-5-fluoro-2,4-dinitrophenyl-L-leucinamide (L-FDLA) or N-5-fluoro-2,4-dinitrophenyl-D-leucinamide (D-FDLA) in acetone, respectively. The reaction mixtures were incubated in a water bath at 85 °C for 3 min. The reaction vials were placed in an ice-water bath for 3 min and then dried in vacuo. Then, the reaction was quenched by the addition to 20 μL of 2 N HCl, and the reaction mixtures were dissolved in 400 μL of HPLC-grade acetonitrile. A 10 μL aliquot of the resulting mixtures was analyzed by LC−MS (Phenomenex, Luna C18 (2), 5 μm, 250 × 4.6 mm, 40 °C, 0.9 mL/ min, UV 340 nm). The gradient elution was as follows: 35-50% aqueous ACN (0-40 min), 50-100% aqueous ACN (40-45 min), and 100% aqueous ACN (45−50 min). The procedures described above

were identically performed for determination of the absolute configuration of the amino acids of compounds 4 and 5. Modified Mosher’s Esters of Compounds 1 and 4. Compound 1 was hydrolyzed by acid to obtain aglycone moiety to establish the absolute configuration of C-29. Prior to reaction, it was dried completely under high vacuum and divided into two vials. Each was dissolved in 600 μL of anhydrous pyridine, and a slight excess of dimethylaminopyridine was added as a catalyst and reacted for 5 min at room temperature. The reaction mixtures were treated with 10 μL of R- and S-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA-Cl), respectively. After reaction for 2 h, the reactions were quenched by the addition of 50 μL of MeOH. The resulting mixtures were purified using semi-preparative HPLC (Phenomenex, Luna C18 (2), 5 μm, 250 × 10.0 mm, room temperature; 2.0 mL/min; UV 210, 254 nm) with a gradient of ACN/H2O (from 10:90 to 100:0) over 40 min. The S-MTPA (1a) and R-MTPA (1b) esters eluted at 31.0 and 32.0 min, respectively. The 1H NMR chemical shifts of 1a and 1b were assigned by the interpretation of 1H and 1H−1H COSY NMR experiments and ΔδS‑R values around the stereogenic centers were calculated. To determine the absolute configuration of C-21 and C-29 of compound 4, identical procedures as above were performed and the difference in 1H NMR chemical shifts between 4a and 4b was compared. Bis-S-MTPA Ester (1a). 1H NMR (500 MHz, DMSO-d6) δ 8.94 (1H, d, J = 9.0 Hz, H-18), 7.53−7.48 (5H, m, MTPA-Ar), 7.28−7.19 (5H, m, H-13, 14, 15, 16, 17), 5.39 (1H, dd, J = 3.5, 7.0 Hz, H-21), 5.09 (1H, dt, J = 6.0, 9.5 Hz, H-10), 5.06 (1H, m, H-29), 4.82 (1H, d, J = 12.0 Hz, H-2), 4.70 (1H, m, H-23), 3.48 (3H, s, MTPA-OCH3), 3.16 (1H, dd, J = 9.5, 12.5 Hz, H-11a), 2.90 (1H, dd, J = 6.5, 13.0 Hz, H-11b), 2.80 (1H, dq, J = 3.5, 6.5 Hz, H-20), 2.65 (3H, s, H-8), 1.97 (1H, m, H-22a), 1.92 (1H, m, H-22b), 1.86 (1H, m, H-3), 1.51 (2H, m, H-24, 25a), 1.44 (1H, m, H-28a), 1.28 (3H, d, J = 6.0 Hz, H-30), 1.26 (1H, m, H-28b), 1.23 (2H, m, H-26a, 26b, 27a), 1.06 (1H, m, H27b), 0.93 (1H, m, H-4a), 0.84 (1H, m, H-25b), 0.80 (3H, d, J = 6.5 Hz, H-6), 0.72 (3H, d, J = 7.0 Hz, H-31), 0.71 (4H, m, H-4b, 5), 0.68 (3H, d, J = 6.5 Hz, H-32). Bis-R-MTPA Ester (1b). 1H NMR (500 MHz, DMSO-d6) δ 8.93 (1H, d, J = 9.0 Hz, H-18), 7.49−7.43 (5H, m, MTPA-Ar), 7.27−7.19 (5H, m, H-13, 14, 15, 16, 17), 5.34 (1H, dd, J = 3.5, 8.0 Hz, H-21), 5.06 (dt, J = 6.0, 9.0 Hz, H-10), 5.01 (1H, m, H-29), 4.80 (d, J = 11.5 Hz, H-2), 4.66 (1H, dt, J = 3.0, 11.0 Hz, H-23), 3.50 (s, MTPAOCH3), 3.14 (dd, J = 9.5, 13.0 Hz, H-11a), 2.90 (2H, dd, J = 6.5, 13.5 Hz, H-11b/dq, J = 3.5, 6.5 Hz, H-20), 2.65 (s, H-8), 1.99 (1H, m, H25a), 1.96 (1H, m, H-22a), 1.85 (1H, m, H-3), 1.84 (1H, m, H-22b), 1.49 (1H, m, H-28a), 1.39 (1H, m, H-28b), 1.31 (1H, m, H-24), 1.25 (3H, m, H-26a, 26b, 27a), 1.17 (3H, d, J = 6.5 Hz, H-30), 1.07 (1H, m, H-27b), 0.97 (1H, m, H-4a), 0.93 (3H, d, J = 7.0 Hz, H-31), 0.85 (1H, m, H-25b), 0.79 (3H, d, J = 6.5 Hz, H-6), 0.71 (1H, m, H-4b/ 3H, d, J = 3.0 Hz, H-5), 0.60 (3H, d, J = 7.0 Hz, H-32). Bis-S-MTPA Ester (4a). 1H NMR (500 MHz, DMSO-d6) δ 8.94 (1H, d, J = 9.0 Hz, H-18), 7.53−7.48 (5H, m, MTPA-Ar), 7.28−7.19 (5H, m, H-13, 14, 15, 16, 17), 5.39 (1H, dd, J = 3.5, 7.0 Hz, H-21), 5.09 (1H, dt, J = 6.0, 9.5 Hz, H-10), 5.06 (1H, m, H-29), 4.82 (1H, d, J = 12.0 Hz, H-2), 4.70 (1H, m, H-23), 3.48 (3H, s, MTPA-OCH3), 3.16 (1H, dd, J = 9.5, 12.5 Hz, H-11a), 2.90 (1H, dd, J = 6.5, 13.0 Hz, H-11b), 2.80 (1H, dq, J = 3.5, 6.5 Hz, H-20), 2.65 (3H, s, H-8), 1.97 (1H, m, H-22a), 1.92 (1H, m, H-22b), 1.86 (1H, m, H-3), 1.51 (2H, m, H-24, 25a), 1.44 (1H, m, H-28a), 1.28 (3H, d, J = 6.0 Hz, H-30), 1.26 (1H, m, H-28b), 1.23 (2H, m, H-26a, 26b, 27a), 1.06 (1H, m, H27b), 0.93 (1H, m, H-4a), 0.84 (1H, m, H-25b), 0.80 (3H, d, J = 6.5 Hz, H-6), 0.72 (3H, d, J = 7.0 Hz, H-31), 0.71 (4H, m, H-4b, 5), 0.68 (3H, d, J = 6.5 Hz, H-32). Bis-R-MTPA Ester (4b). 1H NMR (500 MHz, DMSO-d6) 8.93 (1H, d, J = 9.0 Hz, H-18), 7.49−7.43 (5H, m, MTPA-Ar), 7.27−7.19 (5H, m, H-13, 14, 15, 16, 17), 5.34 (1H, dd, J = 3.5, 8.0 Hz, H-21), 5.06 (dt, J = 6.0, 9.0 Hz, H-10), 5.01 (1H, m, H-29), 4.80 (d, J = 11.5 Hz, H-2), 4.66 (1H, dt, J = 3.0, 11.0 Hz, H-23 δ), 3.50 (s, MTPA-OCH3), 3.14 (dd, J = 9.5, 13.0 Hz, H-11a), 2.90 (2H, dd, J = 6.5, 13.5 Hz, H11b/dq, J = 3.5, 6.5 Hz, H-20), 2.65 (s, H-8), 1.99 (1H, m, H-25a), 1.96 (1H, m, H-22a), 1.85 (1H, m, H-3), 1.84 (1H, m, H-22b), 1.49 G

DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX

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The Journal of Organic Chemistry (1H, m, H-28a), 1.39 (1H, m, H-28b), 1.31 (1H, m, H-24), 1.25 (3H, m, H-26a, 26b, 27a), 1.17 (3H, d, J = 6.5 Hz, H-30), 1.07 (1H, m, H27b), 0.97 (1H, m, H-4a), 0.93 (3H, d, J = 7.0 Hz, H-31), 0.85 (1H, m, H-25b), 0.79 (3H, d, J = 6.5 Hz, H-6), 0.71 (1H, m, H-4b/3H, d, J = 3.0 Hz, H-5), 0.60 (3H, d, J = 7.0 Hz, H-32). Cell Viability Assay. HT22 cells, an immortalized mouse hippocampal cell line, were grown in Dulbecco’s modified Eagle’s medium (Corning, Manassas, VA) including 10% fetal bovine serum (Atlas, Fort Collins, CO) and penicillin/streptomycin (Gibco, Grand Island, NY). The cultures were maintained in a humidified atmosphere supplemented with 5% CO2 at 37 °C. In accordance with the manufacturer’s instruction, cell viability was evaluated by the EZ-CyTox cell viability assay kit (Daeil Lab Service, Seoul, Korea). HT22 cells were put on a 96-well plate at a density of 1x104 cells/well and were left untreated for 24 h. Then, 5 mM glutamate (Sigma, St. Louis, MO) was added to the cells simultaneously with the isolated compounds. After exposure for 24 h, the cells were treated with 10 μL of EZ-CyTox reagent for 30 min. The cell viability was measured at 450 nm on an E-Max microplate reader (Molecular Devices, Sunnyvale, CA) and determined as a percentage of the live control cells. Statistical Analysis. All data were obtained from three individual experiments and presented as the mean ± standard error of the mean. Statistical significance was determined using one-way analysis of variance with the Bonferroni correction for multiple comparisons. Values of p < 0.05 were considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.9b01511. Tables for NMR data of colletotrichamides A−E, LC− MS chromatograms of the FDLA derivatives of compounds 1, 4, and 5, Neuroprotective effect of compounds 1−5, Key 1H−1H DQF COSY and HMBCs of 1−5, NMR and MS spectra of compounds 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-531-82605355 (X.X.). *E-mail: [email protected]. Tel.: +82-2-901-8774 (S.H.S.). ORCID

Ki Sung Kang: 0000-0003-2050-5244 Sang-Jip Nam: 0000-0002-0944-6565 Sang Hee Shim: 0000-0002-0134-0598 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF-2018R1A2B6001733 and NRF2016R1A6A1A03007648).

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REFERENCES

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DOI: 10.1021/acs.joc.9b01511 J. Org. Chem. XXXX, XXX, XXX−XXX