Pyristriatins A and B - Pyridino-cyathane Antibiotics From the Basidiomycete Cyathus cf. striatus Christian Richter†, Soleiman E. Helaly†,‡, Benjarong Thongbai§, Kevin D. Hyde§ and Marc Stadler†* †
Department of Microbial Drugs, Helmholtz Centre for Infection Research; and German
Centre for Infection Research (DZIF), partner site Hannover/Braunschweig, Inhoffenstrasse 7, 38124 Braunschweig, Germany. ‡
Aswan University, Faculty of Science, Department of Chemistry, Aswan 81528, Egypt.
§
Institute of Excellence in Fungal Research and School of Science, Mae Fah Luang
University, Chiang Rai 57100, Thailand.
TABLE OF CONTENTS Taxonomic characterization of the specimen Pyristriatin A (1) Figure S3. Mass chromatogram of Pyristriatin A (1) Figure S4. 1H NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) Figure S5. 13C NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) Figure S6. COSY NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) Figure S7. HSQC NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) Figure S8. HMBC NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) Figure S9. NOESY NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) Pyristriatin B (2) Figure S10. Mass chromatogram of Pyristriatin B (2) Figure S11. 1H NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) Figure S12. 13C NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) Figure S13. COSY NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) Figure S14. HSQC NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) Figure S15. HMBC NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) Figure S16. NOESY NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) Striatin C (3) Figure S17. Mass chromatogram of Striatin C (3) Figure S18. 1H NMR spectrum (700 MHz, CD3OD) of Striatin C (3) Figure S19. 13C NMR spectrum (700 MHz, CD3OD) of Striatin C (3) Figure S20. COSY NMR spectrum (700 MHz, CD3OD) of Striatin C (3) Figure S21. HSQC NMR spectrum (700 MHz, CD3OD) of Striatin C (3) Figure S22. HMBC NMR spectrum (700 MHz, CD3OD) of Striatin C (3) Figure S23. NOESY NMR spectrum (700 MHz, CD3OD) of Striatin C (3)
References
Taxonomic characterization of the specimen Cyathus cf. striatus MFLU 15–1416
Figure S1. 1: a basidiomata. b basidiomata in top view showing peridioles. c peridioles and plications on the inner surface of peridium by longitudinal section. d-e basidiomata in side view showing fluffy hairs. Scale bars: a = 3 mm; b-d = 1 µm. 2: a basidiospores. b purse hyphae showing clamp-connections. Scale bars: a-b = 10 µm.
Description Basidiomata clavate to broadly obconic, without stipe, 5.5-7.0 mm in diam. high and 4.0-6.0 mm in diam. wide at the top, external peridium covered with shaggy or fluffy yellowish brown or buff hairs with age, inner peridium surface gray, darkening with age, distinctly plicate. Peridioles 3.0–3.5 mm in diam. wide, greyish brown to dark gray covered with a minutely grayish to greyish brown hairs. Basidiospores 13.83–17.14 × 8.06–9.72 µm (av. = 15.18 × 8.12, Q = 1.5–1.91, Qm = 1.70, n = 40), ellipsoid to broadly ellipsoid, some ovoid, rarely subglobose, hyaline, smooth, thin-walled (1.5–3 µm). Basidia not observed. Clamp connections present in all tissues. Habitat: On rotten woods in forest with Fagaceae species. Material examined: THAILAND, Chiang Mai Province, near Mushroom Research Center, 12th August 2014, leg. B. Thongbai, C. Richter & Marc Stadler (MFLU 15–1416).
Notes
Cyathus striatus (Huds.) Willd. belongs to the family Nidulariaceae. There are widely distributed in temperate regions.S1,S2 Cyathus cf. striatus MFLU 15–1416 characterized by external peridium covered with shaggy or fluffy yellowish brown or buff hairs with age, inner peridium surface gray, darkening with age, distinctly plicate. Greyish brown to dark gray covered with a minutely grayish to greyish brown hairs of peridioles. Cyathus cf. striatus MFLU 15–1416 looks very much like C. striatus on the account of shaggy or fluffy yellowish brown or buff hairs of peridium, distinctly plicate inner peridium while peridioles are greyish brown to dark gray. Based on morphological comparison between C. striatus and C. cf. striatus MFLU 15–1416. The latter has smaller basidioma and also smaller ellipsoid basidiospores. However, peridioles are covered with a distinctive minutely grayish to greyish brown hairs while peridioles of the C. striatus are smooth. Remarkably, this species was reported ranging pigments from pale to dark with varying significantly in size.S3,S4 Additionally, an initial BLAST search of the nrITS nucleotide sequence from NCBI database (http://www.ncbi.nlm.nih.gov/) gave C. striatus (EU784194), the closest hit with the maximum similarity of 94,5%. The BLAST search of the nrLSU nucleotide sequence of C. cf. striatus MFLU 15–1416 resulted in a high degree similarity of 99% with the only LSU Cyathus sequence of C. striatus DQ071742 that was available in the database.S5 However, these data appear insignificant for identification at the species level. The comparison of the 20 closest ITS BLAST results shows the phylogenetic relationship of MFLU 15–1416, Cyathus striatus, and C. stercoreus (Figure S2). The specimens, however, appeared morphologically much more similar to C. striatus than to the coprophilic C. stercoreus, which also does not normally grow on wood. The closest hit (100%), the sequence of “Cyathus sp. KJ195660” originated from an apparently unpublished study by Chinese researchers. The sequence of the next closest hit (JX082337 with 99,3%) belongs to an unidentifed Cyathus species, which was collected in French Guiana.S6 No morphological data are available for the either of the aforementioned specimens whose ITS sequence data showed the highest homology to MFLU-15-1416. Based on both morphological and molecular data we have addressed the strain as Cyathus cf. striatus MFLU 15–1416. Presently, a literature study on previous reports of Cyathus from warmer climates is being carried out and further morphological studies and molecular phylogenetic work will be carried out to verify that our strain represents a hitherto undescribed species.
Figure S2. Phylogenetic tree of the 20 closest ITS BLAST results
Pyristriatin A (1)
Figure S3. Mass chromatogram of Pyristriatin A (1)
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0
Figure S4. 1H NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) 3.5 3.0 2.5 2.0
0.95 2.70 2.60 2.19
1.93 0.75 0.98 1.99 1.90 3.32
0.71 0.93
2.17 0.23 1.00 8.78
0.88 0.56 9.42 0.53 0.86
1.00
0.83
1.00
0.82 0.82
1.32 1.32 1.18 1.18
3.29 3.29 3.27 3.27 3.16 3.16 3.13 3.13 2.36 2.36 2.35 2.35 2.24 2.24 1.64 1.64 1.56 1.56 1.54 1.54 1.54 1.54
3.50 3.50
5.23 5.23 5.19 5.19 4.92 4.92 4.89 4.89 4.73 4.73 4.72 4.72
5.85 5.85
0.81 0.81
1.31 1.31
3.32 3.32 3.30 3.30
1.07 1.07 1.06 1.06
6.47 6.47
8.45 8.45
3.59 3.59 3.31 3.31
3.31 3.31
4.90 4.90
P5_F1_1H.esp
1.5 Chemical Shift (ppm)
200 180 160 140 120 100
Figure S5. 13C NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1) 90.36 90.36
107.85 107.85
80 60
47.51 43.44 39.88 39.88 43.44 38.49 38.49 33.13 33.13 29.34 29.34 27.17 27.17 24.99 24.99 21.09 21.09
50.49 50.49
57.35 57.35
68.81 68.81 67.22 67.22
140.41 140.41 135.83 139.12 134.46 134.46
151.90 151.90 150.79 150.79 145.95 145.95
200.52 200.52
49.15 49.15
P5_F1_13C.esp
40 Chemical Shift (ppm)
2.19 2.60 2.70 3.32 1.90 1.99 1.93 8.78 1.00 2.17 9.42 1.00 1.00
1.59, 2.40 1.67, 2.40
1.43, 1.43
1.59, 1.59
3.63, 3.63
4
5.26, 4.95 5.90, 5.89 6.51, 6.51
F2 Chemical Shif t (ppm) 7 6 Figure S6. COSY NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1)
2
3.39, 4.77 6
4.95, 5.26
8 5
4
3
2
1
2.39, 28.90
1.10, 21.61
3.38, 43.29 3.63, 56.89 3.32, 26.76
4.94, 67.07
1.08, 22.04
50
2.27, 47.45 5.89, 89.93 4.77, 68.38 6.51, 107.48 100
150 F2 Chemical Shif t (ppm) 7 6 Figure S7. HSQC NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1)
5
4
3
2
1
5.89, 47.36 6.51, 57.20
5.89, 32.97
3.33, 29.14
2.28, 47.36
0.85, 38.35
2.28, 47.72
0.85, 39.74 2.28, 43.30
3.19, 68.66 6.51, 90.22
4.77, 47.36 6.51, 47.36 3.63, 107.71
8.49, 135.71 5.89, 107.71
8.49, 150.64
5.89, 150.64
4.77, 138.95
4.77, 135.71
2.39, 140.29 3.38, 135.71 2.28, 138.95
50
100
1.35, 90.22
150
1.08, 140.29
5.27, 200.39 200
F2 Chemical Shif t (ppm)
7
6
5
Figure S8. HMBC NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1)
4
3
2
1
2.19 2.60 2.70 3.32 1.90 1.99 1.93 8.78 1.01 2.17 9.42 1.00 1.00
5.89, 1.34
4.77, 1.08
3.34, 1.08
2.28, 0.85
0
2.39, 1.67 4.77, 2.28 4.77, 3.19
8.49, 3.38
3.63, 1.52 3.40, 2.28
4.77, 3.34
6.51, 3.63
2.28, 1.22 3.34, 1.34 2.28, 3.38
3.34, 4.77
2
1.34, 1.51
4
1.11, 2.39
2.28, 4.77
6 1.34, 5.89
3.64, 6.51 8 F2 Chemical Shif t (ppm) 7
6
Figure S9. NOESY NMR spectrum (700 MHz, CD3OD) of Pyristriatin A (1)
5
4
3
2
1
Pyristriatin B (2)
Figure S10. Mass chromatogram of Pyristriatin B (2)
8.5 8.0
Figure S11. H NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) 1
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
3.50 3.18 1.35 3.15
1.26 3.51 1.29 3.38
2.39 1.11
3.48 1.86 6.63 1.03 1.12
10.34 1.22
1.06
1.08
1.00
1.00
0.89 0.89
3.29 3.29 3.27 3.27 3.16 3.16 3.12 3.12 2.35 2.35 2.35 2.35 2.19 2.19 1.64 1.64 1.62 1.62 1.55 1.55 1.55 1.55 1.53
3.58 3.58 3.35 3.35 3.30 3.30
5.23 5.23 5.19 5.19 4.94 4.94 4.93 4.93 4.89 4.86 4.86 4.89 4.72 4.72 4.71 4.71
5.92 5.92
6.58 6.58 6.58 6.58
8.46 8.46
1.07 1.07 1.06 1.06 1.04 1.04
3.31 3.31
0.79 0.79
1.32 1.32
3.31 3.31
3.50 3.50
4.90 4.90
P5_F2_1H.esp
Chemical Shift (ppm)
200 190 180 170 160 150 140 130 120 110 100
Figure S12. 13C NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2) 90
89.27 89.27
151.97 151.97
80 70 60 50
21.00 21.00
29.36 29.36
24.92 24.92 22.03 22.03
32.07 32.07
47.78 43.52 43.52 47.78 39.97 39.97 38.38 38.38
50.43 50.43
56.64 56.64
68.69 68.69 67.20 67.20
107.77 107.77
150.99 150.99 146.11 146.11 140.44 139.04 139.04 135.85 135.85 135.10 135.10
200.52 200.52
49.15 49.15
P5_F2_13C.esp
Chemical Shift (ppm)
1.42, 1.59 5.96, 3.18
4.75, 2.23
3.18, 2.23
4.75, 3.38
3.18, 3.38
4.75, 4.75
3.18, 4.75 4.75, 3.18
2.23, 3.18
3.38, 4.75
2.23, 4.75
1.68, 2.38
1.42, 0.92
2
1.59, 2.38 4
6.61, 5.96 6
3.18, 5.96 5.96, 6.61
F2 Chemical Shif t (ppm) 7
3.18, 8.50
6
5
Figure S13. COSY NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2)
4
8 3
2
1
0
1.42, 38.21
3.38, 43.43 3.55, 56.49 4.95, 67.05 3.39, 50.21
40
1.11, 21.88 2.39, 29.23
60
2.23, 48.33
5.96, 89.20
80
4.76, 68.53 6.62, 107.61
100 120 140 160
F2 Chemical Shif t (ppm) 7
6
5
Figure S14. HSQC NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2)
4
3
2
1
5.96, 20.90
3.32, 29.20
5.96, 47.64 6.62, 56.53
1.08, 21.84
1.36, 47.64
0
4.76, 47.64 3.18, 68.52
6.62, 89.10 8.50, 43.36
0.83, 39.82
4.76, 48.31 3.54, 107.63
4.76, 135.76
8.50, 150.81 5.96, 150.80
2.23, 47.64 1.42, 138.89 2.23, 138.89
5.96, 134.96 4.76, 138.89 6.62, 150.81
50 0.93, 89.10
3.38, 135.76 2.39, 140.29
100
1.36, 89.10
2.23, 140.29 1.11, 140.29
150
200 F2 Chemical Shif t (ppm) 7
6
5
Figure S15. HMBC NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2)
4
3
2
1
0
3.15 1.35 3.18 3.50 3.38 1.29 3.51 1.26 2.39 7.11 1.86 3.48 10.34
3.32, 1.08
0.83, 1.42
6.62, 1.58
0.83, 1.68
3.40, 2.23 4.76, 1.08
5.96, 1.36
4.76, 2.23 8.50, 3.37 6.62, 3.54
4.76, 3.37
3.40, 4.76 4.76, 3.19 3.32, 4.76
2.23, 0.83 3.32, 1.36
2.23, 0.93
2.23, 3.37
1.11, 3.32
2
0.93, 1.58
0.83, 2.23 4
1.08, 3.32
2.23, 4.76
1.08, 4.76
6
1.36, 5.96 3.54, 6.62 8 F2 Chemical Shif t (ppm) 7
6
5
Figure S16. NOESY NMR spectrum (700 MHz, CD3OD) of Pyristriatin B (2)
4
3
2
1
0
Striatin C (3)
Figure S17. Mass chromatogram of Striatin C (3)
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5
Figure S18. 1H NMR spectrum (700 MHz, CD3OD) of Striatin C (3) 4.0 3.5 3.0
2.39 0.99 2.22 2.27 1.07 1.09 3.00 6.31 3.20
0.99 1.04 2.82 1.05 0.11 1.20 0.96 4.07 1.00
0.97 10.67 2.10
0.97
1.00
2.5 2.0
1.06 1.06 1.05 1.05 1.01 1.01 1.00 1.00
3.43 3.43 3.36 3.36 3.35 3.35 3.35 3.35 3.34 3.34 3.03 3.03 3.01 3.01 2.38 2.38 2.37 2.37 2.36 2.36 2.27 2.27 1.75 1.75 1.74 1.62 1.62 1.61 1.61 1.60 1.60 1.59 1.59
3.69 3.69
5.28 5.28 5.28 5.28 4.98 4.98 4.69 4.69 4.68 4.68 4.66 4.66 3.92 3.92 3.92 3.92 3.91 3.91
6.15 6.15 6.14 6.14 6.14 6.14 6.13 6.13 6.13 6.13 6.12 6.12 6.12 6.12 6.12 6.12
1.26 1.26 1.06 1.06
3.60 3.60
4.94 4.94
P7_F2_1H.esp
1.5 Chemical Shift (ppm)
180 160 140 120 100
Figure S19. 13C NMR spectrum (700 MHz, CD3OD) of Striatin C (3) 80 60 48.24 48.24 41.44 41.44 40.71 40.71 38.34 38.34 30.28 30.28 29.49 29.49 22.42 22.42 22.21 22.21 21.76 21.76
56.67 56.67
70.23 65.21 65.21
50.57 50.57
81.34 81.34
99.35 99.35 97.99 91.59 91.59
107.25 107.25
140.70 140.70 138.13 138.13 136.37 136.37 133.36 133.36
49.15 49.15
P7_F2_13C.esp
40 Chemical Shift (ppm)
1.57, 1.57
1.61, 1.61
1
2.27, 2.27
4.68, 2.27
2
2.37, 1.74
4.68, 3.02
2.37, 2.37
3.57, 3.69
1.47, 1.80 1.00, 3.43
1.74, 2.37
3
6.13, 3.02 1.06, 3.43 4.98, 4.99
4
3.57, 3.91
6.13, 4.67
5
2.27, 4.67
3.02, 4.68 4.67, 6.13
6 F2 Chemical Shif t (ppm)
5.0
4.5
4.0
Figure S20. COSY NMR spectrum (700 MHz, CD3OD) of Striatin C (3)
3.5
3.0
2.5
2.0
1.5
1.0
21 21
77 99
26 26
25 25
3.43, 26.70
1.27, 21.54 2.37, 29.26
20
1.60, 38.06 3.60, 56.45 4.67, 70.03 (9, 9) 25, 25, 99 7 26 26 21 21
3.02, 48.50
4.69, 91.31 (7, 7) 5.28, 99.08 (26, 26)
40
1.06, 24.36
60
2.27, 48.08
80
3.92, 70.03 (25, 25)
100 4.98, 107.02 (21, 21)
F2 Chemical Shif t (ppm)
5.0
4.5
4.0
Figure S21. HSQC NMR spectrum (700 MHz, CD3OD) of Striatin C (3)
120
3.5
3.0
2.5
2.0
1.5
1.0
3.43, 29.32 2.27, 41.27
4.67, 41.27
1.26, 41.27
20
3.43, 22.28
5.28, 56.55
3.69, 69.99 6.13, 48.09 4.67, 48.09 4.98, 91.40 3.69, 97.80 6.13, 69.99 4.98, 81.15 6.13, 99.24 4.67, 133.24
40 2.27, 69.99 3.02, 41.27
1.06, 40.56 2.27, 50.40
60 80
3.02, 81.15 3.43, 137.95 2.27, 137.95
3.60, 99.24
100
1.26, 91.40
120
1.06, 137.95
140 4.67, 136.25 F2 Chemical Shif t (ppm)
5.0
3.43, 140.56 4.5
4.0
Figure S22. HMBC NMR spectrum (700 MHz, CD3OD) of Striatin C (3)
3.5
2.27, 140.56 3.0
2.5
1.06, 140.56 2.0
1.5
1.0
3.20 6.31 3.00 1.09 1.07 2.27 2.22 2.39 4.07 1.20 2.82 2.10 10.67
1.26, 1.60
3.02, 1.77
2.27, 1.06
4.69, 1.26
2 4.69, 3.02 4.67, 3.43
4.98, 3.57 5.27, 3.60 6.13, 4.67 4.69, 3.93
2.26, 3.02 3.02, 2.26 3.43, 4.67
3.93, 3.69 3.69, 3.93
5.27, 6.13 4.67, 6.13 6.13, 5.27
F2 Chemical Shif t (ppm)
5.0
1
3 4
2.26, 4.67
3.02, 4.69
5
1.26, 4.69
3.60, 5.27 3.02, 5.27
4.5
4.0
Figure S23. NOESY NMR spectrum (700 MHz, CD3OD) of Striatin C (3)
3.5
3.0
2.5
6
2.0
1.5
1.0
References S1
Zhao, R.L.; Jeewon, R., Desjardin, D.E.; Soytong, K.; Hyde, K.D. Mycologia 2007, 99, 385-395.
S2
Cruz, R.H.S.F.; Assis N.M.; Silva, M.A.; Baseia, I.G. Mycosphere 2014, 5 (4), 531540.
S3
Kuo, M. 2014, web site: http://www.mushroomexpert.com/cyathus_striatus.html
S4
Wood, M.; Stevens, F. 2015, web site: http://www.mykoweb.com/CAF/species/Cyathus_striatus.html
S5
Garnica, S.; Weiss, M.; Walther, G.; Oberwinkler, F. Mycological Research 2007, 111, 1019-1029.
S6
Berrin, J.-G.; Navarro, D.; Couturier, M.; Olive, C.; Grisel, S.; Haon, M.; Taussac, S.; Lechat, C.; Courtecuisse, R.; Favel, A.; Coutinho, P.M.; Lesage-Meessen; L. Appl. Environ. Microbiol. 2012.