海洋源真菌Trichoderma brevicompactum NTU439之二次代謝產物之生物活性及化學結構分析

; N

Graduate Institute of Biochemical Sciences College of Life Science

National Taiwan University Master Thesis

Trichoderma brevicompactum NTU439 5 T

Biological Activities and Chemical Structures of Secondary Metabolites Isolated from Marine-Derived Fungus Trichoderma

brevicompactum NTU439

Tzu-Jie Huang

3 ;

Advisor: Shih-Hsiung Wu, Ph.D.

107 7

July 2018

; 2

1 1 ;

0 4 ， N 2

， ： 8 09

0

2 1 1

0 1 1)- N0

0

0 N 0 1

0 1 0

0 paper 8 2

0 ， N0 0 1

0 8 0 ; y0

9 0 、 0 2

2018 8

0 9 U

2 80 0

0 t

4 B 2

。 0 4 、 2

0 Candida albicans Cryptococcus

neoformans

0

brevicompactum

0 U

0 2 ， U

0 e U 0

trichobrevidin A 1 trichobrevidin B alamethicin F50/J)

2 0 、 7(

t 0 2

Trichoderma brevicompactum1

ABSTRACT

Ever since the introduction of antibiotics into clinical practice, the overuse of antibiotics has led to the alarming emergence of drug resistance throughout many infectious microorganisms. Though the occurance of antifungal resistance is less frequent than antibacterial resistance, more than half of antifungal agents fail to efficiently inhibit clinical fungal infections. Therefore, there is an urgent need for new antifungal agents to overcome drug resistance in fungi. Natural products with diverse set of structures have been recognized as a reservoir for new therapeutic entities for given bioactivities. In our study, we utilized fungal pathogens Candida

bioassay-guided screening in order to isolate bioactive and hitherto unreported compounds from marine-derived

identified by spectroscopic analysis, which revealed three new compounds 1 trichobrevidin A , 2 trichobrevidin B and 8 alamethicin F50/J).

Compounds 3 and 5-9 showed biological activities against pathogenic fungi. In addition, compound 2 showed anti-hepatocellular carcinoma activity.

Keywords marine-derived Trichoderma brevicompactum, antifungal

TABLE OF CONTENTS

... ii

... iii

... iv

ABSTRACT ... v

TABLE OF CONTENTS ... vi

LIST OF FIGURES ... ix

LIST OF TABLES ... xiv

LIST OF ABBREVIATIONS ... xvi

1. INTRODUCTION ... 1

1.1 Antifungal Resistance Is a Global Challenge ... 1

1.2 Natural Products Play a Vital Role in Drug Discovery ... 3

1.3 Objective ... 4

2. LITERATURE REVIEW ... 5

3. MATERIALS AND METHODS ... 40

3.1 Fungal Sources ... 40

3.2 Fermentation ... 40

3.2.1 Liquid Fermentation ... 40

3.2.2 Solid Fermentation ... 40

3.3 Extraction ... 41

3.3.1 Extraction of Liquid Fermentation ... 41

3.3.2 Extraction of Solid Fermentation ... 41

3.5 Spectroscopic Tool ... 42

3.5.1 Nuclear Magnetic Resonance ... 42

3.5.2 Mass Spectrometry ... 43

3.5.3 Fourier Transform Infrared Spectroscopy ... 43

3.5.4 Optical Rotation ... 43

3.6 In Vitro Bioactivity Test ... 44

3.6.1 Disk Diffusion Assays ... 44

3.6.2 Minimum Inhibitory Concentrations ... 44

3.6.3 Sulforhodamine B Assays ... 45

4. RESULTS ... 46

4.1 Optimal Fermentation Condition for Trichoderma

4.2 Flow Chart for Separation Process of Trichoderma

4.2.1 Flow Chart of Compound Purification from T.

4.2.2 Flow Chart of Compound Purification from T.

4.3 Structure Elucidation of Compound 1 ... 50

4.4 Structure Elucidation of Compound 2 ... 58

4.5 Structure Elucidation of Compound 3 ... 65

4.6 Structure Elucidation of Compound 4 ... 73

4.7 Structure Elucidation of Compound 5 ... 92

4.8 Structure Elucidation of Compound 6 ... 97

4.9 Structure Elucidation of Compound 7 ... 101

4.10 Structure Elucidation of Compound 8 ... 105

4.11 Structure Elucidation of Compound 9 ... 111

4.13 Cytotoxicity Activities of Compounds 1-2 ... 116

5. DISCUSSION ... 117

5.1 Review of Trichodermin ... 117

5.2 Review of Alamethicin ... 118

5.3 Biosynthetic Pathway of Compounds 1 and 3 ... 119

5.4 Biosynthesis of Compounds 4-9 ... 120

6. REFERENCES ... 122

7. APPENDIX ... 137

LIST OF FIGURES

Figure 1. Morphology of T. brevicompactum cultivated at PDA

medium... 47

Figure 2. Antimicrobial assay against C. albicans in different cultivation conditions of T. brevicompactum ... 47

Figure 3. Mass cultivation of T. brevicompactum by using (a) MY for liquid fermentation and (b) quiano for solid fermentation ... 47

Figure 4. Mass spectrometry of compound 1 ... 54

Figure 5. FTIR spectrum of compound 1 ... 54

Figure 6.

H NMR spectrum of compound 1 ... 55

Figure 7.

C NMR spectrum of compound 1 ... 55

Figure 8.

H-

H COSY spectrum of compound 1 ... 56

Figure 9. HSQC spectrum of compound 1 ... 56

Figure 10. HMBC spectrum of compound 1 ... 57

Figure 11. ROESY spectrum of compound 1 ... 57

Figure 12. Mass spectrometry of compound 2 ... 61

Figure 13. FTIR spectrum of compound 2 ... 61

Figure 14.

H NMR spectrum of compound 2 ... 62

Figure 15.

C NMR spectrum of compound 2 ... 62

Figure 16.

H-

H COSY spectrum of compound 2 ... 63

Figure 17. HSQC spectrum of compound 2 ... 63

Figure 18. HMBC spectrum of compound 2 ... 64

Figure 19. Mass spectrometry of compound 3 ... 69

Figure 21.

H NMR spectrum of compound 3 ... 70

Figure 22.

C NMR spectrum of compound 3 ... 70

Figure 23.

H-

H COSY spectrum of compound 3 ... 71

Figure 24. HSQC spectrum of compound 3 ... 71

Figure 25. HMBC spectrum of compound 3 ... 72

Figure 26. ROESY spectrum of compound 3 ... 72

Figure 27. Mass spectrometry of compound 4 ... 80

Figure 28. CID spectrum of the ion m/z 1189 within 600-1200 of compound 4 ... 80

Figure 29. HCD spectrum of the ion m/z 1189 within 100-700 of compound 4 ... 81

Figure 30. CID spectrum of the ion m/z 774 within 400-800 of compound 4 ... 81

Figure 31. HCD spectrum of the ion m/z 774 within 100-500 of compound 4 ... 82

Figure 32. FTIR spectrum of compound 4 ... 82

Figure 33.

H NMR spectrum of compound 4 ... 83

Figure 34. Detailed view of

H NMR spectrum of compound 4 ... 84

Figure 35.

C NMR spectrum of compound 4 ... 85

Figure 36. Detailed view of

C NMR spectrum of compound 4 ... 86

Figure 37.

H-

H TOCSY spectrum of compound 4 ... 87

Figure 38. Detailed view of

H-

H TOCSY spectrum of compound 4

Figure 40. HSQC spectrum of compound 4 ... 90 Figure 41. HMBC spectrum of compound 4 ... 91 Figure 42. Comparison of

H NMR spectra of compounds 4 and 5 .. 94 Figure 43. Mass spectrometry of compound 5 ... 94 Figure 44. CID spectrum of the ion m/z 1189 within 600-1200 of

compound 5 ... 95 Figure 45. HCD spectrum of the ion m/z 1189 within 100-700 of

compound 5 ... 95 Figure 46. CID spectrum of the ion m/z 774 within 400-800 of

compound 5 ... 96 Figure 47. HCD spectrum of the ion m/z 774 within 100-500 of

compound 5 ... 96 Figure 48. Mass spectrometry of compound 6 ... 98 Figure 49. CID spectrum of the ion m/z 1203 within 600-1200 of

compound 6 ... 98 Figure 50. HCD spectrum of the ion m/z 1203 within 100-700 of

compound 6 ... 99 Figure 51. CID spectrum of the ion m/z 774 within 400-800 of

compound 6 ... 99 Figure 52. HCD spectrum of the ion m/z 774 within 100-500 of

compound 6 ... 100 Figure 53. Mass spectrometry of compound 7 ... 102 Figure 54. CID spectrum of the ion m/z 1203 within 600-1200 of

compound 7 ... 102

compound 7 ... 103 Figure 56. CID spectrum of the ion m/z 775 within 400-800 of

compound 7 ... 103 Figure 57. HCD spectrum of the ion m/z 775 within 100-500 of

compound 7 ... 104 Figure 58. Mass spectrometry of compound 8 ... 107 Figure 59. CID spectrum of the ion m/z 1189 within 600-1200 of

compound 8 ... 107 Figure 60. HCD spectrum of the ion m/z 1189 within 100-700 of

compound 8 ... 108 Figure 61. CID spectrum of the ion m/z 789 within 400-800 of

compound 8 ... 108 Figure 62. HCD spectrum of the ion m/z 789 within 100-500 of

compound 8 ... 109 Figure 63. Chemical shifts of Iva

in DMSO (a)

H-NMR data (b)

13

C-NMR data ... 110 Figure 64. Mass spectrometry of compound 9 ... 112 Figure 65. CID spectrum of the ion m/z 1189 within 600-1200 of

compound 9 ... 112 Figure 66. HCD spectrum of the ion m/z 1189 within 100-700 of

compound 9 ... 113 Figure 67. CID spectrum of the ion m/z 775 within 400-800 of

compound 9 ... 113

Figure 69. Biosynthetic pathway of compounds 1 and 3 ... 120

Figure 70. Representative scheme of substrate activation in the multi-

enzymatic peptide synthesis system ... 121

LIST OF TABLES

Table 1. Phenethyl alcohols ... 5

Table 2. Pyrones ... 5

Table 3. Koninginins ... 5

Table 4. Steroids ... 6

Table 5. Ergosterol derivatives ... 6

Table 6. Viridiofungins ... 7

Table 7. Pyridones ... 7

Table 8. Pirrolidindiones ... 7

Table 9. Butenolides ... 8

Table 10. Lactones ... 8

Table 11. Isocyano metabolites ... 9

Table 12. Epipolythiodiketopiperazines ... 10

Table 13. Aminolipopeptides ... 10

Table 14. Azaphilones ... 11

Table 15. Anthraquinones ... 11

Table 16. Xanthones ... 12

Table 17. Bisabolanes ... 12

Table 18. Decalins ... 12

Table 19. Statins ... 13

Table 23. Daucanes ... 15

Table 24. Trichothecenes ... 15

Table 25. Acoranes ... 15

Table 26. Cyclopentenones ... 16

Tables 27. Cyclonerodiol derivatives ... 16

Table 28. Polyketides ... 16

Table 29. Others ... 17

Table 30. Heptelidic acid ... 17

Table 31. Peptaibols ... 18

Table 32. Antimicrobial activities of liquid-fermentation fractions .. 48

Table 33. Antimicrobial activities of solid-fermentation fractions .... 49

Table 34.

H and

C NMR (CD

OD, 500 and 125 MHz) for compound 1 ... 53

Table 35.

H and

C NMR (CD

OD, 500 and 125 MHz) for compound 2 ... 60

Table 36.

H and

C NMR (CD

OD, 500 and 125 MHz) for compound 3 ... 68

Table 37.

H and

C NMR (DMSO-d

, 500 and 125 MHz) for compound 4 ... 76

Table 38. NMR data used to assess the absolute configuration of Iva residues in a right-handed helical peptide ... 110

Table 39. MIC values of compounds 1-9 ... 115

Table 40. Inhibition of cell proliferation by compounds 1 and 2 at

different concentrations ... 116

LIST OF ABBREVIATIONS

ACN Acetonitrile

ALM Alamethicin

COSY Correlation Spectroscopy

calcd calculated

CD3OD Deuterated Methanol

DMSO-d6 Deuterated Dimethyl Sulfoxide

EtOAc Ethyl Acetate

FTIR Fourier Transform Infrared Spectroscopy HPLC High Performance Liquid Chromatography HSQC Heteronuclear Single Quantum Coherence HMBC Heteronuclear Multiple Bond Coherence

HR-ESI-MS High-resolution Electrospray Ionization Mass Spectrometry

MeOH Methanol

MIC Minimum Inhibitory Concentration

NOESY Nuclear Overhauser Effect Spectroscopy

NMR Nuclear Magnetic Resonance

PFP Pentafluorophenyl

ppm parts per million

ROESY Rotating Frame Overhauser Effect Spectroscopy

RP Reversed Phase

RI Refractive Index

[α]D Specific Rotation

TOCSY Total Correlation Spectroscopy

TLC Thin Layer Chromatography

UV Ultraviolet

1. INTRODUCTION

1.1 Antifungal Resistance Is a Global Challenge

Since the discovery of Penicillin in 1929 by Alexander Fleming, a growing

number of antibiotics has prevented countless cases of infectious diseases and improved

the quality of life for people. However, the fight against infections never ends as the

excessive use of antibiotics continues to accelerate the emergence of antibiotic

resistance. In past decades, multidrug resistant (MDR) bacteria have become a threat to

public health¹.

In addition, antibiotic resistance also occurs in pathogenic fungi². Invasive fungal

infections have killed at least 1.5 millions of people every year³. The most prevalent

fungal pathogens are Candida, Aspergillus and Cryptococcus spp⁴. The

immunosuppressive patients, including those that deal with HIV/AIDS and organ

transplantation, are highly susceptible to be infected by these pathogens. With the

dramatic increasing number of the immunosuppressive patients in recent years, the

widespread use of antifungal agents further aggravates drug resistance.

There are four available major classes of antifungal agents⁵, including polyenes,

Polyenes, such as amphotericin B (AmB), bind to ergosterol and thus disrupt the

membranes, leading to leakage of cellular contents. 5-Fluorocytosine (5-FC),

pyrimidine analogs, are converted into 5-fluorouracil by fungal cells, which destabilize

nucleic acids and therefore result in growth arrest. Echinocandins block the cell wall

biosynthesis via noncompetitive inhibition of the !-(1,3)-D-glucan synthase. Azoles

can specifically target the crucial biosynthetic enzyme of ergosterol, which catalyzes

lanosterol 14"-demethylation.

Along with the overuse of these antifungal agents, drug-resistant strains have been

gradually emerged⁶. In 1977, Aspergillus fumigatus was reported to show resistance to

itraconazole⁷. Nowadays, more investigations have indicated that Aspergillus species

no longer respond to azole and 5-FC. In addition, Aspergillus terreus often shows

intrinsic resistance to AmB. Candidasis is one of the most common infectious diseases.

Among Candida species, C. albican is widely known for its resistance to fluconazole, and this mechanism has already been identified. C. krusei and C. lusitaniae are intrinsically resisted to fluconazole and AmB, respectively. Recently, C. auri, a new

According to the research in 1999, echinocandins failed to treat cryptococcosis⁹.

Overall, because of the limited therapeutic options for invasive fungal infections, the

development of new antifungal drugs is in urgent need.

1.2 Natural Products Play a Vital Role in Drug Discovery

Natural products (NPs) often provide the distinctive bioactive compounds for drug discovery¹⁰. Statistically, more than half of FDA-approved drugs are unaltered NPs and NPs derivatives or mimetics. With extensive exploration of terrestrial resources, scientists have begun to develop marine natural products (MNPs)¹¹. Since 1985, more than 4000 bioactive compounds from marine organisms have been reported¹². For example, in 2004, the first MNPs approved by FDA was ziconotide, which was extracted from cone snail, Conus magus¹³. Furthermore, these MNPs have been reported to exhibit the potential of treating drug-resistant fungal infections.¹⁴

Over the past few years, marine fungi have been largely emphasized due to the increasing number of new compounds¹⁵. It is believed that marine-derived fungi, thriving in the high salt environment, may produce a large variety of metabolites compared to terrestrial-derived ones. In contrast to the terrestrial Trichoderma spp.,

marine-derived Trichoderma spp. were isolated from mussels¹⁶, followed by a new polyketide compound was extracted from marine-derived Trichoderma harzianum¹⁷ in the same year. Owing to the numerous applications of secondary metabolites from Trichoderma spp., it is believed that marine-derived Trichoderma genus are a potential source of unique bioactive compounds.

1.3 Objective

The secondary metabolites of Trichoderma genus have been extensively investigated. However, Trichoderma brevicompactum is a relatively new species published in 2004¹⁸. The information on this species is still in scarcity. Preliminary screening has revealed that the extraction from marine-derived Trichoderma brevicompactum possess outstanding antimicrobial activity against both C. albicans and C. neoformans. In this study, our goal is to isolate the bioactive and unexplored- secondary metabolites for the development of next-generation of antifungal drugs.

2. LITERATURE REVIEW

Until now, Trichoderma spp. have been discovered to metabolize approximately

240 compounds, which are categorized into 31 different groups (Tables1-13)

according to their structural similarity¹⁹.

Table 1. Phenethyl alcohols

Compounds Species

2-Phenylethanol (10) Trichoderma harzianum²⁰ 4-Hydroxyphenethyl alcohol (11) Trichoderma koningii²¹

Table 2. Pyrones

Compounds Species

6-Pentyl-"-pyrone (12) Trichoderma viride²² 6-(Pent-1-enyl)-"-pyrone (13) Trichoderma viride²³ Viridepyronone (14) Trichoderma viride²⁴ Trichoderpyrone (15) Trichoderma gamsii²⁵ Cytosporone S (16) Trichoderma sp. FKI-6626²⁶

Table 3. Koninginins

Compounds Species

Koninginin A (17) Trichoderma koningii²⁷

Koninginin C (19) Trichoderma koningii²⁹ Koninginin D (20) Trichoderma koningii³⁰ 7-O-Methylkoninginin D (21) Trichoderma koningii³¹ Koninginin E (22) Trichoderma koningii³² Koninginin L (23) Trichoderma koningii³³ Koninginin M (24) Trichoderma koningii³³ Koninginin G (25) Trichoderma aureoviride³⁴ Koninginin I (26) Trichoderma neokongii³⁵ Koninginin J (27) Trichoderma neokongii³⁵ Koninginin K (28) Trichoderma neokongii³⁵

Table 4. Steroids

Compounds Species

Viridin (29) Trichoderma viride³⁶

Viridiol (30) Trichoderma viride³⁷

Virone (31) Trichoderma virens³⁸

Table 5. Ergosterol derivatives

Compounds Species

Lanosterol (32) Trichoderma pseudokoningii³⁹

Ergokonin B (36) Trichoderma koningii⁴⁰

Table 6. Viridiofungins

Compounds Species

Viridiofungin A (37) Trichoderma viride⁴¹ Viridiofungin B (38) Trichoderma viride⁴¹ Viridiofungin C (39) Trichoderma viride⁴¹ Viridiofungin A1 (40) Trichoderma viride⁴² Viridiofungin A2 (41) Trichoderma viride⁴² Viridiofungin A3 (42) Trichoderma viride⁴² Viridiofungin A4 (43) Trichoderma viride⁴² Viridiofungin B2 (44) Trichoderma viride⁴² Viridiofungin Z2 (45) Trichoderma viride⁴²

Table 7. Pyridones

Compounds Species

Harzianopyridone (46) Trichoderma harzianum⁴³

Trichodin A (47) Trichoderma sp. MF-106⁴⁴

Trichodin B (48) Trichoderma sp. MF-106⁴⁴

Pyridoxatin (49) Trichoderma sp. MF-106⁴⁴

Table 8. Pirrolidindiones

Harzianic acid (50) Trichoderma harzianum⁴⁵ Demethylharzianic (51) Trichoderma harzianum⁴⁶ Homoharzianic (52) Trichoderma harzianum⁴⁶

MR93B (53) Trichoderma harzianum⁴⁷

Melanoxadin (54) Trichoderma sp. ATF-606⁴⁸

Melanoxazal (55) Trichoderma sp. ATF-451⁴⁹

Table 9. Butenolides

Compounds Species

Harzianolide (56) Trichoderma harzianum⁴⁵

Dehydroharzianolide (57) Trichoderma harzianum⁵⁰ T39butenolide (58) Trichoderma harzianum⁵¹ 5-Hydroxyvertinolide (59) Trichoderma longibrachiatum⁵² Trichocyalide A (60) Trichoderma sp. FKI-5513⁵³ Trichocyalide B (61) Trichoderma sp. FKI-5513⁵³

Table 10. Lactones

Compounds Species

#-Decalactone (62) Trichoderma harzianum⁵⁴ Harzialactone A (63) Trichoderma harzianum⁵⁵

Cremenolide (67) Trichoderma cerinum⁵⁷ 5-Hydroxy-4-hydroxymethyl-2H-pyran-

2-one (68)

Trichoderma sp.⁵⁸

(5-Hydroxy-2-oxo-2H pyran-4-yl) methyl acetate (69)

Trichoderma sp.⁵⁸

Table 11. Isocyano metabolites

Compounds Species

Dermadin-type

Dermadin (70) Trichoderma viride⁵⁹

Isonitrinic acid F (71) Trichoderma hamatum⁶⁰

Spirolactone (72) Trichoderma hamatum⁶⁰

(E)-3-(1,2-Dihydroxy-4-isocyano cyclopent-3-en-1-yl) acrylic acid (73)

Trichoderma hamatum⁶⁰

Methyl 3-(3-isocyano-6oxabicyclo [3.1.0]hex-2-en-5-yl) acrylate (74)

Trichoderma hamatum⁶¹

Methyl 3-(3-isocyanocyclopent-2- enylidine) propionate (75)

Trichoderma hamatum⁶¹

Trichoviridin-type

Tirchoviridin (76) Trichoderma sp. Tk-1⁶²

Isonitrins A (77) Trichoderma hamatum⁶³

Isonitrins B (78) Trichoderma hamatum⁶³

MR304A (80) Trichoderma harzianum⁶⁴

MR566A (81) Trichoderma harzianum⁴⁷

MR566B (82) Trichoderma harzianum⁴⁷

Homothallin I (83) Trichoderma koningii⁶⁵ Homothallin II (84) Trichoderma koningii⁶⁶ 3-Dimethylamino-5-hydroxy-5-vinyl-2-

cyclopenten-1-one (85)

Trichoderma koningii⁶⁷

Table 12. Epipolythiodiketopiperazines

Compounds Species

Trichodermamide A (86) Trichoderma virens⁶⁸ Trichodermamide B (87) Trichoderma virens⁶⁸

Dithioaspergillazine A (88) Trichoderma brevicompactum⁶⁹ Aspergillazine A (89) Trichoderma brevicompactum⁶⁹ Pretrichodermamide A (90) Trichoderma sp. BCC5926⁷⁰

Table 13. Aminolipopeptides

Compounds Species

Trichoderin A (91) Trichoderma sp.⁷¹

Trichoderin A1 (92) Trichoderma sp.⁷¹

Compounds Species

Harziphilone (94) Trichoderma harzianum⁷²

Fleephilone (95) Trichoderma harzianum⁷²

T22azaphilone (96) Trichoderma harzianum⁵¹

Table 15. Anthraquinones

Compounds Species

Trichodermaol (97) Trichoderma sp.⁷³

Pachybasin (98) Trichoderma viride⁷⁴

Chrysophanol (99) Trichoderma viride⁷⁴

Emodin (100) Trichoderma viride⁷⁴

1,3,6,8-Tetrahydroxyanthraquinone (101)

Trichoderma viride⁷⁵

1-Acetyl-2,4,5,7-tetrahydroxy anthraquinone (102)

Trichoderma viride⁷⁵

Trichodermaquinone (103) Trichoderma aureoviride⁷⁶ Coniothranthraquinone 1 (104) Trichoderma aureoviride⁷⁶ Isorhodoptilometrin (105) Trichoderma aureoviride⁷⁶ 2-Methylquinizarin (106) Trichoderma aureoviride⁷⁶ ω-Hydroxyemodin (107) Trichoderma aureoviride⁷⁶ 1-Hydroxy-3-methyl-anthraquinone

(108)

Trichoderma harzianum⁷⁷

(109)

1,5-Dihydroxy-3-hydroxymethyl-9,10- anthraquinone (110)

Trichoderma harzianum⁷⁸

1,7-Dihydroxy-3-hydroxymethyl-9,10- anthraquinone (111)

Trichoderma harzianum⁷⁸

ω-Hydroxypachybasin (112) Trichoderma harzianum⁷⁸

Table 16. Xanthones

Compounds Species

Trichodermaxanthone (113) Trichoderma aureoviride⁷⁶

Aloesone (114) Trichoderma aureoviride⁷⁶

2-(2'S-Hydroxypropyl)-5-methyl-7- hydroxychromone (115)

Trichoderma aureoviride⁷⁶

5-Hydroxy-3-hydroxymethyl-2-methyl- 7-methoxychromone (116)

Trichoderma harzianum⁷⁹

Table 17. Bisabolanes

Compounds Species

Trichoderic acid (117) Trichoderma sp. PR-35⁸⁰ (+)-12-Hydroxysydonic acid (118) Trichoderma brevicompactum⁶⁹

Trichoharzin (119) Trichoderma harzianum¹⁷ Trichoharzianol (120) Trichoderma harzianum⁷⁹ Eujavanicol A (121) Trichoderma harzianum⁷⁹

Tandyukisin (122) Trichoderma harzianum⁸¹

Tandyukisin B (123) Trichoderma harzianum⁸² Tandyukisin C (124) Trichoderma harzianum⁸² Tandyukisin D (125) Trichoderma harzianum⁸² Tandyukisin E (126) Trichoderma harzianum⁸³ Tandyukisin F (127) Trichoderma harzianum⁸³

Trichosetin (128) Trichoderma harzianum⁸⁴

Table 19. Statins

Compounds Species

Compactin (129) Trichoderma longibrachiatum⁸⁵ Trichoderma pseudokoningii⁸⁵

Table 20. Diketopiperazines

Compounds Species

Gliotoxin (130) Trichoderma viride⁸⁶

Gliovirin (131) Trichoderma virens⁸⁷

Table 21. Sorbicillinoids

Sorbicillin (132) Trichoderma longibrachiatum⁸⁸ Epoxysorbicillinol (133) Trichoderma longibrachiatum⁸⁹ Sorbicillinol (134) Trichoderma sp. USF-2690⁹⁰ Oxosorbicillinol (135) Trichoderma sp. USF-2690⁹¹ Demethylsorbicillin (136) Trichoderma sp. USF-2690⁹¹ (E)-6-(2,4-Dihydroxyl-5-methylphenyl)-

6-oxo-2-hexenoic acid (137)

Trichoderma sp.⁹²

6-(2,4-Dihydroxyl-5-methylphenyl)- 6-oxohexanoic acid (138)

Trichoderma sp.⁹²

Table 22. Bisorbicillinoids

Compounds Species

Bisvertinolone (139) Trichoderma longibrachiatum⁸⁸ Bisvertinol (140) Trichoderma longibrachiatum⁸⁸ Trichodimerol (141) Trichoderma longibrachiatum⁸⁸ Trichodermolide (142) Trichoderma longibrachiatum⁹³ Sorbiquinol (143) Trichoderma longibrachiatum⁹³ Demethyltrichodimerol (144) Trichoderma sp. USF-2690⁹⁴ Bisorbicillinol (145) Trichoderma sp. USF-2690⁹⁴ Bisorbibetanone (146) Trichoderma sp. USF-2690⁹⁵

96

Dihydrotrichotetronine (150) Trichoderma sp.⁹⁷

Table 23. Daucanes

Compounds Species

CAF-603 (151) Trichoderma virens⁹⁸

L-735,334 (152) Trichoderma virens⁹⁹

Trichocarane A (153) Trichoderma virens¹⁰⁰ Trichocarane B (154) Trichoderma virens¹⁰⁰ Trichocarane C (155) Trichoderma virens¹⁰⁰ Trichocarane D (156) Trichoderma virens¹⁰⁰

Table 24. Trichothecenes

Compounds Species

Trichodermin (157) Trichoderma viride¹⁰¹

T-2 toxin (158) Trichoderma lignorum¹⁰²

Harzianum A (159) Trichoderma harzianum¹⁰³

Table 25. Acoranes

Compounds Species

Trichoacorenol (160) Trichoderma koningii²¹ 15-Hydroxyacorenone (161) Trichoderma harzianum¹⁰⁴

80

dec-8-ene-2!,7"-diol (163)

1"-Isopropyl-4",8-dimethylspiro[4.5]

dec-8-ene-3!,7"-diol (164)

Trichoderma sp. YMF1.02647¹⁰⁵

2!-Hydroxy-1"-isopropyl-4",8-

dimethylspiro[4.5]dec-8-en-7-one (165)

Trichoderma sp. YMF1.02647¹⁰⁵

Table 26. Cyclopentenones

Compounds Species

Trichodenone A (166) Trichoderma harzianum⁵⁵ Trichodenone B (167) Trichoderma harzianum⁵⁵ Trichodenone C (168) Trichoderma harzianum⁵⁵

Table 27. Cyclonerodiol derivatives

Compounds Species

Cyclonerodiol (169) Trichoderma koningii¹⁰⁶

Lignoren (170) Trichoderma lignorum¹⁰⁷

Cyclonerodiol oxide (171) Trichoderma polysporum¹⁰⁸ Epicyclonerodiol oxide (172) Trichoderma polysporum¹⁰⁸

Table 28. Polyketides

Trichodermatide C (175) Trichoderma reesei¹⁰⁹ Trichodermatide D (176) Trichoderma reesei¹⁰⁹ Trichodermaketone A (177) Trichoderma koningii³¹ Trichodermaketone B (178) Trichoderma koningii¹¹⁰ Trichodermaketone C (179) Trichoderma koningii¹¹⁰ Trichodermaketone D (180) Trichoderma koningii¹¹⁰

Table 29. Others

Compounds Species

Harziandione (181) Trichoderma harzianum¹¹¹ Valinotricin (182) Trichoderma polysporum¹⁰⁸

Ceramide (183) Trichoderma koningii²¹

Cyclo-(L-Pro-L-Leu) (184) Trichoderma koningii²¹

Epifriedelinol (185) Trichoderma pseudokoningii³⁹ 2’,4’-Dihydroxy-3’-methoxymethyl-5’-

methylacetophenone (186)

Trichoderma pseudokoningii¹¹²

2’,4’-Dihydroxy-3’,5’-dimethyl acetophenone (187)

Trichoderma pseudokoningii¹¹²

3,4-Dihydro-8-hydroxy-3-methyl isocoumarin (188)

Trichoderma aggressivum¹¹³

Table 30. Heptelidic acid

Heptelidic acid (189) Trichoderma viride¹¹⁴

Table 31. Peptaibols

Compounds Species

Atroviridin A (190) Trichoderma atroviride¹¹⁵ Atroviridin B (191) Trichoderma atroviride¹¹⁵ Atroviridin C (192) Trichoderma atroviride¹¹⁵ Neoatroviridin A (193) Trichoderma atroviride¹¹⁶ Neoatroviridin B (194) Trichoderma atroviride¹¹⁶ Neoatroviridin C (195) Trichoderma atroviride¹¹⁶ Neoatroviridin D (196) Trichoderma atroviride¹¹⁶ Trichobrachin A I (197) Trichoderma longibrachiatum¹¹⁷ Trichobrachin A II (198) Trichoderma longibrachiatum¹¹⁷ Trichobrachin A III (199) Trichoderma longibrachiatum¹¹⁷ Trichobrachin A IV (200) Trichoderma longibrachiatum¹¹⁷ Trichobrachin B I (201) Trichoderma longibrachiatum¹¹⁷ Trichobrachin B II (202) Trichoderma longibrachiatum¹¹⁷ Trichobrachin B III (203) Trichoderma longibrachiatum¹¹⁷ Trichobrachin B IV (204) Trichoderma longibrachiatum¹¹⁷ Trichogin A IV (205) Trichoderma longibrachiatum¹¹⁸

119

Paracelsin B (209) Trichoderma reesei¹²⁰ Paracelsin C (210) Trichoderma reesei¹²⁰

Paracelsin E (211) Trichoderma saturnisporum¹²¹ Pseudokonin KL III (212) Trichoderma pseudokoningii¹²² Pseudokonin KL VI (213) Trichoderma pseudokoningii¹²² Aspereline A (214) Trichoderma asperellum¹²³ Aspereline B (215) Trichoderma asperellum¹²³ Aspereline C (216) Trichoderma asperellum¹²³ Aspereline D (217) Trichoderma asperellum¹²³ Aspereline E (218) Trichoderma asperellum¹²³ Aspereline F (219) Trichoderma asperellum¹²³ Trichopolyn I (220) Trichoderrna polysporurn¹²⁴ Trichopolyn II (221) Trichoderrna polysporurn¹²⁴ Trichotoxin A-40 (222) Trichoderma viride¹²⁵ Trichodecenin-I (223) Trichoderma viride¹²⁶ Trichodecenin-II (224) Trichoderma viride¹²⁶ Trichokonin V (225) Trichoderma koningii¹²⁷ Trichokonin VI (226) Trichoderma koningii¹²⁷ Trichokonin VII (227) Trichoderma koningii¹²⁷ Trichokonin VIII (228) Trichoderma koningii¹²⁷ Trichokonin-Ia (229) Trichoderma koningii¹²⁸ Trichokonin-Ib (230) Trichoderma koningii¹²⁸

Trichorzin PA II (232) Trichoderma harzianum¹²⁹ Trichorzin PA IV(233) Trichoderma harzianum¹²⁹ Trichorzin PA V (234) Trichoderma harzianum¹²⁹ Trichorzin PA VI (235) Trichoderma harzianum¹²⁹ Trichorzin PA VII (236) Trichoderma harzianum¹²⁹ Trichorzin PA VIII (237) Trichoderma harzianum¹²⁹ Trichorzin PA IX (238) Trichoderma harzianum¹²⁹ Trichorzin HA I (239) Trichoderma harzianum¹³⁰ Trichorzin HA II (240) Trichoderma harzianum¹³⁰ Trichorzin HA III (241) Trichoderma harzianum¹³⁰ Trichorzin HA V (242) Trichoderma harzianum¹³⁰ Trichorzin HA VI (243) Trichoderma harzianum¹³⁰ Trichorzin HA VII (244) Trichoderma harzianum¹³⁰ Trichorzin MA I (245) Trichoderma harzianum¹³⁰ Trichorzin MA II (246) Trichoderma harzianum¹³⁰ Trichorzin MA II (247) Trichoderma harzianum¹³⁰ Harzianin HB I (248) Trichoderma harzianum¹³¹ Trichofumin A (249) Trichoderma sp. HKI 0276¹³² Trichofumin B (250) Trichoderma sp. HKI 0276¹³²

OH OH

OH

O O

O O O O

O

O

OMe

OH OH O

HO O

NH₂ O

O HO

H H

O

OH OH O

O

O

OH OH

O OH O

HO

O

O

OH OH

OH

H O

O

OH OH

O H

10 11 12

13 14 15

16 17 18

19 20 21

O

O

OH OH

H O

O

HO O

O

O

HO O

O

OH

OH OH

OH

H

H O

O

OH OH

O

H O

HO

OH

O

O

O COOH

HO

OH

O

OH MeO

O

O

O O

OH MeO

HO

O

O O

O O

O H

O

HO

H

HO

H H R

33 R = β-Me 34 R = α-Me

22 23 24

25 26 27

28 29 30

31 32

OH

RO O

HOOC

35 R = COCH(NH2)CH(OSO3H)CHMe2

36 R = H

HOOC HO COOH

O R

O

37 R=

38 R=

39 R=

HN

COOH OH HN

COOH

HN

COOH NH

HOOC HO COOH

O NH

OH

HOOC HO COOH

O R

41 R =

42 R = HN

COOH OH HN

COOH

45 R = OH

HOOC HO COOH

O NH

O

HOOC HO COOH

O NH

O COOH OH

40

COOH OH

43

COOH OH

44

NH MeO

MeO

OH O

O

NH RO O

O H

H

47 R = H

48 R = O

HO

OH OH

N O

OH OH

N HO COOH O

OH O

N HO COOH H

O OH O

N HO COOH H

O OH O

O N

OH

O OH

N OH

OH

O

N H

OH O

OH O

O O O

46 49

50 51

52 53 54

55 56 57

O O

O O O

OHOH O

O HO

O

O O

OH

O O

H HO

O

OH

O O O

HO O O

H H

H

O O H OH

H

O

O

O O O

O O

O

OH OH

O O

O OH

O

58 59 60

61 62 63

64 65 66

67 68 69

O O

HO

OH

NC O

MeOOC NC

O HOOC

NC

MeOOC NC

HOOC

NC

O O

NC

HOOC HO HO

O

O

O

HO

OH

NC NC

NC

O OH

NC

NC HO

HO

OH OH

NC HO

HO Cl

OH

70 71 72

73 74 75

76 77 78

79 80 81

NC HO

OH OH OH

NC O

NC O

HO

N O

HO

O N O

NH O

O

OCH₃ OCH₃

H OH

OH OH

O N O

NH O

O

OCH3

OCH₃

H OH

OH Cl

O NH O

NH O

O

OCH₃ OCH3

H OH

OH

O NH O

NH O

O

OCH₃ OCH₃

H OH

OH

S S S

O N NH O

O OHH OHOH

OCH₃ OCH₃ H OH

S S

H

82 83 84

85 86 87

88 89

90

N C8H17 O

NH

O R₁

HN

O

NH O

O HN

R2

NH

O H

N

O

NH

O H

N

O

N OH

O OH

O

O OH 91 R1 =

92 R₁ =

93 R₁ =

R₂ =

R2 =

R₂ = CH3

CH₃

H

O O HO

HO

OH

O O

O O

N

O

OH O

O

O O O HO

OH O

O O

O OH

H OHH HO

R₁

R₂ OH

OH O

O OH

HO

R

94 95 96

97 98 R₁ = R₂ = H

99 R₁= OH, R₂ = H 100 R₁ = R₂ = OH

101 R = H 102 R = COCH3

HO

HO

O

O OH

OH R₄

OH O

O R₁

R₂

R3

O

O OH R

O

O OH

OH

R₂ R₁

O OH

OH

HO

O O

O HO

O

OH

O HO

O

O

113 114

115

H

H H

COOH

HO OH O

OH OH

118

117

OH

O

H

OHH H

H

O O

HO O

119

OH

O

O H O

H

120 O

OH

O HO

116 O 103 R = CH2OH

104 R = CH

105 R₁ = R₂ = OH, R₃ = H, R₄ = CH₂CH(OH)CH₃ 106 R₁ = R₂ = H, R₃ = OH, R₄ = CH₃

107 R1 = R2 = OH, R3 = H, R4 = CH2OH

108 R = H 109 R = OH

110 R₁ = H, R₂ = OH 111 R₁ = OH, R₂ = H 112 R1 = H, R2 = H

O O

O O

H H

OHH

H OH

O OH

O

HO OHH

H H

O OH

H

O O

O O

H

HO H H

O OH

H

O

O H

HO H H

124 HO O

O O

H H

OHH

H OH

O

125 O

HO

O OH

H

O O

O O

H

HO H H

126

HO O

O

H H

OHH

H OH

O

127 O

O

O

H H

OHH O H O

O O

121 122 123

HO O NH

O OH

O H

O O

OH O

H

N N

OHH O

O

OH S S

O N

NH

HO OMe

OMe

O OHH

S

O O

S HH

O

HO

OH

O

O

OH O OH

O

O OH

OH

HO O

OH O

OH O

HO OH

HO

OH O

COOH

HO OH

COOH

O O OH

O H HO OH O

O HO OH

128 129 130

131 132 133

134 135 136

137 138 139