A Multiple Dereplication Approach for the Discovery of Plant and Fungal Natural Products and Their Biological Activities = 선택적 표적화 기법을 이용한 식물 및 균류 유래 대사체 발굴과 생리활성

국문 초록 (Abstract)

식물과 균류에서 유래한 천연물은 오랫동안 제약 산업에서 중요한 역할을 해왔다. 이러한 생물체들은 다양한 생리활성을 가진 천연물들의 풍부한 공급원이며, 이 중 많은 물질이 감염성 질환부터 암 치료에 이르기까지 다양한 질병의 치료제로 개발되었다. 하지만 새로운 천연물을 발견하는 과정은 쉽지 않다. 주요한 문제 중 하나는 천연물 혼합물의 복잡성으로, 이는 수백에서 수천 가지의 다양한 화합물을 포함할 수 있다. Dereplication기법은 이미 알려진 화합물을 빠르게 식별하여 연구자들이 새로운 화합물을 발견하는 데 집중할 수 있도록 돕는다. 다양한 Dereplication방법들이 천연물 분리를 가속화하는 데 활용되었으며, 이를 통해 새로운 대사산물을 신속하게 규명할 수 있게 되었다.
본 논문에서는 식물 및 균류에서 새로운 천연물을 탐색하기 위해 두 단계의 Dereplication 전략을 적용하였다. 첫 번째 단계에서는 Feature-Based Molecular Networking(FBMN)과 데이터베이스 검색을 이용하여 untargeted(비표적) 천연물을 탐색하기 위한 후보 물질을 선별하였다. 두 번째 단계에서는 HPLC-DAD, NMR, MS 필터링 방법(생성 이온 필터링, 중성 손실 필터링, 질량 결함 필터링) 등의 분석 도구를 활용하여 분리 전략을 설정하였다. 이러한 두 단계 전략을 통해 다수의 새로운 천연물을 발견할 수 있었으며 아울러, 화합물들의 화학 구조를 규명하고 다양한 변형체를 생성하기 위해 화학 합성도 병행하였다. 연구 결과는 다음과 같이 요약된다.

제1부: Cleistocalyx operculatus 꽃봉오리에서 1H-NMR 및 Molecular Networking을 이용한 플로로글루시놀 메로테르펜의 Dereplication 과Neuraminidase 저해 활성 연구
Cleistocalyx operculatus (동의어: Syzygium nervosum)는 도금양과(Myrtaceae)에 속하는 식물로, 동남아시아의 열대 및 아열대 지역에 널리 분포한다. 베트남에서는 이 식물의 꽃봉오리와 잎을 차로 이용하여 감기 및 위장장애 증상을 완화하는 데 사용해왔다. 연구에서는 1H-NMR과 Molecular Networking을 기반으로 한 Dereplication 전략을 적용하여 해당 식물의 꽃봉오리에서 희귀한 decahydro-2H-cyclopenta[i]chromene 골격을 가진 두 개의 특이한 플로로글루시놀 유도체(1, 2)와 새로운 플로로글루시놀 메로테르펜5종(3-7)을 분리하였다. 이 화합물의 구조는 1D 및 2D NMR, ECD, X선 결정학, DP4+ 확률 계산을 통해 규명하였다. 분리된 화합물 1-7은 천연적으로 광학이성질체 쌍으로 존재하며, 이를 카이랄성 HPLC를 이용해 성공적으로 분리하였다. 또한, 해당 화합물들의 생합성 경로를 분석한 결과, 2,2,4-트라이메틸신나밀-β-트리케톤 중심부가 acetoacetyl-CoA의 후반부 결합을 거쳐 여러 기능화 과정을 통해 화합물 1-4가 형성되는 것으로 추정되었다. 생리활성 평가 결과, 화합물 1과 2는 H1N1 및 H9N2 인플루엔자 바이러스의 Neuraminidase 효소 저해 활성을 나타냈으며, 화합물 1은 비경쟁적 저해 기전을 보였다.
제2부: Cleistocalyx operculatus 꽃봉오리 유래 천연 및 합성 챨콘-모노테르펜 유도체의 PTP1B 저해 활성 연구
C. operculatus의 화학적 구성을 지속적으로 연구한 결과, 라세미 혼합물의 챨콘-모노테르펜 하이브리드 화합물(1-3)과 하나의 챨콘(9)을 포함한 총 4개의 신규 화합물과 9개의 기재 화합물(4-8, 10-13) 을 분리하였다. 화합물들의 구조규명은 NMR 데이터 분석 및 컴퓨터 시뮬레이션(ECD 계산)을 이용해 결정하였으며, 생합성을 모방한 합성 방법을 적용하여 천연 유사 화합물을 재구성하였다. Diels-Alder 반응을 활용한 생합성 모방적 합성을 통해 두 개의 천연 전구체(챨콘 과 모노테르펜)를 기반으로 해당 천연물의 합성이 가능함을 확인하였다. 이런 접근법을 활용하여 분리한 챨콘 화합물들을 통해 총 20개의 비천연 유도체를 합성하였다. PTP1B(Protein Tyrosine Phosphatase 1B) 저해 활성을 평가한 결과, 화합물 4, 9, 13, 16b는 우르솔산(IC50 = 3.0 ± 0.3 μM)보다 우수한 IC50 값(0.9 ± 0.2 ~ 3.9 ± 0.7 μM)을 보였다. 또한, (+)-4 및 (−)-16b 광학이성질체가 각각 대응하는 반대 광학이성질체보다 높은 활성을 보였다. 효소저해 기전 연구 결과, 이들 활성 화합물은 혼합형 저해(mixed inhibition) 기전을 따르며, Molecular Docking 시뮬레이션이 실험적 결과와 일치함을 확인하였다. 연구결과에 따르면, Cleistocalyx operculatus 꽃봉오리에서 유래한 챨콘-모노테르펜 하이브리드 화합물은 PTP1B 효소 저해 활성을 부분적인 기전으로 하여 당뇨병 치료제 후보로서의 가능성을 가진다.
제3부: 담수 유래 곰팡이 Talaromyces gwangjuensis에서 분리한 Cyclodepsipeptides 및 지방산 락톤과 자가포식 활성 유도 가능성 연구
Talaromyces 속은 Penicillium 속의 유성형(teleomorphic) 종을 포함하며, 얇은 세포벽을 가진 자낭포자들을 생성하고, 서로 얽혀 있는 균사로 덮여 있다. 현재까지 이 속에는 총 203종이 보고되었으며, 토양, 담수, 실내 환경 및 다양한 식품들에서 분리되는 등 전 세계적으로 널리 분포하고 있다. 본 연구에서는 GFP-tagged LC3를 안정적으로 발현하는 인간 신장 세포주인 HEK293A 세포에서 자가포식 조절 활성을 평가하기 위해 200개의 곰팡이 추출물을 스크리닝하였다. 이를 통해, 담수 유래 곰팡이 Talaromyces gwangjuensis에서 유래한 잠재적 자가포식 조절 인자를 확인하였다. Feature-based Molecular Networking (FBMN) 기법을 활용하여, 활성 분획에서 7종의 cyclodepsipeptides (1–7) 및 4종의 락톤 유도체 (8–11)를 분리하였다. 새롭게 분리된 화합물인 arthrichitin E-H (1–4)와 gwangjupone A-D (8–11)의 화학 구조는 1D 및 2D NMR 분광법, Marfey’s 분석, ECD 계산, DP4+ 확률 계산을 통해 규명되었다. 특히, 화합물 1, 4, 6은 adeno-associated virus로 mCherry-GFP-LC3를 발현한 HEK293A 세포에서 autophagic flux를 유도하는 것으로 밝혀졌으며, 이는 자가포식 활성 촉진제로서의 가능성을 시사한다. 이번 연구는 첨단 분광학 기술과 molecular networking을 활용하여 새로운 천연물 탐색의 가능성을 보여준 사례라 할 수 있다.
제 4 부: Molecular Networking을 이용한 Penicillium sp. CNUFC-EML-48에서의 세스퀴테르펜-프롤린 결합체 및 페날레논의 선택적 표적화 연구 및 세포독성 활성
Penicillium 속은 350종 이상의 균주로 이루어진 대표적인 곰팡이 속으로, 토양, 부패한 유기물, 실내 환경 등 다양한 환경에서 흔히 발견된다. 이 속에 속한 곰팡이들은 바이오테크놀로지 및 의약학적으로 중요한 생리활성 2차 대사산물을 생성하는 능력을 지니고 있다. The NATURAL PRODUCTS ATLAS 데이터베이스에 따르면, 현재까지 곰팡이에서 보고된 천연물 중 Penicillium 유래 화합물이 약 19%를 차지하고 있으며, 이는 Penicillium 종이 다양한 2차 대사산물을 다작하는 생산자임을 시사한다. 본 연구에서는 Feature-based Molecular Networking (FBMN) 기법을 활용하여 곰팡이 유래 신규 천연물 탐색을 진행하였으며, 이를 통해 Penicillium kyesullee에서 17종의 화합물을 확인하였다. 이 중, 4개의 신규 세스퀴테르펜-프롤린 유도체 (1–4) 와 6개의 새로운 페날레논 유도체 (9–14) 를 분리하였다. 분리된 화합물들의 화학 구조는 1D 및 2D NMR 분광법, ECD 분석, DP4+ 확률 계산 및 MS/MS 조각화 분석을 통해 구조 규명되었다. 화합물 1-4는 프롤린 모획이 포함된 희귀한 세스퀴테르펜-아미노산 골격을 가지고 있으며, 화합물 1의 반합성을 통해 이러한 구조적 모티프가 자연에서 존재함이 확인되었다. 합성된 화합물은 세스퀴테르펜-프롤린 1-4가 용액 상태에서 두 가지 이성질체 (conformer)의 혼합물로 존재함을 입증한다. 새롭게 분리된 6개의 페날레논(phenalenone) 화합물들은 자연적으로 C-8 위치에서 세 쌍의 diastereomer 형태로 얻어졌으며, 이는 생합성 과정에서 비효소적(non-enzymatic) 경로가 작용했음을 시사한다. 합물 1-17의 세포독성 효과는 MC38 마우스 대장암 세포에서 평가되었다. 그 중 화합물 9-11과 17이 암세포의 증식을 유의하게 억제하였고, 이러한 결과는 페날레논(phenalenone) 화합물이 대장암 치료제 개발에 잠재력이 있음을 강조한다.

다국어 초록 (Multilingual Abstract)

Natural products derived from plants and fungi have acted as an essential part of the pharmaceutical industry for many years. These organisms are a prolific source of bioactive substances, many of which have been developed into pharmaceuticals for the treatment of many diseases, ranging from microbial infections to cancer. Despite their potential, the discovery of new natural products is not a straightforward task. One of the main challenges is the complexity of natural product mixtures, which can contain hundreds or even thousands of different compounds. Dereplication facilitates the rapid identification of known compounds, enabling researchers to narrow down their efforts on the discovery of novel molecules. Diverse dereplication approaches have been utilized for accelerating the purification of natural products, enabling rapid identification of previously uncharacterized metabolites from various sources.
In this thesis, a two-stage strategy in dereplication natural products was employed in searching for novel natural products from plants and fungi. The first-stage dereplication used FBMN in conjunction with database searching in selecting potential material for untargeted natural products. In the second-stage dereplication, to navigate the isolation strategy, various analytical tools and techniques such as HPLC-DAD, NMR, MS filtering method (product ions filtering, neutral loss filtering, mass-defect filtering) were employed. The implementation of two-stage strategies has led to the discovery of multiple new natural products. In parallel, chemical synthesis was used to prove the chemical structure and diversify natural products. Those results are summarized in the following parts as follows.
Part 1: 1H-NMR and Molecular Networking-Guided Dereplication of Phloroglucinol Meroterpenoids from Cleistocalyx operculatus Buds as Neuraminidase Inhibitors
Cleistocalyx operculatus (synonym: Syzygium nervosum) belongs to the Myrtaceae family and is widely distributed in tropical and subtropical regions, particularly in Southeast Asia. Its buds and leaves in Vietnam have traditionally been used as tea to relieve symptoms of the common cold and gastrointestinal disorders.
A dereplication strategy guided by 1H-NMR and molecular networking resulted in the isolation of two unusual phloroglucinols (1 and 2), which possess a rare decahydro-2H-cyclopenta[i]chromene skeleton, along with five undescribed phloroglucinol meroterpenoids (3-7) from the buds of Cleistocalyx operculatus. The chemical structures of isolated compounds were determined by 1D and 2D NMR spectroscopy, ECD, X-ray crystallography, and DP4+ probability calculations. Compounds 1-7 were naturally found as enantiomeric pairs, which were successfully separated using chiral phase HPLC. Structural analysis of these compounds suggested a plausible biosynthetic pathway, involving the late-stage incorporation of acetoacetyl-CoA into the 2,2,4-trimethylcinnamyl-β-triketone core, followed by a series of functionalization steps leading to compounds 1-4. Compounds 1 and 2 exhibited moderate neuraminidase inhibitory activity against neuraminidase enzymes from both H1N1 and H9N2 influenza strains. Kinetic experiments suggested a non-competitive binding mechanism for compound 1.
Part 2: Natural and Synthetic Chalcone-Monoterpene Derivatives from the Buds of Cleistocalyx operculatus as PTP1B Inhibitors
In a continuous effort to discover chemical composition of C. operculatus, four new compounds, racemic chalcone-monoterpene hybrids (1-3) and a chalcone (9), along with nine known compounds (4-8, 10-13), have been isolated from the buds of Cleistocalyx operculatus. The chemical structures of the isolated compounds were identified through NMR data analysis and confirmed by computational methods, including ECD calculations, and further synthetic approaches. Using a biomimetic synthetic strategy, we successfully reconstructed these natural analogues using two natural precursors, a chalcone and a monoterpene, under thermally induced Diels-Alder conditions. Expanding on this approach allowed us to synthesize 20 unnatural analogues, starting from the isolated chalcones. The inhibitory effects on protein tyrosine phosphatase 1B (PTP1B) of both naturally isolated and synthetically obtained compounds were evaluated. Compounds 4, 9, 13, and 16b exhibited potent PTP1B inhibitory activity, with IC50 values ranging from 0.9 ± 0.2 to 3.9 ± μM, compared to ursolic acid, used as positive control, with an IC50 of 3.0 ± 0.3 μM. The enantiomers (+)-4 and (−)-16b showed enhanced activity compared to their respective enantiomers. Kinetic studies indicate that all active compounds inhibit PTP1B through mixed mechanisms, and molecular docking simulations agree with the experimental assays on PTP1B. Our results suggest that chalcone-meroterpene adducts from the buds of C. operculatus exhibit potential as antidiabetic agents, partly due to their PTP1B enzyme inhibition.
Part 3: Cyclodepsipeptides and Fatty Acid Lactones from the Freshwater-Derived Fungus, Talaromyces gwangjuensis, and Their Potential as Autophagy Activators
The genus Talaromyces includes teleomorphic Penicillium species, which produce soft-walled ascomata covered with interwoven hyphae. To date, 203 species have been reported to belong to this genus. These species have a cosmopolitan distribution and can be isolated from various environments such as soil, freshwater, indoor environments, and various food products. In this study, we screened 200 fungal extracts for their ability to modulate autophagy in HEK293A cells, a human kidney cell line that stably expresses GFP-tagged LC3, a marker of autophagy. Our screening identified a potential autophagy regulator from the freshwater-derived fungus, Talaromyces gwangjuensis. Through the implementation of Feature-based Molecular Networking (FBMN), seven cyclodepsipeptides (1-7) and four lactone derivatives (8-11) were isolated from the bioactive fractions. The chemical structure of the newly isolated compounds, arthrichitin E-H (1-4) and gwangjupone A-D (8-11), were elucidated using 1D and 2D NMR spectroscopy, Marfey’s analysis, ECD and DP4+ probability calculations. Compounds 1, 4, and 6 were found to stimulate autophagic flux in HEK293A cells infected with an adeno-associated virus carrying an mCherry-GFP-LC3 construct, highlighting their potential as autophagy activators. This approach demonstrates the potential of integrating advanced spectrometry techniques with molecular networking to uncover new and potentially valuable natural products.

Part 4: Dereplication of Sesquiterpene-Proline Conjugates and Phenalenones from Penicillium sp. CNUFC-EML-48 Using Molecular Networking and Cytotoxic Activities
Penicillium is a diverse genus of filamentous fungi, comprising over 350 species. These fungi are ubiquitously found in various environments, from soil and decaying organic matter to indoor settings. Their significance in biotechnology and medicine cannot be overstated, particularly due to their ability to produce an array of bioactive secondary metabolites. According to The Natural Products Atlas, the number of secondary metabolites derived from Penicillium species accounts for 19% of fungal natural products. Penicillium species are prolific producers of secondary metabolites. In the effort to discover new natural products from fungi, chemical investigation using dereplication tools of Feature-based Molecular Networking (FBMN) have led to identify 17 compounds including four new sesquiterpene-prolines (1-4) and six new phenalenones (9-14) were isolated from Penicillium kyesullee. The chemical structures of the isolated compounds were elucidated through extensive spectroscopic analyses, using 1D and 2D NMR, electronic circular dichroism (ECD), and MS/MS fragmentation analysis. Compounds 1-4 possessed a rare sesquiterpene-amino acid scaffold incorporating a proline moiety, and the natural occurrence of this structural motif was confirmed through the semisynthesis of compound 1. The synthesized product confirms the natural characteristics of sesquiterpene-prolines 1-4 as a mixture of two conformers in solution. The six new phenalenones were naturally obtained as three pairs of diastereomer at C-8, proposing the non-enzymatic pathway during biosynthesis process. The cytotoxic effects of compounds 1-17 were evaluated in MC38 murine colorectal cancer cells. Among them, compounds 9–11 and 17 significantly inhibited cancer cell proliferation.

목차 (Table of Contents)

• Abstract i
• Table of contents v
• List of figures x
• List of tables xvi
• List of abbreviations xviii
• INTRODUCTION PART: Dereplication methods in natural product discovery: tools and strategies 1
• 1. Targeted and untargeted strategies in natural product discovery 2
• 1.1 Targeted strategy 2
• 1.2 Untargeted strategy and dereplication 3
• 2. Molecular networking 5
• 2.1 Introduction about molecular networking 5
• 2.2 Generation of molecular networking 6
• 2.3 Network analysis 7
• 2.4 Application in natural products discovery 8
• 3. Other dereplication methods 9
• 3.1 HPLC-DAD 9
• 3.2 Product ion and neutral loss filtering 10
• 3.3 Mass defect filtering 11
• 3.4 Nuclear Magnetic Resonance (NMR) 12
• 4. A two-stage strategies approach in discovery of new natural products 15
• 5. Research purpose 16
• PART 1: 1H-NMR and Molecular Networking-Guided Dereplication of Phloroglucinol Meroterpenoids from Cleistocalyx operculatus Buds as Neuraminidase Inhibitors 17
• 1. Introduction 18
• 2. Dereplication strategy 19
• 3. Structural elucidation 22
• 3.1 Compound 1 22
• 3.2 Compound 2 26
• 3.3 Compound 3 29
• 3.4 Compound 4 32
• 3.5 Compound 5 36
• 3.6 Compound 6 39
• 3.7 Compound 7 42
• 3.8 Compound 8 and 9 45
• 4. Tautomerism of phloroglucinols 3-7 46
• 5. Proposed biosynthetic pathway 46
• 6. Neuraminidase inhibitory activities 48
• 7. Experimental Sections 50
• 7.1 Chemicals and instruments 50
• 7.2 Plant materials 50
• 7.3 Extraction and isolation 50
• 7.4 X-ray crystallography analysis of (±)-1 and (±)-2 54
• 7.5 Computational ECD analysis 55
• 7.6 Influenza neuraminidase inhibition assay 56
• 8. Conclusion 56
• PART 2: Natural and Synthetic Chalcone-Monoterpene Derivatives from the Buds of Cleistocalyx operculatus as PTP1B Inhibitors 57
• 1. Introduction 58
• 1.1 Diels-Alder reaction 58
• 1.2 PTP1B enzyme 59
• 2. Dereplication strategy 59
• 3. Structural elucidation 62
• 3.1 Compound 1 62
• 3.2 Compound 2 66
• 3.3 Compound 3 69
• 3.4 Compound 4 72
• 3.5 Compound 5 73
• 3.6 Compound 9 74
• 3.7 Compound 6-8, 10-13 76
• 4. Synthesis of chalcone-monoterpene derivatives 80
• 4.1 Synthesis natural chalcone-monoterpenes 1-5 80
• 4.2 Synthesis chalcone-monoterpene analogues 81
• 5. Synthetic chalcone-monoterpene analogues are natural products 83
• 5.1 Detection of isolated and synthetic compounds in crude extract 83
• 5.2 Stability test of extract under different isolation conditions 85
• 6. PTP1B inhibitory 86
• 7. Experimental sections 87
• 7.1 Plant materials 87
• 7.2 Chemicals and reagents 87
• 7.3 Extraction and isolation 88
• 7.4 Physiochemical properties of isolated compounds 89
• 7.5 Synthesis of chalcone-monoterpenes 93
• 7.6 Physiochemical properties of synthetic compounds 14-23 94
• 7.7 Computational ECD analysis 98
• 7.8 PTP1B inhibitory assay 99
• 8. Conclusion 99
• PART 3: Cyclodepsipeptides and Fatty Acid Lactones from the Freshwater-Derived Fungus, Talaromyces gwangjuensis, and Their Potential as Autophagy Activators 101
• 1. Introduction 102
• 2. Bioactivity screening and dereplication strategy 103
• 3. Structural Elucidation 105
• 3.1 Compound 1 105
• 3.2 Compound 2 113
• 3.3 Compound 3 116
• 3.4 Compound 4 119
• 3.5 Compound 8 122
• 3.6 Compound 9 126
• 3.7 Compound 10 129
• 3.8 Compound 11 132
• 3.9 Compound 5-7, 12 134
• 4. Autophagy regulation of isolated compounds 1-12 138
• 4.1 Screening autophagy regulation 138
• 4.2 Cyclodepsipeptides promote autophagy 139
• 5. Experimental Sections 141
• 5.1 General experimental procedures 141
• 5.2 Fungal strain, fermentation and extraction 142
• 5.3 Isolation and purification 142
• 5.4 Physiochemical properties 143
• 5.5 Marfey’s analysis for compounds 1-5 146
• 5.6 Synthesis of (R)- and (S)-MTPA esters of compound 11 146
• 5.7 ECD calculations 147
• 5.8 NMR calculations 147
• 5.9 Data-Dependent UHPLC-QTof-MS/MS acquisition 147
• 5.10 Feature-Based Molecular Networking 148
• 5.11 Cell culture 149
• 5.12 Cell viability assay 149
• 5.13 Confocal microscope imaging 149
• 5.14 Statistical Analysis 150
• 6. Conclusion 150
• PART 4: Dereplication of Sesquiterpene-Proline Conjugates and Phenalenones from Penicillium sp. CNUFC-EML-48 Using Molecular Networking and Cytotoxic Activities 151
• 1. Introduction 152
• 2. Dereplication strategy 153
• 3. Structural elucidation 156
• 3.1 Compound 1 156
• 3.2 Compound 2 161
• 3.3 Compound 3 164
• 3.4 Compound 4 167
• 3.5 Compound 9-10 170
• 3.6 Compound 11-12 174
• 3.7 Compound 13-14 177
• 3.8 Compound 5-8, 17 182
• 4. Antiproliferative activity 185
• 5. Experimental sections 186
• 5.1 General experimental procedures 186
• 5.2 Fungal strain, fermentation and extraction 187
• 5.3 Isolation and purification 187
• 5.4 Physiochemical properties 188
• 5.5 ECD calculations 193
• 5.6 Synthesis of aculenamide A (1) 193
• 5.7 Marfey’s analysis of compounds 1–4. 194
• 5.8 Cell proliferation assay 194
• 5.9 Trypan blue exclusion assay 195
• 5.10 Statistical analysis 195
• 6. Conclusion 195
• List of References 196
• 초록 209
• Acknowledgements 214