울릉도 토양 방선균으로부터 신규 생리활성 이차대사산물의 발굴 : Discovery of Novel Bioactive Secondary Metabolites Produced by Terrestrial Actinomycetes from Ulleung Island

국문 초록 (Abstract)

미생물 유래 이차대사산물은 다양한 화학구조와 특이적 생리활성을 기반으로 매우 중요한 선도물질 및 약학 조성물로서 이용되어 왔다. 미생물 유래 이차대사산물의 지속적인 활용을 위해서는 기존에 발굴되지 않은 신규 화합물을 효과적으로 탐색하고 동정하는 것이 매우 중요하다. 본 논문은 신규 이차대사산물을 발굴하는 노력의 일환으로 수행된 실험들, 즉 동해의 화산섬인 울릉도의 토양에서 분리한 방선균으로부터 새로운 이차대사산물의 탐색과 발굴된 신규 화합물들의 구조 및 생리활성적 특성에 대해 논한다.
미생물로부터 신규 이차대사산물을 발굴할 가능성이 높은 육지에서 멀리 떨어진 환경의 섬인 울릉도의 토양으로부터 220여종의 방선균을 분리하였으며, 분리한 균주를 다양한 배양조건을 검토하여 순수배양하였다. 액체크로마토그래피 질량분석법(LC-MS) 및 활성검정법을 동반한 화학적 및 생리활성적 검색을 이용해 대사산물의 특성을 일차적으로 조사하여 3가지의 균주를 선정하였다. 추후 대량배양과 크로마토그래피 기법을 통한 물질 분리 및 분광학 기법을 기반으로 한 화학구조 분석을 수행한 결과, 다양한 계열의 신규 화합물을 도출할 수 있었다. 분리된 신규 화합물은 각각 cyclic depsipeptide 계열의 ulleungamides A–B, glycoside 계열의 ulleungoside, 2-methylaminobenzoyl 6-deoxy-α-L-talopyranoside, naphthomycinoside, macrolide 계열의 catenulisporolides A–D, cyclic peptide 계열의 ulleungmycins A–B로 명명하였다. 분리된 화합물은 각각의 구조적 특징에 따라 항균, 항말라리아, 세포독성 등을 포함한 다양한 생리활성을 나타냈다.
본 논문은 방선균이 생산하는 신규 이차대사산물의 화학구조와 생리활성을 밝힘으로써 자연계에서 유래하는 저분자 화합물의 화학적 다양성을 확장시키는 의의를 지닌다. 특히 기존에 알려진 이차대사산물과 구조적으로 상이한 물질의 경우, 이차대사산물의 새로운 생합성 경로를 밝히는 것을 목적으로 하는 후속 연구에 이용될 수 있을 것으로 기대된다. 또한 본 논문에서 신규 이차대사산물을 발굴하는 방법으로서 새로운 자연환경의 탐색, 희소 방선균의 분리배양, 유전체 분석 등을 효과적으로 활용함으로써 해당 접근법들의 폭넓은 적용 가능성을 제시할 수 있었다.

다국어 초록 (Multilingual Abstract)

Microbial secondary metabolites, which possess diverse chemical structures and intriguing biological activities, have been considered as an important resource for therapeutic agents and drug leads. The discovery of compounds with new chemical structures is of significance for successful and continuous utilization of microbial metabolites. This thesis discusses the results of research aimed at discovering previously unknown secondary metabolites produced by the actinomycetes from Ulleung Island in Korea by applying a variety of strategies and their chemical structures as well as bioactivities.
To increase the possibility of discovering new microbial secondary metabolites, ~220 actinomycetes were isolated from a biodiversity hot spot, Ulleung island which is located far from the continent, and cultured pure strains under various culture conditions. Secondary metabolic profiles were then investigated by chemical and biological screenings using a combination of LC-MS and bioassay systems. Further scale-up culture of the strains with promising potential to produce unknown metabolites and subsequent chromatographic purification led to pure compounds. Their chemical structures were assigned to be new compounds belonging to multiple structural classes by combined spectroscopic and chemical analyses. The isolated new compounds were designated as follows: cyclic depsipeptides, ulleungamides A–B; 6-deoxy-α-L-talopyranosides, ulleungoside, 2-methylaminobenzoyl 6-deoxy-α-L-talopyranoside, naphthomycinoside; glycosylated macrolides, catenulisporolides A–D; chlorinated cyclic peptides, ulleungmycins A–B. These compounds were found to exhibit various biological activities including antibacterial, antimalarial, and cytotoxic activities.
The results reported in this thesis contribute to extending the chemical diversity of the small molecules derived from natural sources. Especially, the compounds with an unprecedented moiety may provide early steps towards elucidating new biosynthetic pathways, which would allow generation of new compounds with interesting bioactivity through a combinatorial biosynthesis. This thesis also suggests various strategies (i.e., exploration of uninvestigated sites, utilization of rare actinomycetes, and genome mining) and supports the possibility of their broad application for discovering novel secondary metabolites.

목차 (Table of Contents)

• 국문 초록 iii
• ABSTRACT v
• Contents vii
• List of Figures x
• List of Tables xiii
• 1. Ulleungamides A and B, Modified α,β-Dehydropipecolic Acid Containing Cyclic Depsipeptides from Streptomyces sp. 1
• 1.1. Introduction 1
• 1.2. Results and Discussion 3
• 1.2.1. Structure determination of ulleungamide A (1) 3
• 1.2.2. Determination of the absolute configuration of 1 5
• 1.2.3. Structure determination of ulleungamide B (2) 8
• 1.2.4. Determination of the absolute configuration of 2 9
• 1.2.5. Biological activities of 1 and 2 11
• 1.2.6. Discussion 12
• 1.3. Materials and Methods 15
• 2. Polyketides and Anthranilic Acid Possessing 6-Deoxy-α-L-talopyranose from Streptomyces sp. 27
• 2.1. Introduction 27
• 2.2. Results and Discussion 30
• 2.2.1. Strain prioritization 30
• 2.2.2. Structure determination of ulleungoside (3) 32
• 2.2.3. Structure determination of 2-methylaminobenzoyl 6-deoxy-α-L-talopyranoside (4) 34
• 2.2.4. Determination of the absolute configuration of the sugar 35
• 2.2.5. Structure determination of naphthomycinoside (5) 36
• 2.2.6. Determination of the absolute configuration of 5 39
• 2.2.7. Structure determination of piericidin A1 3′-O-6-deoxy-α-L-talopyranoside (7) 41
• 2.2.8. Biological activities of 3–9 43
• 2.2.9. Discussion 45
• 2.3. Materials and Methods 47
• 3. Catenulisporolides A–D, 26-Membered Glycosylated Macrolides from a Rare Actinomycete Catenulispora sp. 61
• 3.1. Introduction 61
• 3.2. Results and Discussion 64
• 3.2.1. Structure determination of catenulisporolide A (10) 64
• 3.2.2. Determination of the absolute configuration of 10 66
• 3.2.3. Structure determination of catenulisporolides B–D (11–13) 71
• 3.2.4. Bioinformatics-based configurational assignment 72
• 3.2.5. Biological activities of 10–13 73
• 3.2.6. Discussion 74
• 3.3. Materials and Methods 75
• 4. Ulleungmycins A and B, Chlorinated Cyclic Peptides Discovered by a Genome Mining Approach 86
• 4.1. Introduction 86
• 4.2. Results and Discussion 88
• 4.2.1. In silico analysis of the putative halogenase containing NRPS gene cluster 88
• 4.2.2. Structure determination of ulleungmycin A (14) 91
• 4.2.3. Determination of the absolute configuration of 14 93
• 4.2.4. Structure determination of ulleungmycin B (15) 95
• 4.2.5. Biosynthetic gene cluster analysis for ulleungmycins 95
• 4.2.6. Bioactivities of 14 and 15 99
• 4.2.7. Discussion 101
• 4.3. Materials and Methods 103
• 5. Conclusions 113
• 6. Summary 114
• Bibliography 115