Nitrogenous Compounds from Marine and Dung Beetle-Associated Bacteria

Abstract

Part A. 해양 방선균이 생산하는 생리활성 펩타이드 이차대사물질 연구 성산핀은 제주도 근해 심층 토양에서 분리된 스트렙토마이세스 박테리아로 부터 생산되는 15개의 아미노산 잔기로 이루어진 펩타이드이다. 성산핀의 평면구조는 핵자기공명분석법, 질량분석법, 자외선분석법 등을 활용하여 결정되었다. 성산핀의 입체화학구조는 마피반응과 GITC반응 후 LC-MS분석을 통하여 결정되었다. ROESY NMR spectrum을 통하여 성산핀의 3차 구조를 예측하였고, 7개의 아미노산으로 연결된 꼬리 부분이(-Ser9-Phe-Gly-Leu-Ser-Trp-Leu15) 이 8개의 아미노산으로 이루어지는 고리 부분으로(-Gly1-Phe-Gly-Ser-Lys-Pro-Ile-Asp8-) 들어가는 형태의 Lasso peptide 계열의 물질로 확인되었다. 성산핀은 해양 박테리아 기원의 최초의 Lasso peptide로, Human lung cancer cell invasion assay에서 효과를 보였다. 오명사마이신 A와 B는 제주도 신양해변에서 채집된 토양에서 분리된 스트렙토마이세스 균주가 생산하는 물질이다. 오명사마이신의 평면구조는 핵자기공명, 자외선, 적외선, 질량분석법등의 분광기술을 이용하여 결정되었다. 오명사마이신 A와 B는 N-methyl-4-methoxytrytophan, β-hydroxyphenylalanine, and N,N-dimethylvaline와 같은 특이 아미노산을 가지고있는 싸이클릭 펩타이드이다. 마피반응을 통하여 오명사마이신을 이루는 아미노산의 알파탄소의 입체구조응 규명하였고, Threonine과 N-Me-Threonine의 베타카본 입체구조는 GITC반응을 통하여 결정되었다. 베타하이드록시페닐알라닌은 모셔반응을 통하여 베타카본위치의 입체 구조를 규명하였다. N,N-dimethylvaline의 알파카본의 입체구조을 알기위해 PGME derivatization을 이용한 새로운 분석기법을 개발하여 적용하였다. 오명사마이신 A와 B는 고무적인 항암활성과 항균효과를 보였다.

Part B. 소똥구리 공생 미생물이 생리활성 이차대사물질 연구 코프리스아마이드 A와 B는 애기뿔소똥구리 (Copris tripartitus) 장내에서 분리된 박테리아가 생산하는 물질이다. 코프리스아마이드 A와 B의 평면구조는 핵자기공명법과 질량분석법을 기초로하여 규명되었고, β-methylaspartic acid 와 valine이 2,3-diaminopropanoic acid에 붙어있고, 2-heptatrienyl cinnamic acid등 특이한 구조를 가지는 신규 싸이클릭 펩타이드로 확인되었다. 코프리스아마이드의 입체구조는 마피반응과 으로 규명되었다. 코프리스아마이드는 quinone reductase 유발에 효과를 보였다. 코프리시딘 A와 B는 애기뿔소똥구리 (Copris tripartitus) 장내에서 분리된 박테리아가 생산하는 물질이다. 코프리시딘 A와 B의 평면구조는 핵자기공명법과 질량분석법, 자외선분광법등을 이용하여 규명되었다. 코프리시딘의 입체구조는 ECD계산을 통하여 결정지었고, 자연계에서 첫번째로 발견된 naphthoquinone-oxindole alkaloids로 확인되었다. 코프리시딘 A는Na+/K+-ATPase활성을 억제하였고, 코프리시딘 B는 quinone reductase 유발에 효과를 보였다.

During my doctoral course, I studied on bacterial secondary metabolites, which have been recently more highlighted as unique sources of novel small molecules with useful pharmaceutical potential. My overall goals of my study were to discover new bioactive bacterial secondary metabolites from marine bacteria (Part A) and dung beetle, Copris tripartitus, associated bacteria (Part B).

Part A. Discovery of new bioactive peptide secondary metabolites from marine Streptomyces spp.

In my search for novel bioactive molecules, I collected deep-sea sediments as well as seashore sediments around Jeju Island, Republic of Korea. Extensive isolation and chemical screening of marine Streptomyces strains from these sea sediments, I discovered two strains, SNJ013 and SNJ042, producing structurally-novel peptides. Sungsanpin, a new 15-amino-acid peptide, was discovered from a Streptomyces species isolated from deep- sea sediment collected off Jeju Island, Korea. Also, ohmyungsamycins A and B, which are new cyclic peptides, were isolated from a marine bacterial strain belonging to the Streptomyces genus collected from a sand beach on Jeju. The planar structures of the compounds were determined by 1D and 2D NMR spectroscopy, mass spectrometry and UV spectroscopy. Sungsanpin is the first example of a lasso peptide isolated from a marine-derived microorganism while ohmyungsamycins A and B were elucidated as cyclic depsipeptides bearing unusual amino acid units, including N-methyl-4- methoxytrytophan, b-hydroxyphenylalanine, and N,N-dimethylvaline. The absolute configurations of the stereocenters in the compounds were assigned by derivatization of the hydrolysate of sungsanpin with Marfeys reagents and GITC (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate), followed by LC-MS analysis. I have developed a new method utilizing PGME (phenylglycine methyl ester) derivatization coupled with chromatographic analysis to determine the absolute configuration of N,N-dimethylvaline in ohmyungsamycin B. Sungsanpin displayed inhibitory activity in a cell invasion assay of the human lung cancer cell line A549. Ohmyungsamycins A and B showed significant inhibitory activities against diverse cancer cells as well as antibacterial effects.

Part B. Discovery of new bioactive secondary metabolites from gut- associated bacteria in the dung beetle, Copris tripartitus

A large majority of animals and plants rely on bacterial symbionts in order to survive and thrive. Yet despite the pervasiveness of these mutualistic interactions, the molecular underpinnings of most either remain unexplored or are just beginning to be understood. Recently, several studies have highlighted insect-microbial associations as particularly promising sources for new bioactive small molecules, many of which may have pharmaceutical potential. In my search for novel bioactive molecules, I collected the dung beetle, Copris tripartitus, in Jeju Island from 2012 to 2016. Coprisamides and coprisidins were isolated from a bacterial strain, SNU533 in the gut of the dung beetle Copris tripartitus. Spectroscopic analysis revealed that the planar structures of coprisamides A and B are novel cyclic heptapeptides bearing unusual units, such as β-methylaspartic acid and 2,3-diaminopropanoic acid branched to valine and 2-heptatrienyl cinnamic acid. Absolute configurations were established by chemical derivatization and chiroptical spectroscopy. The coprisamides displayed significant activity for induction of quinone reductase. Also, I discovered new naphthoquinone-oxindole alkaloids, coprisidins A and B from a gut-associated Streptomyces sp. strain, SNU607, in the dung beetle Copris tripartitus. Using a combination of spectroscopic techniques, the planar structures of the compounds were determined to be the first examples of natural naphthoquinone-oxindole alkaloids, with the absolute configurations of coprisidins A and B determined by electronic circular dichroism (ECD). Coprisidin A was found to inhibit the action of Na+/K+-ATPase and coprisidin B showed activity for the induction of NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II detoxifying enzyme that plays an important role in cancer prevention and provides a potential target for the prevention of carcinogenesis.