Dinoflagellates, a type of unicellular phytoplankton, are a rich source of secondary metabolites, which fascinates us a lot for their structural features and potent biological activities. From genus Prorocentrum, okadaic acid (OA) was confirmed as the causative substance of diarrheic shellfish poisoning and later turned out to be a potent inhibitor of protein phosphatase 2A. In addition, genus Prorocentrum produces hoffmanniolide, prorocentrolides, and spiro-prorocentrimine, having diverse structural features as polyether carboxylic acid, macrolactones, and cyclicimines.

During our continuing search for new secondary metabolites from marine dinoflagellates, we have identified a new polyene-polyol with a molecular weight of 1474, named prorocentrol, in the cell extracts of Prorocentrum hoffmannianum. Prorocentrol possesses unique structural features such as diene, triene, hemiacetal, ketone moieties, and 30 hydroxy groups. With regards to the biological activities, prorocentrol inhibits growth of murine leukemia cells P388 at 15μg/mL and diatom Nitzchia sp. at 50μg/mL. In addition, inhibition of spore formation against the fungs Aspergillus niger was observed at 100μg/disc.

Structural elucidation of prorocentrol has not been completed because of a ketone-hemiacetal tautomerism during the course of NMR measurements. In this research, to obtain a single tautomer for NMR analysis was the first target in order to investigate its stereochemistry related to further biological properties.

Isolation and planar structure of prorocentrol

The dinoflagellate P. hoffmannianum was grown for 30-45 days at 25 °C in a seawater medium enriched with f/2 nutrients. The cultured cells were harvested by centrifugation and then extracted with methanol. The extracts were subjected to 4 steps of biphasic partition as shown in Fig. 2. Since the 1-butanol extracts contained a lot of okadaic acid, this extract was partitioned between ethyl acetate and 0.15% acetic acid, resulting in effective removaI of okadaic acid from prorocentrol. The 0.15% AcOH extracts were passed through an ODS column, HW-40(S) column, and finally purification using ODS MPLC afforded prorocentrol as colorless amorphous solid. During the above purification procedures, prorocentrol in eluates were monitored with a photodiode array detector at 271 nm and also by ion peak at 1497 [M+Na]} on MALDI-MS spectra.

The high resolution ESI-MS gave [M+Na]' at m/z 1497.7089 (Δ-1.5 mmu), and its molecular formula was deduced to be C68H114034. The UV and IR absorption spectra suggested the presence of conjugated diene (λmax 232 nm) and conjugated triene (λmax 260, 270 and 281 nm) chromophores, and hydroxy groups (vmax 3365 cm I) and ketones (v max1711 cm-I). The 13C-NMR spectra in methanol-d4 and DMF-d7 were very complicated with more than 68 signals observed. This phenomenon is explained by the keto-enol tautomerism or ring opening acetal exchange at cyclic hemiacetal moiety. To resolve this problem, NMR measuring conditions such as solvents, pH, or temperature were examined to lead to the solvent system of pyridine-d5/D20 (6/1) giving simplified and improved spectrum at room temperature as shown in Fig. 3. Each signal around the acetal and the ketone was observed as a single peak in the 13C-NMR spectrum. All carbon signals were successfully assigned as 2 methyls, 12 aliphatic methylenes, 2 aliphatic methines, I oxymethylene, 33 oxymethines, 1 acetal, 16 olefins including one quaternary olefinic carbon, and I ketone. The numbers of hydroxy groups were estimated by measuring the 13C-NMR deuterium shift on the hydroxy bearing carbons between pyridine-d5/H2O (6/1) and pyridine-d5/D20 (6/1) solvent systems. Of all the monooxygenated carbons (64.6-100.9ppm), only the 5 oxymethines did not show the deuterium induced shift. These experimental data suggested prorocentrol possesses one hemiacetaI and one ketone moieties as a major tautomer in this solvent system.

A detailed analysis of the DQF-COSY, TOCSY, and HSQC-TOCSY spectra allowed us to elucidate 5 partial structures, C1-C15, C18-C34, C36-C38, C40-C41 and C42-C65. In the HMBC spectra, a pair of hemiacetal and ketone signals was confirmed with corresponding correlation signals. This result supported the assignment of the one hemiacetaI and one ketone structure as the major tautomer in pyridine-d5/D20 (6/1). In a similar way, two partial structures were connected to a quaternary olefinic carbon. The presence of tetrahydrofurane and tetrahydropyrane rings was also confirmed.

Relative configurational analysis on prorocentrol

The relative configuration of the tetrahydropyrane ring (C10-C14) of prorocentrol was elucidated based on vicinal proton coupling constants and NOE correlations from E. COSY and 2D-NOESY spectra, respectively. Coupling constants and NOE correlations between protons on this ring suggested that this ring had a chair conformation as shown in Fig. 5. Although the relative configuration of the above cyclic structure was determined by the use of conventional NMR experiments, the problem was more complicated for contiguous polyol units on a long acyclic carbon chain. Therefore the author applied the JBCA (J based configuration analysis), a method developed by Murata et. aI. This method leads to the correct diastereomer of 1,2- or 1,3-methine systems through its major conformation based on estimated multi-bonded hetero and homonuclear coupling constants and thus indicated dihedral angles. For the C52-055 portion, 3JH,H and 2'3Jc,H values were obtained from E. COSY and HETLOC spectra, and dominant conformation and relative stereoconfiguration was shown as Newman projections and stereo projection in Fig. 6., respectively.

Likewise, the longest polyol segment of prorocentrol, C1-C9 portion was assigned by JBCA method as C2-C3, C3-C4, C4-05, C5-C6, C6-C7, C7-C9, and C9-C10 into erythro, threo, threo, threo, threo, syn, and threo as shown in Fig. 7. By using JBCA method, relative stereoconfiguration at contiguous polyol segment of prorocentrol in both termini was elucidated.

Molecular interaction between prorocentrol and okadaic acid

As mentioned in the isolation part, okadaic acid (OA) which should hardly be partitioned in water, was observed in 1H-NMR spectra of the prorocentrol enriched fraction. Interestingly, some proton signals of okadaic acid were shifted under the coexistence of prorocentrol.

Considering the observed 1H-NMR signal shifts as shown in Fig. 8., specific association of prorocentrol and okadaic acid was speculated. Since this phenomenon was suspected to affect the biologicaI activity of okadaic acid, cytotoxicity of okadaic acid against P388 cells, which is 1000 times more potent than that of prorocentrol, was examined under co-existence of OA/prorocentrol. The obtained dose-response curves of a solution of OA /prorocentrol 1/5 eq. were shown in Fig. 9. From this result, significant change in cytotoxicity of OA was not observed.

Fig.1. Planar structure of prorocentrol

Fig. 2 Isolation scheme of prorocentrol

Fig. 3.(13)C-NMR spectra of prorocentrol,(a): in methanol-di, 125 MHz,(b): in py-d5/ D2o 6/1,100 MHz.

Fig. 4.NMR assignments of prorocentrol.

Fig. 5.Relative stereoconfiguration of C10-C14

Fig. 6.Relative stereoconfiguration of C52-053

Fig. 7.Relative stereoconfiguration of C2 to C14

Fig. 8.Above: 500MHz1H-NMR spectra in methanol-d4,(a) OA and prorocentrol mixture,(b) OA, (c) prorocentrol.Below: structure of okadaic acid (OA).

Fig. 9 Dose-response curve of cytotoxicity assay.

本論文の研究材料である海洋性渦鞭毛藻からは、赤潮毒素を始めとする生理活性低分子化合物の単離・構造決定が近年になり盛んに報じられてきており、これらは主としてエーテル環構造を含むポリケチドと呼ばれる直鎖炭素鎖からなる化合物である。本論文ではこのうちProrocentrum hoffmannianumと命名された渦鞭毛藻が産生しポリエン・ポリオール化合物で分子式C(689H(114)o(34)(分子量1474,下図)でprorocentro1と名付けた化合物についての化学構造式の決定に関するものである。

本論文は全5章からなり、第1章では序論として上記の背景と、かつて本論文提出者の研究室にて過去に本化合物が単離された経緯が紹介されており、この研究を引き継いだ論文提出者の寄与する部分が明確になっている。第2章では、互変異性(共有結合が分子内にて短時間で組み変わる性質)により水素核磁気共鳴(1H-NMR)による構造決定が困難であった本化合物の化学構造式の決定における問題を、これら異性体の一方のみが優位になる測定溶媒の検索により克服することでこの平面構造を確定した経緯が述べられている。

第3章ではさらに、NMR測定に関して近年開発された1Hと(13)Cとの遠隔結合定数による三次元立体配座解析を経由した立体配置決定(J-based configurational analysis:JBCA法)、および合成された一連の部分構造モデル化合物ライブラリーの1H-NMR解析データベースの適用を駆使することにより、本化合物の立体配置を可能な範囲で決定した経緯が述べられており、こうした手法の有用性とともに立体配座が複数存在する化合物へのこれらの適用における現時点での限界を指摘している。

第4章では研究対象として用いた渦鞭毛藻が産生する強力な細胞毒で貝の摂食による食中毒の原因の一つと考えられているオカダ酸(C44からなるポリ環状工一テル化合物)との親和性が1H-NMRにて観測される事実に関し、この毒性をこの渦鞭毛藻においてprorocentro1が中和している可能性について論じている。細胞毒性試験ではこれらが否定される実験結果を得たことに踏まえて、本化合物の他の生態的意義の可能性を論じている。

第5章は実験項であり、行った実験の方法と結果が追試可能な内容で詳細に記されており、この後に本研究の結論と測定された生の実験データが付記添付されている。

以上、本論文の研究内容は従来困難であった溶液中で相互変換する化合物のNMRによる化学構造解析に関して、ここでの問題を克服するための手段を示唆するとともに、これらの立体配座決定における論文提出者が用いた手法の有用性および限界を示しており、今後の本分野での問題解決における指針を示しているとの判断が審査委員全員の賛同により認められた。

なお、本研究は本化合物を当初単離した北村嘉章、村田道雄と、その後の研究における佐竹真幸、橘和夫の助言により遂行されたが、本論文に記された実験の立案と実施、およびこれらの結果の解析と考察は論文提出者のものであり、その寄与は十分であると判断できる。

従って、本論文提出者である菅原孝太郎は、博士(理学)の学位を授与できるものと認める。