Agrocybynes A to E from the culture broth of Agrocybe praecox

Five novel compounds (1-3, 5 and 6), and two known ones (4 and 7) were isolated from the culture broth of Agrocybe praecox. Their structures were determined by the interpretation of spectroscopic data. Compounds 1 to 4 inhibited hypocotyl growth of lettuce, and 1, 3 and 4 inhibited the root growth. 1. Introduction In 2007, abnormal enlargement of strawberry fruits was observed and a kind of mushroom grew near the stimulated fruits in a greenhouse in Niigata prefecture, Japan. It was identified as Agrocybe praecox (English name, Spring Fieldcap; Japanese name, Fumizukitake) . On the other hand, it has been reported that an Agrocybe sp. caused cringing of strawberry fruits. These widely varying phenomena related to growth stimulation and suppression, suggest that Agrocybe genus produces plant growth regulator(s). A. praecox is edible and widespread in the northern temperate zone throughout the world. The mushroom is litter degrading fungus that is able to grow in the forest soil and produces non-specific extracellular enzymes, which decompose soil detritus (including polymers such as cellulose and hemicellulose) into monomers and oligomers, which in turn are made available to microbes and plants. The fungus has been in focus for various application due to the hydrolytic and ligninolytic enzymatic activities for bioremediation, however it has not yet been reported as environmental adaptation by coexisting with plants. In this article we focused on plant growth regulators from the fungus A. praecox. Here, we describe the isolation, structural determination, and biological activity of five novel compounds (1-3, 5 and 6), and two known ones (4 and 7) from the culture broth of the fungus. 2. Results Culture broth of A. praecox was partitioned between EtOAc and water, and then n-BuOH and water. Each fraction was tested to plant growth regulatory activity using lettuce and the EtOAc-soluble part showed significant inhibitory activity. The EtOAc-soluble part was fractionated by repeated chromatography, guided by the bioassay results leading to discovery of five novel compounds (1-3, 5 and 6), and two known ones (4 and 7) were purified. Agrocybyne A (1) was purified as colorless needle shaped. Its molecular formula was determined as C8H9NO by HRESIMS m/z 158.05586 [M+Na] (calcd for C8H9NNaO, 158.05818), indicating the presence of five degrees of unsaturation in the molecule. Structure of 1 was elucidated by interpretation of NMR spectra including DEPT, COSY, HMQC, and HMBC (Figure 1). The DEPT experiment indicated the presence of a methyl, two methylenes, and five quaternary carbons. The molecular formula, the unsaturation degrees, and the DEPT data suggested the presence of two substituted triple bonds and a carboxyamide in the molecule. The complete assignment of the protons and carbons of NMR was accomplished as shown in Table 1. The structure of propyl moiety (C6 to C8) was elucidated by the COSY correlations (H7/H6, H8) and the HMBC correlations (H6/C7, C8; H7/C6, C8; H8/C6, C7). The presence and position of butadiyne moiety (C2 to C5) was confirmed by the HMBC correlations (H6/C2 to C5; H7/C5) and the characteristic
chemical shifts (δC 63.7, 67.0, 71.7, 87.3). As a result, structure of 1 was supposed to be octa-2,4-diynamide and was confirmed by X-ray crystallography (Figure 2).