However, despite this symmetry, the methylene groups have a diastereotopic relationship with each other, and therefore display different chemical shifts in the 1H-NMR. In addition, each proton on the methylene groups also has a diastereotopic relationship with each other, and this results in the appearance of a large geminal coupling constant (15.3 Hz) between these protons. The symmetrical nature of 1 is also supported by the presence of only five signals in
the 13C-NMR. The other possible isomer of dimethyl citrate, with a terminal carboxylic acid, would possess a center of chirality, and as a result, there would be two methyl signals in the 1H-NMR, as well as possibly eight signals in the 13C-NMR (Anet & Park, 1992). The second compound that eluted from the column (187 mg) displayed two EPZ015666 ic50 singlets in the 1H-NMR (δ 3.76, 3H, and 3.65, 6H), which suggested the presence of two unique methyl ester groups. A pattern of doublets similar to that observed in 1, at δ 2.94 and 2.82 (J=15.3 Hz), suggested that this compound was trimethyl citrate (2). This was further reinforced by the 13C-NMR, where the two carbonyl groups (δ 175.3 and 171.8) were evident along with a signal for an oxygenated quaternary carbon (δ 74.8), and
two signals (δ 53.3 and 52.4) consistent with methyl esters and an additional signal (δ 44.4) suggested a methylene attached to an electron-withdrawing group. The EI-MS http://www.selleckchem.com/products/bgj398-nvp-bgj398.html suggested a molecular formula of C9H14O7 consistent with the proposed
structure of 2. The symmetrical nature of 2 was evident from the 1H- and 13C-NMR Adenosine and the pattern of signals can be explained using a discussion similar to that for 1. The least polar compound (198 mg) had a rather simple set of spectra, displaying only a single peak in the 1H-NMR at δ 3.76 and only two peaks in the 13C-NMR spectrum at δ 157.6 and 53.1. Based on these data, the structure of this compound was assigned as dimethyl oxalate (3). All of the structural assignments described were confirmed by comparison with spectra in the literature for the compounds. Additionally, a repeat fermentation of this organism using newly propagated spores led to the production of these compounds at a level comparable to our first fermentation. Despite the scale of global citric acid fermentation, there appear to have been no reports of methylated derivatives being produced by fungal cultures. To the best of our knowledge, the strain of A. niger described here is the first report of a filamentous fungus capable of producing methylated citric acid derivatives. Dimethyl citrate (1) and trimethyl citrate (2) have been reported previously as secondary metabolites in a variety of other organisms, but mainly in higher plants such as Prunus mume (Miyazawa et al., 2003), an apricot variety; Gastrodia elata (Pyo et al., 2000), an orchid; Dioscorea opposite (Bai et al., 2008), the Chinese yam; Opuntia ficus-indica (Han et al.