Two dimerized cyclic hexapeptides, antatollamides A (1) and B (2), were isolated from the colonial ascidian Didemnum molle collected in Pohnpei. The amino acid compositions and sequences were determined by interpretation of MS and 1D and 2D NMR data. Raney Ni reduction of antatollamide A cleaved the dimer to the corresponding monomeric cyclic hexapeptide with replacement of Cys by Ala. The amino acid configuration of 1 was established, after total hydrolysis, by derivatization with a new chiral reagent, (5-fluoro-2,4-dinitrophenyl)-N-l-tryptophanamide (FDTA), prepared from l-Trp, followed by LCMS analysis; all amino acids were found to be l-configured except for d-Ala.
Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in E. coli each of the woodrat proteins gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.
The 2S configuration
of the marine natural product
distaminolyne A was recently disputed based upon total synthesis,
yet paradoxically supported by a second independent total synthesis
from a different research group. We now verify the 2S configuration of distaminolyne A by extensive chiroptical studies
and support the veracity of the EC ECD method originally used to prove
it. The origin of the apparent paradox appears to lie in the limits
of precision of polarimetry in the context of weakly rotatory molecules,
which strikes a cautionary note on the reliability of “reassignment”
of natural product configurations based solely on specific rotation.
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