A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey’s reagent, and liquid chromatography–mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.

Background: L-to-D conversion of an amino acid in a neuropeptide can be required for bioactivity. Results: A new D-amino acid-containing peptide (DAACP), GdFFD, shows stereospecific bioactivity in the feeding circuit. Conclusion: Our findings broaden the importance of this unusual post-translational modification, providing new methods to accelerate DAACP discovery. Significance: GdFFD is the first DAACP showing bioactivity in a well defined circuit.

Intermolecular annulation reactions of 1-aryl-2-naphthols with internal alkynes proceed efficiently in the presence of a Ru catalyst and a Cu oxidant to generate spirocyclic compounds by sequential cleavage of the C(sp(2))-H bond, migratory insertion of the alkyne, and dearomatization of the naphthyl ring. Various spirocyclic molecules bearing an all-carbon quaternary stereocenter could be obtained by this novel method with good yields and excellent regioselectivity, and the current process tolerates a variety of synthetically important functional groups.

RNA-based protein synthesis produces L-amino acid-containing proteins and peptides. D-amino acid-containing peptides (DAACPs) can be generated from L-amino acid peptides via post-translational modification. In the nervous system, the conformational change of a single L-amino acid in a peptide to its D-form results in altered bioactivity, with some DAACPs having orders-of-magnitude enhanced efficacy. However, this modification is often overlooked when characterizing endogenous peptides. Here, using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry, neuropeptides that have the second residue isomerized to the D-isoform are distinguished from their L-epimers via differences in the relative amounts of specific fragment ions during tandem MS. By choosing the appropriate fragment ions, and in some cases with the use of metal adducts, epimer discrimination is optimized. Specifically, the cardioexcitatory peptide Asn-DTrp-Phe-amide (NdWFa) was assayed directly from neurons isolated from the sea slug Aplysia californica; the fraction of the peptide with the second residue (W) in the D- versus L-form was 90 ± 10%. We demonstrate that this approach is well suited for confirming DAACPs directly from cells and tissue, advancing our understanding of the L to D modification and the role it plays in cell-to-cell signaling.