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.

Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use L-amino acids to make proteins, a group of D-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the D-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-L sequence. Because this L-to-D post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides.

Within the parent protein molecule, most peptides are inactive, and they are released with biofunctionalities after enzymatic hydrolysis. Marine algae have high protein content, up to 47% of the dry weight, depending on the season and the species. Recently, there is an increasing interest in using marine algae protein as a source of bioactive peptides due to their health promotion and disease therapy potentials. This review presents an overview of marine algae-derived bioactive peptides and especially highlights some key issues, such as in silico proteolysis and quantitative structure-activity relationship studies, in vivo fate of bioactive peptides, and novel technologies in bioactive peptides studies and production.

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To examine and quantify the potential relation between diabetic retinopathy (DR) and risk of all-cause mortality, stroke and heart failure (HF).The resources of meta-analysis of epidemiological observational studies were from Pub-med, EMBASE, CINAHL, Cochrane Library, conference, and proceedings.Random/fixed effects models were used to calculate pooled subgroup analysis stratified by different grades of DR was performed to explore the potential source of heterogeneity. Statistical manipulations were undertaken using program STATA.Of the included 25 studies, comprising 142,625 participants, 19 studies were concluded to find the relation of DR to all-cause mortality, 5 for stroke, and 3 for HF. Risk ratio (RR) for all-cause mortality with the presence of DR was 2.33 (95% CI 1.92–2.81) compared with diabetic individuals without DR. Evidences showed a higher risk of all-cause mortality associated with DR in patients with T2D or T1D (RR 2.25, 95% CI 1.91–2.65. RR 2.68, 95% CI 1.34–5.36). According to different grades of DR in patients with T2D, RR for all-cause mortality varied, the risk of nonproliferative diabetic retinopathy (NPDR) was 1.38 (1.11–1.70), while the risk of proliferative diabetic retinopathy (PDR) was 2.32 (1.75–3.06). There was no evidence of significant heterogeneity (Cochran Q test P = 0.29 vs 0.26, I2 = 19.6% vs 22.6%, respectively). Data from 5 studies in relation to DR and the risk of stroke showed that DR was significantly associated with increased risk of stroke (RR = 1.74, 95%CI: 1.35–2.24), compared with patients without DR. Furthermore, DR (as compared with individuals without DR) was associated with a marginal increased risk of HF in patients with diabetes mellitus (DM) (n = 3 studies; RR 2.24, 95% CI 0.98–5.14, P = 0.056).Our results showed that DR increased the risk of all-cause mortality, regardless of the different stages, compared with the diabetic individuals without DR. DR predicted increased risk of stroke and HF. Although only 3 studies about HF were available, the association between DR and HF should be careful.