Aspartame and Phe-Containing Degradation Products in Soft Drinks across Europe.

Phenylketonuria and tyrosinemia type 1 are treated with dietary phenylalanine (Phe) restriction. Aspartame is a Phe-containing synthetic sweetener used in many products, including many 'regular' soft drinks. Its amount is (often) not declared; therefore, patients are advised not to consume aspartame-containing foods. This study aimed to determine the variation in aspartame concentrations and its Phe-containing degradation products in aspartame-containing soft drinks. For this, an LC-MS/MS method was developed for the analysis of aspartame, Phe, aspartylphenylalanine, and diketopiperazine in soft drinks. In total, 111 regularly used soft drinks from 10 European countries were analyzed. The method proved linear and had an inter-assay precision (CV%) below 5% for aspartame and higher CVs% of 4.4-49.6% for the degradation products, as many concentrations were at the limit of quantification. Aspartame and total Phe concentrations in the aspartame-containing soft drinks varied from 103 to 1790 µmol/L (30-527 mg/L) and from 119 to 2013 µmol/L (20-332 mg/L), respectively, and were highly variable among similar soft drinks bought in different countries. Since Phe concentrations between drinks and countries highly vary, we strongly advocate the declaration of the amount of aspartame on soft drink labels, as some drinks may be suitable for consumption by patients with Phe-restricted diets.

Thermally-induced formation of taste-active 2,5-diketopiperazines from short-chain peptide precursors in cocoa.

2,5-diketopiperazines (DKPs) are cyclic dipeptides responsible for the specific bitter taste of cocoa formed during roasting. The 2,5-diketopiperazine and peptide composition of four different cocoa bean samples from different origins were studied using LC-MS techniques. 34 diketopiperazines were identified, of which 10 are newly reported in cocoa. Their formation was followed during two different roasting time-series using a zero-order and an alternative Prout-Tompkins solid-state kinetic models. The activation energies of diketopiperazine formation showed a distribution close to normal with individual values depending on the nature of the substituents. The relative concentrations of the DKPs were correlated with their putative peptide precursors in unroasted cocoa bean samples. The results showed a significant positive correlation, indicating that oligopeptides formed in cocoa bean fermentation are taste-precursors for bitter tasting diketopiperazines. Unexpectedly, for most diketopiperazines, a single major peptide precursor could be suggested.

Roasted Barley Extract Affects Blood Flow in the Rat Tail and Increases Cutaneous Blood Flow and Skin Temperature in Humans.

Roasted barley extract (RBE, "Mugicha") is a traditional Japanese beverage reported to improve blood viscosity and affect food functionality. RBE is suggested to contain 2,5-diketopiperazines, which are the functional component with neuroprotective and immunostimulatory effects that are produced in food through roasting. In this study, we investigated the effects of RBE on blood circulation, both clinically and in rats. At first, we confirmed five 2,5-diketopiperazine derivatives in RBE by LC-MS analysis. Secondarily, we revealed that RBE affects blood flow in the rat tail and compared the efficacy on rat tail blood flow among five 2,5-diketopiperazines in RBE. Especially, cyclo(d-Phe-l-Pro) was the most effective in increasing blood flow in the rat tail. We also researched the mechanism of cyclo(d-Phe-l-Pro) with rat aorta study. As a result, we confirmed that cyclo(d-Phe-l-Pro) has an effect on vasodilatation through the release of nitric oxide in the vascular endothelium. Finally, we also confirmed that RBE affects cutaneous blood flow and increases skin temperature in humans.

Investigating on the Correlation Between Some Biological Activities of Marine Sponge-Associated Bacteria Extracts and Isolated Diketopiperazines.

Marine organisms have been considered as the richest sources of novel bioactive metabolites, which can be used for pharmaceutical purposes. In the last years, the interest for marine microorganisms has grown for their enormous biodiversity and for the evidence that many novel compounds isolated from marine invertebrates are really synthesized by their associated bacteria. Nevertheless, the discovery of a chemical communication Quorum sensing (QS) between bacterial cells and between bacteria and host has gained the researchers to expand the aim of their study toward the role of bacteria associated with marine invertebrates, such as marine sponge. In the present paper, we report the evaluation of biological activities of different extracts of bacteria Vibrio sp. and Bacillus sp. associated with marine sponges Dysidea avara and Ircinia variabilis, respectively. Moreover, we evaluated the biological activities of some diketopiperazines (DKPs), previously isolated, and able to activate QS mechanism. The results showed that all extracts, fractions, and DKPs showed low scavenging activity against DPPH and superoxide anion, low cytotoxic and anti-tyrosinase activities, but no antimicrobial and acetylcholinesterase inhibitory activities. One DKP [cyclo-(trans-4-hydroxy-L-prolyl-L-leucine)] has the highest α-glucosidase inhibitory activity even than the standard acarbose.

Chiral Recognition of Diketopiperazine Cyclo(Pro-Gly) and Propranolol Using (-)-Epigallocatechin-3-O-gallate.

In the (1)H-NMR spectrum of a solution containing an equimolecular amount of cyclo(L-Pro-Gly), cyclo(D-Pro-Gly) and (-)-epigallocatechin-3-O-gallate (EGCg) in a D2O, a difference in the chemical shift of (1)H-NMR signal for H7α, H7ß,8α of the Pro residue was observed. Judging from the crystal structures of the 2 : 2 complexes of EGCg and cyclo(L-Pro-Gly), cyclo(D-Pro-Gly), the difference in the chemical shift resulted mainly from a magnetic anisotropic shielding effect by the ring current from the B ring of EGCg. Therefore, it was considered that chirality of cyclo(Pro-Gly) was recognized by EGCg in the D2O solution. Furthermore, in the (1)H-NMR spectrum of a solution containing an equimolecular amount of racemic propranolol ((R)- and (S)-propranolols) and EGCg in D2O, the (1)H-NMR signal for H2 of the naphthalene group was observed as two doublets, suggesting that the racemic propranolol formed diastereomers of complexes with EGCg; as a result, chirality of propranolol was recognized by EGCg in the D2O solution.

Liquid chromatography coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry and post-column addition of metal salt solutions as a powerful tool for the metabolic profiling of Fusarium oxysporum.

Fusarium oxysporum L11 is a non-pathogenic soil-borne fungal strain that yielded an extract that showed antifungal activity against phytopathogens. In this study, reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry (API-QTOF-MS) was applied for the comprehensive profiling of the metabolites from the extract. The employed sources were electrospray (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Post-column addition of metal solutions of Ca, Cu and Zn(II) was also tested using ESI. A total of 137 compounds were identified or tentatively identified by matching their accurate mass signals, suggested molecular formulae and MS/MS analysis with previously reported data. Some compounds were isolated and identified by NMR. The extract was rich in cyclic peptides like cyclosporins, diketopiperazines and sansalvamides, most of which were new, and are reported here for the first time. The use of post-column addition of metals resulted in a useful strategy for the discrimination of compound classes since specific adducts were observed for the different compound families. This technique also allowed the screening for compounds with metal binding properties. Thus, the applied methodology is a useful choice for the metabolic profiling of extracts and also for the selection of metabolites with potential biological activities related to interactions with metal ions.

IR action spectroscopy shows competitive oxazolone and diketopiperazine formation in peptides depends on peptide length and identity of terminal residue in the departing fragment.

The interplay between the entropically and enthalpically favored products of peptide fragmentation is probed using a combined experimental and theoretical approach. These b2 ion products can take either an oxazolone or diketopiperazine structure. Cleavage after the second amide bond is often a favorable process because the products are small ring structures that are particularly stable. These structures are structurally characterized by action IRMPD spectroscopy and semi-quantified using gas-phase hydrogen-deuterium exchange. The formation of the oxazolone and diketopiperazine has been thought to be largely governed by the identity of the first two residues at the N-terminus of the peptide. We show here that the length of the precursor peptide and identity of the third residue play a significant role in the formation of the diketopiperazine structure in peptides containing an N-terminal asparagine residue. This is additionally the first instance showing an N-terminal residue with an amide side chain can promote formation of the diketopiperazine b2 ion structure.

Chiral separation of a diketopiperazine pheromone from marine diatoms using supercritical fluid chromatography.

The proline derived diketopiperazine has been identified in plants, insects and fungi with unknown function and was recently also reported as the first pheromone from a diatom. Nevertheless the stereochemistry and enantiomeric excess of this natural product remained inaccessible using direct analytical methods. Here we introduce a chiral separation of this metabolite using supercritical fluid chromatography/mass spectrometry. Several chromatographic methods for chiral analysis of the diketopiperazine from the diatom Seminavis robusta and synthetic enantiomers have been evaluated but neither gas chromatography nor high performance liquid chromatography on different chiral cyclodextrin phases were successful in separating the enantiomers. In contrast, supercritical fluid chromatography achieved baseline separation within four minutes of run time using amylose tris(3,5-dimethylphenylcarbamate) as stationary phase and 2-propanol/CO2 as mobile phase. This very rapid chromatographic method in combination with ESI mass spectrometry allowed the direct analysis of the cyclic dipeptide out of the complex sea water matrix after SPE enrichment. The method could be used to determine the enantiomeric excess of freshly released pheromone and to follow the rapid degradation observed in diatom cultures. Initially only trace amounts of c(d-Pro-d-Pro) were found besides the dominant c(l-Pro-l-Pro) in the medium. However the enantiomeric excess decreased upon pheromone degradation within few hours indicating that a preferential conversion and thus inactivation of the l-proline derived natural product takes place.

Cyclo-(His,Leu): a new microbial diketopiperazine from a terrestrial Bacillus subtilis strain B38.

In continuation of our search for bioactive secondary metabolites from terrestrial Bacillus spp., a new microbial diketopiperazine, cis-cyclo-(His,Leu) (1) was isolated from the ethyl acetate extract of a strain B. subtilis B38, together with cis-cyclo-(Phe,Phe) (2), tryptophane (3), cis-cyclo-(Leu,Tyr) (4), cis-cyclo-(Trp,Tyr) (5) and macrolactin A (6). The chemical structures of the isolated compounds were identified by comparison of their 1D, 2D NMR and HRESIMS data with authentic spectra and literatures. To the best of our knowledge, this is the first time that cyclo-(His,Leu) has been isolated from natural products.

Structure elucidation of a new natural diketopiperazine from a Microbispora aerata strain isolated from Livingston Island, Antarctica.

A new natural diketopiperazine (1) was obtained from the culture broth of Microbispora aerata strain imbas-11A, isolated from penguin excrements collected on the Antarctic Livingston Island. Compound 1 was purified consecutively by solvent extraction, silica gel column chromatography and preparative HPLC. The structure of the compound was elucidated by 1D and 2D NMR experiments and mass spectrometric investigations. The absolute configuration of compound 1 was determined by amino acid analysis and NOESY correlations. A low antiproliferative and cytotoxic effect of trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) was determined with L-929 mouse fibroblast cells, K-562 human leukemia cells and HeLa human cervix carcinoma. Trans-cyclo-(D-tryptophanyl-L-tyrosyl) (1) did not show antimicrobial activity at a concentration of 50 µg per disc against Bacillus subtilis, Staphylococcus aureus, Streptomyces viridochromogenes, Escherichia coli, Candida albicans and Mucor miehei.

[New producers of biologically active compounds--fungal strains of the genus Penicillium isolated from permafrost].

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazines, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxiagroclavine-I and quinocitrinins. The main physiological and biochemical characteristics of this producer were investigated.

Synthesis and characterization of ester-based prodrugs of glucagon-like peptide 1.

Peptides represent a rich natural source of potential medicines with one notable pharmaceutical limitation being their relatively short duration of action. A particularly good example of this phenomenon is glucagon-like peptide 1 (GLP), a hormone of appreciable interest for the treatment of type II diabetes. In the native form, GLP demonstrates an extremely short half-life in plasma and a relatively narrow therapeutic index with gastrointestinal adverse pharmacology. We envisioned a prodrug of GLP as a means to extend the duration of action and broaden the therapeutic index of this peptide hormone. We designed, synthesized, and characterized ester-based prodrugs of GLP that differentially convert to the parent drug under physiological conditions driven by their inherent chemical instability. In a set of dipeptide extended GLP-analogs we explored the rate of diketopiperazine (DKP) and diketomorpholine (DMP) formation, and the release of the active peptide. The rate of cleavage was observed to be a function of the conformation of the dipeptide promoiety and the strength of the cyclization nucleophile. Through the careful selection of chemical functionality, a set of GLP ester prodrugs of variable half-lives has been identified.

Determination of diketopiperazines of Burkholderia cepacia CF-66 by gas chromatography-mass spectrometry.

Bacteria communicate with each other by a process termed "quorum sensing" (QS), and diffusible, low-molecular-weight chemicals, called signal molecules, are used as the communication languages. In cell-free Burkholderia cepacia CF-66 culture supernatants, five compounds suspected of being signal molecules were identified. The gene (cepI) related with AHLs synthesis were not detected by polymerase chain reaction (PCR) using specific primers. Gas chromatography-mass spectrometry (GC-MS) revealed that these compounds were not AHLs but the diketopiperazines (DKPs) cyclo(Pro-Phe), cyclo(Pro-Tyr), cyclo(Ala-Val), cyclo(Pro-Leu), and cyclo(Pro-Val), all of which were both D and L-type. Four kinds of DKPs had been isolated from other gram-negative bacteria, but the other was a novel kind discovered in CF-66, and L-cyclo (Pro-Phe) was quantified by GC-MS. It was found that exogenous DKPs had a negative effect on the candidacidal activity of the culture supernatant extracts.

Detection and quantitation of 2,5-diketopiperazines in wheat sourdough and bread.

Liquid chromatography mass spectrometry (LC-MS) was used to quantify the levels of the 2,5-diketopiperazines cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) in acidified dough and bread. Dough acidification led to a significant increase in the level of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) over 48 h compared to a nonacidified dough. However, no differences were found between chemically (mix of lactic and acetic acid in the presence of antibiotics) and biologically acidified doughs. On examination of the levels of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) in bread crumb and crust, it was found that temperature is the main causative agent of 2,5-diketopiperazine formation during the baking process. Bread crumb and crust contained almost 100 and 2000 times respectively the levels found in dough prior to baking. cis-Cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) were also found to be at sensorally active levels in bread crust, however both 2,5-diketopiperazines were found to be below the minimum inhibitory concentration for antifungal activity in bread.

2, 5-diketopiperazines (cyclic dipeptides) in beef: identification, synthesis, and sensory evaluation.

Stewed beef and grilled dry aged beef were analyzed as part of an in-depth analytical program, with the aim of creating new flavors incorporating only compounds identified in the target foods and identifying new synthesis targets. In-house GC-MS analyses of several types of cooked beef have identified over 1000 volatile and semivolatile components; many for the 1st time. Among the semivolatiles detected were ten 2, 5-diketopiperazines (cyclic dipeptides) previously unreported in beef. These cyclic dipeptides are cis-cyclo(L-Ile-L-Pro), cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Pro-L-Pro), cis-cyclo(L-Pro-L-Val), cis-cyclo(L-Ala-L-Pro), cyclo(Gly-L-Pro), cyclo(Gly-L-Leu), cis-cyclo(L-Met-L-Pro), cis-cyclo(L-Phe-L-Pro), and cis-cyclo(L-Phe-L-Val). All 10 cyclic dipeptides were synthesized and evaluated organoleptically. Among them cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Met-L-Pro), and cis-cyclo(L-Phe-L-Pro) were found to be of particular organoleptic interest.

Detection of amoxicillin-diketopiperazine-2', 5' in wastewater samples.

The short half-life of aminopenicillin antibiotics in the aquatic environment put to the challenge the detection of their degradation products among environmental hydro-chemists. In a quest to study the occurrence of a new emerging micro-pollutant in the aquatic environment we attempted this by analyzing samples from a wastewater treatment plant for a major degradation product of amoxicillin (i.e., amoxicillin-diketopiperazine-2', 5') using a high-performance liquid chromatography technique coupled with tandem mass spectrometry method. ADP was repeatedly detected in all wastewater and effluent samples (18) from which it was extracted. To the best of our knowledge, this is the first study that evidently proves the occurrence of the chemically stable form of AMX, its Diketopiperazine-2', 5', in wastewater and effluent samples. Furthermore, penicillins are known to cause most allergic drug reactions. There is a risk that residues of hypersensitivity-inducing drugs, such as penicillins and their degradation products, may elicit allergic reactions in human consumers of water and food of animal origin.

Rapid screening for cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. using liquid chromatography/tandem mass spectrometry.

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products.