Anti-Quorum-Sensing Activity of Tryptophan-Containing Cyclic Dipeptides.

Quorum sensing (QS) can regulate the pathogenicity of bacteria and the production of some virulence factors. It is a promising target for screening to find anti-virulence agents in the coming post-antibiotics era. Cyclo (L-Trp-L-Ser), one variety of cyclic dipeptides (CDPs), isolated from a marine bacterium Rheinheimera aquimaris, exhibited anti-QS activity against Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PAO1. Unlike the CDPs composed of phenylalanine or tyrosine, the anti-QS activity has been widely studied; however, cyclo (L-Trp-L-Ser) and derivatives, containing one tryptophan unit and one non-aromatic amino acid, have not been systematically explored. Herein, the cyclo (L-Trp-L-Ser) and seven derivatives were synthesized and evaluated. All tryptophane-contained CDPs were able to decrease the production of violacein in C.violaceum CV026 and predicted as binding within the same pocket of receptor protein CviR, but in lower binding energy compared with the natural ligand C6HSL. As for P. aeruginosa PAO1, owning more complicated QS systems, these CDPs also exhibited inhibitory effects on pyocyanin production, swimming motility, biofilm formation, and adhesion. These investigations suggested a promising way to keep the tryptophan untouched and make modifications on the non-aromatic unit to increase the anti-QS activity and decrease the cytotoxicity, thus developing a novel CDP-based anti-virulence agent.

Serine-glycine-betaine, a novel dipeptide from an endophyte Macrophomina phaseolina: isolation, bioactivity and biosynthesis.

AIMS: Endophytes are a rich source for structurally complex chemical scaffolds with interesting biological activities. Endophytes associated with Brugmansia aurea L. (family: Solanaceae), a medicinal plant, have not yet explored for the bioactive metabolites. METHOD AND RESULTS: Hence, Macrophomina phaseolina, a fungal endophyte, was isolated from the roots of the plant. Its methanolic extract was found active against human cancer cell lines with IC50 <20 µg ml-1 . Later, a di-peptide compound, serine-glycine-betaine, was isolated and characterized. Serine-glycine-betaine consists of a unit of an N-trimethyl glycine attached to serine. It exhibited potent activity against MIA PaCa-2 and HCT-116 cell lines with IC50 8·9 and 15·16 µmol l-1 , respectively. Furthermore, it induced apoptosis in MIA PaCa-2 cells confirmed by microscopy. The apoptotic cell death in MIA PaCa-2 cells was evidenced biochemically with the generation of intracellular reactive oxygen species level and leading to loss of mitochondrial membrane potential due to activation of the intrinsic pathway. This study describes the plausible biosynthesis of serine-glycine-betaine based on genomics (genome sequencing, annotation and genes alignment). CONCLUSIONS: A novel di-peptide, serine-glycine-betaine isolated from M. phaseolina induced apoptosis in MIA-Pa-Ca-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that dipeptides like serine-glycine-betaine and tyrosine-betaine might be specific to fungal genera, hence being used for diagnostic purposes.

Biotechnological and Ecological Potential of Micromonospora provocatoris sp. nov., a Gifted Strain Isolated from the Challenger Deep of the Mariana Trench.

A Micromonospora strain, isolate MT25T, was recovered from a sediment collected from the Challenger Deep of the Mariana Trench using a selective isolation procedure. The isolate produced two major metabolites, n-acetylglutaminyl glutamine amide and desferrioxamine B, the chemical structures of which were determined using 1D and 2D-NMR, including 1H-15N HSQC and 1H-15N HMBC 2D-NMR, as well as high resolution MS. A whole genome sequence of the strain showed the presence of ten natural product-biosynthetic gene clusters, including one responsible for the biosynthesis of desferrioxamine B. Whilst 16S rRNA gene sequence analyses showed that the isolate was most closely related to the type strain of Micromonospora chalcea, a whole genome sequence analysis revealed it to be most closely related to Micromonospora tulbaghiae 45142T. The two strains were distinguished using a combination of genomic and phenotypic features. Based on these data, it is proposed that strain MT25T (NCIMB 15245T, TISTR 2834T) be classified as Micromonospora provocatoris sp. nov. Analysis of the genome sequence of strain MT25T (genome size 6.1 Mbp) revealed genes predicted to responsible for its adaptation to extreme environmental conditions that prevail in deep-sea sediments.

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial ß-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata.

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of ß-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of ß-Ala-Tyr and its admixtures have been converted into preparative purification of ß-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the ß-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Silica Monolith Nested in Sponge (SiMNS): A Composite Monolith as a New Solid Phase Extraction Material for Environmental Analysis.

We report a new material of a composite silica monolith nested in sponge (SiMNS) and demonstrate an application in the trace analysis of environmental contaminants in water. SiMNS is prepared through sponge absorption of a hydrolyzed mixture of siloxanes and in situ gel formation within the pores. Images obtained using scanning electron microscopy show that the silica and sponge skeletons are mutually nested in SiMNS. This nested composite structure of SiMNS enhances the mechanical flexibility of the material, allowing for reproducible production of desirable sizes and shapes for solid phase extraction (SPE) cartridges without the need to use frits. Functionalization of SiMNS provides appropriate SPE options for selective and efficient extraction of specific contaminants. SPE cartridges packed with functionalized SiMNS-SO3Na have high extraction capacity, good stability in the pH range of 2 to 11, and efficient enrichment of dipeptides in water. Extraction of six dipeptides from water using these new SiMNS-SO3Na SPE cartridges followed by HPLC-MS/MS analysis results in improved method detection limits (MDLs) of 0.02-1.3 ng/L and method quantification limits (MQLs) of 0.05-4.3 ng/L. Successful identification and quantification of three dipeptides, Tyr-Gly, Phe-Gly, and Tyr-Ala, from raw water demonstrates a useful application of the new SPE materials for environmental analysis of trace contaminants. On the basis of this work, a range of functionalized SiMNS materials can be produced and tailored for various environmental and exposomic analyses.

Isolation and characterization of collagen type I crosslink from skin: high-resolution NMR reveals diastereomers of hydroxylysinonorleucine crosslink.

Skin is made up of mainly collagen type I and its structure is stabilised by the formation of covalent immature and mature crosslinks. In this study, collagen immature crosslink hydroxylysinonorleucine (HLNL) was isolated from bovine skin in high purity using two sequential purification steps. These consisted of preparative fibrous cellulose and size exclusion chromatography. The purified crosslink was then analysed using tandem mass spectrometry and high-resolution nuclear magnetic resonance (NMR) spectroscopy. The mass of singly and doubly charged ions of HLNL was 292.1865 and 146.5970 m/z and their optimised fragmentation energy was 17 keV and 5 keV, respectively. The 13C NMR of HLNL showed a doubled-up peak at 67.84 and 67.91 ppm which corroborated a diastereomeric form of collagen immature crosslink HLNL and both are chiroptically indistinguishable. The chemical structure was fully resolved using 1H, 13C and DEPT-135 high-resolution NMR spectroscopy and compared with other previous studies. We also obtained for the first time the 2D NMR spectra COSY and HSQC of HLNL. We therefore suggested that collagen organization into specific fibrils' orientation may be affected by the different configuration of these diastereomers of HLNL.

Callyspongiamides A and B, sterol O-acyltransferase inhibitors, from the Indonesian marine sponge Callyspongia sp.

Callyspongiamides A (1) and B (2), two new sterol O-acyltransferase (SOAT) inhibitors, were isolated from the Indonesian marine sponge Callyspongia sp. together with a known congener, dysamide A (3). The structures of 1 and 2 were elucidated to be polychlorine-containing modified dipeptides based on their spectroscopic data. Compounds 1-3 inhibited both of the SOAT isozymes, SOAT1 and SOAT2, in cell-based and enzyme-based assays.

Enantiomeric resolution and modeling of DL-alanine-DL-tryptophan dipeptide on amylose stationary phase.

The enantiomeric resolution of DL-alanine-DL-tryptophan dipeptide is described on amylose stationary phase. The eluent used was CH3 OH─CH3 COONH4 (10mM)─CH3 CN (50: 40, 10) at 0.8-mL/min flow, 230-nm detection, 25-minute run time, and 25°C ± 1°C temperature. The chiral phase was amylose [AmyCoat RP (15 cm × 0.46 cm × 5 micron)]. The magnitudes of the retention factors (k) were 2.71, 3.52, 5.11, and 7.75. The magnitudes of separation factor (α) were 1.19, 1.57, and 1.51 while the resolution factors (Rs) were 3.25, 14.84, and 15.76. The limits of detection and quantitation were of 2.5 to 5.4 and 12.8 to 27.5 µg/mL. The enantiomeric resolution is controlled by hydrogen, hydrophobic, π-π, steric, etc interactions. The elution order of the enantiomer was supported by the modeling data. The described method is fast, reproducible, precise, and selective, which can be used successfully for evaluating the enantiomers of the reported dipeptide.

A New Lignanamide from the Root of Lycium yunnanense Kuang and Its Antioxidant Activity.

A new lignanamide (1), lyciumamide K, together with four known analogues (2-5), was isolated from the root of Lycium yunnanense Kuang. Based on HR-ESI-MS, NMR spectral data and quantum chemistry ECD calculations, the structure of this new compound was confirmed, including its absolute configuration. Evaluation of the antioxidant activity of compounds 1-5 in the oxygen radical absorption capacity (ORAC) assay showed that they all exhibited significant antioxidant activities. Particularly, compound 1 showed the best activity with ORAC values (U/mol) of 7.90 ± 0.52. Thus, the new lignanamide may be a good source of bioavtive and protective compounds.

Separation and Characterization of Antioxidative and Angiotensin Converting Enzyme Inhibitory Peptide from Jellyfish Gonad Hydrolysate.

The gonad of jellyfish (RhopilemaesculentumKishinouye), containing high protein content with a rich amino acid composition, is suitable for the preparation of bioactive peptides. Jellyfish gonad was hydrolysed with neutral protease to obtain jellyfish gonad protein hydrolysate (JGPH), which was then purified sequentially by ultrafiltration, gel filtration chromatography, and RP-HPLC. The peptides were characterized with HPLC-MS/MS. One peptide with amino acid sequence Ser-Tyr (SY) was identified and synthesized, which showed good ACE inhibitory and antioxidant activity. The IC50 of this peptide on DPPH, ·OH, super oxygen anion scavenging activities, and ACE inhibitory activity are 84.623 µM, 1177.632 µM, 456.663 µM, and 1164.179 µM, respectively. The anchor in the binding site of SY and ACE C-domain (ACE-C) was obtained by molecular simulations. The results showed that the dipeptide purified from jellyfish gonad protein hydrolysates can be used as functional food material and is helpful in the study of antioxidant and inhibition of ACE.

Identification of Cyclic Dipeptides from Escherichia coli as New Antimicrobial Agents against Ralstonia Solanacearum.

Ralstonia solanacearum is a causative agent of bacterial wilt in many important crops throughout the world. How to control bacterial wilt caused by R. solanacearum is a major problem in agriculture. In this study, we aim to isolate the biocontrol agents that have high efficacy in the control of bacterial wilt. Three new bacterial strains with high antimicrobial activity against R. solanacearum GMI1000 were isolated and identified. Our results demonstrated that these bacteria could remarkably inhibit the disease index of host plant infected by R. solanacearum. It was indicated that strain GZ-34 (CCTCC No. M 2016353) showed an excellent protective effect to tomato under greenhouse conditions. Strain GZ-34 was characterized as Escherichia coli based on morphology, biochemistry, and 16S rRNA analysis. We identified that the main antimicrobial compounds produced by E. coli GZ-34 were cyclo(l-Pro-d-Ile) and cyclo(l-Pro-l-Phe) using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) analysis. The two active compounds also interfered with the expression levels of some pathogenicity-contributors of R. solanacearum. Furthermore, cyclo(l-Pro-l-Phe) effectively inhibited spore formation of Magnaporthe grisea, which is a vital pathogenesis process of the fungal pathogen, suggesting cyclic dipeptides from E. coli are promising potential antimicrobial agents with broad-spectrum activity to kill pathogens or interfere with their pathogenesis.

Affinity-Based Purification of Polyisocyanopeptide Bioconjugates.

Water-soluble polyisocyanopeptides (PICs) are a new class of synthetic polymers that mimic natural protein-based filaments. Their unique semiflexible properties combined with a length of several hundred nanometers have recently enabled a number of biomedical applications ranging from tissue engineering to cancer immunotherapy. One crucial step toward the further development of PICs for these applications is the efficient and controlled synthesis and purification of PIC-biomolecule conjugates. Considering the large size of PICs and the biomolecules to be conjugated, conjugation reactions do usually not proceed to completion due to steric effects. As a consequence, purification of the reaction mixture is necessary to separate the obtained bioconjugates from unreacted biomolecules. As a direct result of the semiflexible nature of PICs, standard polymer and protein purification methods based on molecular weight have not been successful. Here, we introduce a new affinity-based purification method utilizing biotin as an affinity tag. PICs decorated with a controlled and tunable density of biotin molecules (biotinPICs) were efficiently bound to and eluted from a monoavidin resin in buffered aqueous solution. Using these biotinPICs, two different protein conjugates were synthesized, one carrying the enzyme alkaline phosphatase (PhoA) and the other T-cell activating anti-CD3 antibodies. The resulting biotinPIC-protein conjugates were successfully obtained in high purity (>90%) and without any loss of protein activity. The high purity greatly simplifies the analysis of biotinPIC bioconjugates, such as the determination of the average number of biomolecules conjugated per biotinPIC chain. Most importantly, it allows for the direct and straightforward application of the obtained bioconjugates in the desired applications. The new method developed may further be adapted for the purification of other advanced bioconjugates that are difficult to obtain in high purity with the available standard methods.

N-Me-trichodermamide B isolated from Penicillium janthinellum, with antioxidant properties through Nrf2-mediated signaling pathway.

A new trichodermamide-like alkaloid, N-Me-trichodermamide B (compound 1), was isolated from a marine fungus Penicillium janthinellum HDN13-309. The structure and absolute configuration of compound 1 were determined by extensive NMR analysis and the modified Mosher's method. This new alkaloid exhibited cellular protection from the H2O2-induced oxidative damage, and the mechanism study revealed that this antioxidant activity was regulated through Nrf2-mediated signaling pathway in HaCaT human keratinocytes. In addition, the inhibitor of p38 abrogated compound 1-induced phosphorylation of p38, up-expression of HO-1, and the nuclear localization of Nrf2. As a result, it suggested that this new alkaloid-induced antioxidant signaling pathway might be initiated through activation of p38.

Resolution of the Confusion in the Assignments of Configuration for the Ciliatamides, Acylated Dipeptides from Marine Sponges.

Direct comparison of authentic ciliatamide A with four synthetic isomers (1-4) by means of NMR and chiral-phase HPLC revealed that ciliatamide A possesses the 12R (d-N-MePhe residue) and 22S (l-Lys residue) configurations, which were not identical with either our previous assignment or those proposed by others through total synthesis. The absolute configuration of the methionine sulfoxide residue in ciliatamide D was also revised to be d.

Cytotoxicity and Mechanism of Action of the Marine-Derived Fungal Metabolite Trichodermamide B and Synthetic Analogues.

The trichodermamides are modified dipeptides isolated from a wide variety of fungi, including Trichoderma virens. Previous studies reported that trichodermamide B (2) initiated cytotoxicity in HCT-116 colorectal cancer cells, while trichodermamide A (1) was devoid of activity. We recently developed an efficient total synthesis for the trichodermamides A-C (1-3). Multiple intermediates and analogues were produced, and they were evaluated for biological effects to identify additional structure-activity relationships and the possibility that a simplified analogue would retain the biological effects of 2. The antiproliferative effects of 18 compounds were evaluated, and the results show that 2 and four other compounds are active in HeLa cells, with IC50 values in the range of 1.4-21 µM. Mechanism of action studies of 2 and the other active analogues revealed different spectra of activity. At the IC85 concentration, 2 caused S-phase accumulation and cell death in HeLa cells, suggesting response to DNA double-strand breaks. The analogues did not cause S-phase accumulation or induction of DNA damage repair pathways, consistent with an alternate mode of action. The mechanistic differences are hypothesized to be due to the chlorohydrin moiety in 2, which is lacking in the analogues, which could form a DNA-reactive epoxide.

Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

The Antiproliferative Effect of Cyclodipeptides from Pseudomonas aeruginosa PAO1 on HeLa Cells Involves Inhibition of Phosphorylation of Akt and S6k Kinases.

Pseudomonas aeruginosa PAO1, a potential pathogen of plants and animals, produces the cyclodipeptides cyclo(l-Pro-l-Tyr), cyclo(l-Pro-l-Phe), and cyclo(l-Pro-l-Val) (PAO1-CDPs), whose effects have been implicated in inhibition of human tumor cell line proliferation. Our purpose was to investigate in depth in the mechanisms of HeLa cell proliferation inhibition by the PAO1-CDPs. The results indicate that PAO1-CDPs, both purified individually and in mixtures, inhibited HeLa cell proliferation by arresting the cell cycle at the G0-G1 transition. The crude PAO1-CDPs mixture promoted cell death in HeLa cells in a dose-dependent manner, showing efficacy similar to that of isolated PAO1-CDPs (LD50 of 60-250 µM) and inducing apoptosis with EC50 between 0.6 and 3.0 µM. Moreover, PAO1-CDPs showed a higher proapoptotic activity (~10³-105 fold) than their synthetic analogs did. Subsequently, the PAO1-CDPs affected mitochondrial membrane potential and induced apoptosis by caspase-9-dependent pathway. The mechanism of inhibition of cells proliferation in HeLa cells involves inhibition of phosphorylation of both Akt-S473 and S6k-T389 protein kinases, showing a cyclic behavior of their expression and phosphorylation in a time and concentration-dependent fashion. Taken together our findings indicate that PI3K-Akt-mTOR-S6k signaling pathway blockage is involved in the antiproliferative effect of the PAO1-CDPs.

Chiral HPLC Separation and Modeling of Four Stereomers of DL-Leucine-DL-Tryptophan Dipeptide on Amylose Chiral Column.

Chiral high-performance liquid chromatography (HPLC) separation and modeling of four stereomers of DL-leucine-tryptophan DL-dipeptide on AmyCoat-RP column are described. The mobile phase applied was ammonium acetate (10 mM)-methanol-acetonitrile (50:5:45, v/v). The flow rate of the mobile phases was 0.8 mL/min with UV detection at 230 nm. The values of retention factors for LL-, DD-, DL-, and LD- stereomers were 2.25, 3.60, 5.00, and 6.50, respectively. The values of separation and resolution factors were 1.60, 1.39, and 1.30 and 7.76, 8.05, and 7.19. The limits of detection and quantitation were ranging from 1.0-2.3 and 5.6-14.0 µg/mL. The simulation studies established the elution orders and the mechanism of chiral recognition. It was seen that π-π connections and hydrogen bondings were the main forces for enantiomeric resolution. The reported chiral HPLC method may be applied for the enantiomeric separation of DL-leucine-DL-tryptophan in unknown matrices. Chirality 28:642-648, 2016. © 2016 Wiley Periodicals, Inc.

Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract.

2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, have been known to occur in various foods. Recently, DKPs have attracted attentions as bioactive components. There were some reports on analytical methods for DKPs, but the number of analyzed DKPs was only a part of all DKPs and the quantitative performance was not studied in detail. In this study, we selected 31 kinds of DKPs and developed a quantitative and simultaneous analytical method using LC-MS/MS. This method was applied to DKPs determination in Pu-erh tea, post-fermentation tea, and 18 kinds of DKPs were determined at concentration of 0.0017-0.11 ppm. As a result of spiked test, it was concluded that the developed method using LC-MS/MS was useful for estimating DKPs concentration in tea.

Isolation and characterization of anti-inflammatory peptides derived from whey protein.

The present study was conducted to isolate and characterize anti-inflammatory peptides from whey protein hydrolysates using alcalase. Nine subfractions were obtained after sequential purification by ultrafiltration, Sephadex G-25 gel (GE Healthcare, Uppsala, Sweden) filtration chromatography, and preparative HPLC. Among them, subfraction F4e showed the strongest inhibitory activity on interleukin-1ß (IL-1ß), cyclooxygenase-2, and tumor necrosis factor-α (TNF-α) mRNA expression in lipopolysaccharide-induced RAW 264.7 mouse macrophages. Eight peptides, including 2 new peptides-Asp-Tyr-Lys-Lys-Tyr (DYKKY) and Asp-Gln-Trp-Leu (DQWL)-were identified from subfractions F4c and F4e, respectively, using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Peptide DQWL showed the strongest inhibitory ability on IL-1ß, cyclooxygenase-2, and TNF-α mRNA expression and production of IL-1ß and TNF-α proteins at concentrations of 10 and 100µg/mL, respectively. Additionally, DQWL treatment significantly inhibited nuclear factor-κB activation by suppressing nuclear translocation of nuclear factor-κB p65 and blocking inhibitor κB kinase phosphorylation and inhibitor κB degradation together with p38 mitogen-activated protein kinase activation. Our study suggests that peptide DQWL has anti-inflammatory potential; further confirmation using an in vivo model is needed.