Diketopiperazine Research Articles

Non-isothermal dehydration kinetic study of aspartame hemihydrate using DSC, TGA and DSC-FTIR microspectroscopy

Three thermal analytical techniques such as differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) using five heating rates, and DSC-Fourier Transform Infrared (DSC-FTIR) microspectroscopy using one heating rate, were used to determine the thermal characteristics and the dehydration process of aspartame (APM) hemihydrate in the solid state. The intramolecular cyclization process of APM anhydrate was also examined. One exothermic and four endothermic peaks were observed in the DSC thermogram of APM hemihydrate, in which the exothermic peak was due to the crystallization of some amorphous APM caused by dehydration process from hemihydrate to anhydride. While four endothermic peaks were corresponded to the evaporation of absorbed water, the dehydration of hemihydrate, the diketopiperazines (DKP) formation via intramolecular cyclization, and the melting of DKP, respectively. The weight loss measured in TGA curve of APM hemihydrate was associated with these endothermic peaks in the DSC thermogram. According to the Flynn–Wall–Ozawa (FWO) model, the activation energy of dehydration process within 100–150 °C was about 218 ± 11 kJ/mol determined by TGA technique. Both the dehydration and DKP formation processes for solid-state APM hemihydrate were markedly evidenced from the thermal-responsive changes in several specific FTIR bands by a single-step DSC-FTIR microspectroscopy.

Development of Novel Functional Molecules Based on the Molecular Structure Characteristics of Diketopiperazines

This article focuses on our investigation of the molecular structure characteristics of diketopiperazines (DKPs), and application of these findings to the development of novel functional molecules. DKPs bearing a benzyl moiety are known to adopt a folded conformation, in which the benzyl moiety is folded over the DKP ring. In order to investigate the driving force behind the folded conformation, we synthesized DKPs bearing a benzyl moiety with different para-substituents, and demonstrated that the folded conformation likely arose from intramolecular CH/π interactions, based on the electronic effects of para-substituents on the benzyl group in 1H NMR spectroscopy. On the other hand, N4-methylation of DKPs bearing a benzyl moiety was found to change their folded conformation to an extended conformation, based on single crystal X-ray crystallography and 1H NMR spectroscopy analysis. Next, we attempted to synthesize both hydroxamate-type siderophores containing the DKP ring: rhodotorulic acid and erythrochelin. Facile synthesis of rhodotorulic acid and its N,N'-dimethylated derivative was achieved by microwave-assisted cyclization of the corresponding dipeptide precursors. Interestingly, N,N'-dimethylated rhodotorulic acid was found to be more soluble in various organic solvents than rhodotorulic acid. Moreover, erythrochelin was synthesized for the first time, and its metal-chelating ability with not only Fe(III) but also Mg(II) was confirmed based on electrospray ionization mass spectrometry (ESI-MS) analysis. Finally, we synthesized DKPs bearing a primary amino group, and found that they could catalyze the asymmetric aldol reaction between hydroxyacetone and p-nitrobenzaldehyde.

Solid-state structure of cyclic dipeptides: an X-ray and computational study of cis- and trans-diketopiperazines of N-methyl-phenylalanine with the thia-pipecolic acids and thia-prolines

Ten new crystal structures of cis and trans bicyclic diketopiperazines (DKPs) of thia-pipecolic acid (with sulfur in the β, γ or δ position) or thia-proline (with sulfur in the β or γ position) and N-methyl phenylalanine [(NMe)Phe]: cyclo[(β-S)Pip-(NMe)Phe], cyclo[(γ-S)Pip-(NMe)Phe], cyclo[(δ-S)Pip-(NMe)Phe], cyclo[(β-S)Pro-(NMe)Phe] and cyclo[(γ-S)Pro-(NMe)Phe] were determined with X-ray crystallography. Density functional theory calculations of these molecules in the gas phase succeed in reproducing the observed molecular conformations in the crystal remarkably well. This illustrates the weak to moderate impact of intermolecular packing forces in the absence of classical N—H...O hydrogen bonds. The effect of sulfur on the geometry of the DKP ring and details of amide bond non-planarity are discussed. Molecular flexibility of the DKP ring, as estimated from the calculated deformation energies of its endocyclic ring torsion angles, is not in general the decisive factor for the occurrence of multiple symmetry independent molecules in the unit cell (Z′ > 1), though in some cases a correlation is observed.

Identification of the Biosynthetic Pathway for the Antibiotic Bicyclomycin.

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene clusterand establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

Isolation, Purification, and Characterization of Five Active Diketopiperazine Derivatives from Endophytic Streptomyces SUK 25 with Antimicrobial and Cytotoxic Activities.

In our search for new sources of bioactive secondary metabolites from Streptomyces sp., the ethyl acetate extracts from endophytic Streptomyces SUK 25 afforded five active diketopiperazine (DKP) compounds. The aim of this study was to characterize the bioactive compounds isolated from endophytic Streptomyces SUK 25 and evaluate their bioactivity against multiple drug resistance (MDR) bacteria such as Enterococcus raffinosus, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp., and their cytotoxic activities against the human hepatoma (HepaRG) cell line. The production of secondary metabolites by this strain was optimized through Thornton's medium. Isolation, purification, and identification of the bioactive compounds were carried out using high-performance liquid chromatography, high-resolution mass liquid chromatography-mass spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance, and cryopreserved HepaRG cells were selected to test the cytotoxicity. The results showed that endophytic Streptomyces SUK 25 produces four active DKP compounds and an acetamide derivative, which were elucidated as cyclo-(L-Val-L-Pro), cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Phe), and N-(7-hydroxy-6-methyl-octyl)-acetamide. These active compounds exhibited activity against methicillin-resistant S. aureus ATCC 43300 and Enterococcus raffinosus, with low toxicity against human hepatoma HepaRG cells. Endophytic Streptomyces SUK 25 has the ability to produce DKP derivatives biologically active against some MDR bacteria with relatively low toxicity against HepaRG cells line.

Click strategy using disodium salts of amino acids improves the water solubility of plinabulin and KPU-300

Plinabulin and KPU-300 are promising anti-microtubule agents; however, the low water solubility of these compounds (<0.1µg/mL) has limited their pharmaceutical advantages. Here, we developed five water-soluble derivatives of plinabulin and KPU-300 with a click strategy using disodium salts of amino acids. The mother skeleton, diketopiperazine (DKP), was transformed into a monolactim-type alkyne and a copper-catalyzed alkyne azide cycloaddition (CuAAC) combined azides that was derived from amino acids as a water-solubilizing moiety. The conversion of carboxyl groups into disodium salts greatly improved the water solubility by 0.8 million times compared to the solubility of the parent molecules. In addition, the α-amino acid side chains of the water-solubilizing moieties affected both the water solubility and the half-lives of the compounds during enzymatic hydrolysis. Our effort to develop a variety of water-soluble derivatives using the click strategy has revealed that the replaceable water-solubilizing moieties can alter molecular solubility and stability under enzymatic hydrolysis. With this flexibility, we are approaching to the in vivo study using water-soluble derivative.

Biomimetic Synthesis of Insulin Enabled by Oxime Ligation and Traceless "C-Peptide" Chemical Excision.

For decades, insulin has represented a preeminent synthetic target. Recently introduced "biomimetic" strategies based on convertible single-chain precursors require incorporation of a chemical linker or a unique proteolytic site, which limits their practicality. In this approach the A- and B-chains are linked by two sequential oxime ligations followed by disulfide bond formation under redox conditions and linker excision by diketopiperazine (DKP) formation and ester hydrolysis, yielding native two-chain insulin. The method is expected to be applicable to any member of the insulin superfamily.

Pyrolysis characteristics and pathways of protein, lipid and carbohydrate isolated from microalgae Nannochloropsis sp.

Microalgal components were isolated gradually to get lipid-rich, protein-rich and carbohydrate-rich components. The aim of this work was to study pyrolysis mechanism of microalgae by real isolated real algae components. Thermogrametric analysis (DTG) curve of microalgae was fitted by single pyrolysis curves of protein, lipid and carbohydrate except special zones, which likely affected by cell disruption and hydrolysis mass loss. Experimental microalgae liquefaction without water index N was 0.6776, 0.3861 and 0.2856 for isolated lipid, protein and carbohydrate. Pyrolysis pathways of lipid are decarboxylation, decarbonylation, fragmentation of glycerin moieties and steroid to form hydrocarbons, carboxylic acids and esters. Pyrolysis pathways of protein are decarboxylation, deamination, hydrocarbon residue fragmentation, dimerization and fragmentation of peptide bonds to form amide/amines/nitriles, esters, hydrocarbons and N-heterocyclic compounds, especially diketopiperazines (DKPs). Pyrolysis pathways of carbohydrate are dehydrated reactions and further fragmentation to form ketones and aldehyde, decomposition of lignin to form phenols, and fragmentation of lipopolysaccharides.

Anti-HCV protease of diketopiperazines produced by the Red Sea sponge-associated fungus Aspergillus versicolor

Hepatitis C virus (HCV) infection is a global problem due to the difficulties in developing a protective vaccine. In this work, we demonstrated that the ethyl acetate extract of the endophytic fungus Aspergillus versicolor exhibited significant activity against HCV NS3/4A protease with IC50 value of 30 μg/mL. The fungus was isolated from the Red Sea black sponge Spongia officinalis and identified by its morphology and 18S rDNA. Large-scale fermentation of the fungus followed by chromatographic purification with silica gel, Sephadex LH-20 and semipreparative HPLC of the active extract led to isolation of some known metabolites related to cyclodipeptides and the so-called diketopiperazines (DKPs). The DKP, cyclo(L-Tyr-L-Pro), displayed strong effect as HCV protease inhibitor with IC50 value of 8.2 μg/mL. A computational docking study of cyclo(L-Tyr-L-Pro) against HCV protease was used to formulate a hypothetical mechanism for the inhibitory activity of the active compound on the tested enzyme.

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.

Determination of 2,5-Diketopiperazines Iin Greek Processed Olives by Liquid Chromatography/Mass Spectrometry Analysis

Diketopiperazines (DKPs) are cyclic dipeptides which have been detected in a variety of natural products, especially in thermally treated or fermented foods and beverages, providing a metallic bitter taste. DKPs, mainly due to their characteristic heterocyclic system, have been reported to exhibit a broad spectrum of biological activities including antimicrobial, antiviral, antitumor, antihyperglycaemic and antimutagenic. In the present study, several DKPs were identified in seven different Greek varieties of processed olives using HR-LC-MSn. The identification of DKPs in olive samples was achieved by comparison of their retention time and fragmentation pattern with reference DKP standards. The MSn spectra were identical to confirm the presence of specific compounds because their results associate both fragmentation pattern and fragments’ intensity. Nine compounds were found out of a total of 19 standard DKPs. The most prominent diketopiperazine was the cyclo(Phe-Phe) followed by and cyclo(Phe-Pro). Varieties where most DKPs were identified were Kothreiki, Kalamon, Throumpoelies and Helidoni.

Cyclic Dipeptide Shuttles as a Novel Skin Penetration Enhancement Approach: Preliminary Evaluation with Diclofenac.

This study demonstrates the effectiveness of a peptide shuttle in delivering diclofenac into and through human epidermis. Diclofenac was conjugated to a novel phenylalanyl-N-methyl-naphthalenylalanine-derived diketopiperazine (DKP) shuttle and to TAT (a classical cell penetrating peptide), and topically applied to human epidermis in vitro. DKP and TAT effectively permeated into and through human epidermis. When conjugated to diclofenac, both DKP and TAT enhanced delivery into and through human epidermis, though DKP was significantly more effective. Penetration of diclofenac through human epidermis (to receptor) was increased by conjugation to the peptide shuttle and cell penetrating peptide with enhancement of 6x by DKP-diclofenac and 3x by TAT-diclofenac. In addition, the amount of diclofenac retained within the epidermis was significantly increased by peptide conjugation. COX-2 inhibition activity of diclofenac was retained when conjugated to DKP. Our study suggests that the peptide shuttle approach may offer a new strategy for targeted delivery of small therapeutic and diagnostic molecules to the skin.

Fragmentation Patterns of Aromatic 2,5-diketopiperazines Using Liquid chromatography/Mass Spectrometry

Diketopiperazines (DKPs) are cyclic dipeptides which have been detected in a variety of natural products, especially in thermally treated or fermented foods and beverages, providing a metallic bitter taste. DKPs, mainly due to their characteristic heterocyclic system, have been reported to exhibit a broad spectrum of biological activities including antimicrobial, antiviral, antitumor, antihyperglycaemic and antimutagenic. DKPs are formed from unprotected linear dipeptides under basic conditions especially if the necessary head-to-tail folding is not prevented by steric constraints. A methodology to identify DKPs in complex matrices is of high importance. Up to now, only a few studies have reported the MS fragmentation patterns on a certain number of DKPs. The purpose of this study was to develop a liquid chromatography method for separation and identification of DKPs in complex matrices (i.e. food and beverages) as well as a high resolution mass spectrometry method providing accurate full scan MS and MSn data in order to investigate the fragmentation pattern and confirm the chemical formula and structure of DKPs containing aromatic amino acids. Our results which were supported by in silico DFT energy calculations, revealed different and common fragmentation pathways as well as a series of characteristic fragment ions which are representative of the amino acid residues. The role of MSn was signified by the results since it provided a clear picture of the fragmentation cascades. The obtained fragments are diagnostic and could be used to distinguish cyclic dipeptides in different matrices. Keywords: 2, 5-diketopiperazines, cyclic dipeptides, LC-MSn, fragmentation pattern, bioactive compounds.

Inhibition of biofilm in Bacillus amyloliquefaciens Q-426 by diketopiperazines.

Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas-liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04mgml(-1) DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04mgml(-1) DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.

Nematode-trapping fungi and fungus-associated bacteria interactions: the role of bacterial diketopiperazines and biofilms on Arthrobotrys oligospora surface in hyphal morphogenesis.

In soil, nematode-trapping fungi and bacteria often share microhabitats and interact with each other, but effects of fungus-associated bacteria on its trap formation are underestimated. We have ascertained the presence of Stenotrophomonas and Rhizobium genera associated with A. oligospora GJ-1. After A. oligospora GJ-1 without associated bacteria (cured Arthrobotrys) was co-cultivated with Stenotrophomonas and its supernatant extract, microscopic study of hyphae from co-cultivation indicated that bacterial biofilm formation on hyphae was related to trap formation in fungi and Stenotrophomonas supernatant extract. Four diketopiperazines (DKPs) were purified from Stenotrophomonas supernatant extract that could not induce traps in the cured Arthrobotrys. When cured Arthrobotrys was cultured with Stenotrophomonas and one of DKPs, polar attachment, bacterial biofilms on hyphae and trap formation in fungi were observed. After cured Arthrobotrys with bacterial biofilms was consecutively transferred several times on nutrient poor medium, trap formation disappeared with the disappearance of bacterial biofilms on hyphae. DKPs could facilitate chemotaxis of Stenotrophomonas towards fungal extract which was suggested to contribute to bacterial biofilms on hyphae. Furthermore, when cured Arthrobotrys was cultured with Stenotrophomonas and DKPs in soil, trap formation in fungi and bacterial biofilms on hyphae were also observed, and the fungal activity against nematode was enhanced.

Inhibition of biofilm development and spoilage potential of Shewanella baltica by quorum sensing signal in cell-free supernatant from Pseudomonas fluorescens

The objective of this study was to in vitro evaluate the effect of a cell-free supernatant (CFS) containing quorum sensing (QS) signal of Pseudomonas fluorescens on the growth, biofilm development and spoilage potential of Shewanella baltica, and preliminarily assess the interactive influences of various chemically synthesized autoinducers on spoilage phenotypes of S. baltica. PF01 strain isolated from spoiled Pseudosciaen crocea was identified P. fluorescens. The addition of 25% and 50% CFS to S. baltica culture had no effect on the growth rate during the lag and exponential phase, however, caused cell decline during the stationary phase. The presence of CFS from P. fluorescens significantly inhibited biofilm development, and greatly decreased the production of trimethylamine (TMA) and biogenic amino in S. baltica. Various signal molecules of QS in the CFS of P. fluorescens culture were detected, including seven N-acyl-l-homoserine lactones (AHLs), autoinducer-2 (AI-2) and two diketopiperazines (DKPs). Exogenous supplement of synthesized seven AHLs containing in the CFS decreased biofilm formation and TMA production in S. baltica, while exposure to exogenous cyclo-(l-Pro-l-Leu) was showed to promote spoilage potential, which revealed that S. baltica also sense the two QS molecules. Furthermore, the stimulating effect of cyclo-(l-Pro-l-Leu) was affected when AHL was simultaneously added, suggesting that the inhibitory activity of spoilage phenotypes in S. baltica might be attributed to a competitive effect of these QS compounds in the CFS of P. fluorescens. The present studies provide a good basis for future research on the role of QS in the regulation of spoilage microbial flora.

ChemInform Abstract: Synthesis of Bridged Diketopiperazines by Using the Persistent Radical Effect and a Formal Synthesis of Bicyclomycin.

AbstractBy taking advantage of the persistent radical effect, a new approach to bridged diketopiperazines (DKP) with variable ring sizes is developed.

Antifungal Activity of Pseudomonas frederiksbergensis CMAA 1323 Isolated from the Antarctic Hair Grass Deschampsia antarctica

Aims: Epiphytic bacteria, isolated from Deschampsia antarctica, were screened for their potential to inhibit the plant pathogen Botrytis cinerea, the causal agent of gray mold disease of strawberry pseudofruits. This phytopathogenic fungus is more active and the disease is more serious in temperate climate where the temperatures are lower. Methodology: The approach involved the isolation and characterization of bacteria from the phyllosphere of D. antarctica and the evaluation of their antifungal activity. Results: A total of 56 bacterial strains were isolated and only one of them, identified by the sequencing of 16S rDNA gene, as Pseudomonas frederiksbergensis CMAA 1323, inhibited the mycelial growth and the conidial germination of the fungus. Its ethyl acetate extract also inhibited Original Research Article Melo et al.; BMRJ, 14(3): 1-11, 2016; Article no.BMRJ.25314 2 the mycelial growth. Analysis of the bioactive fraction by using liquid chromatography coupled to a mass spectrometry (LC-MS) revealed the presence of cyclo(Pro-Val), a compound belonging to the class of diketopiperazines (DKPs). Conclusion: These findings support the potential use of the psychrophilic bacterium P. frederiksbergensis as promising antifungal agent, and highlight the need for more studies with this bacterium in the biological control of plant pathogenic fungi.

Inhibition of quorum sensing, biofilm, and spoilage potential in Shewanella baltica by green tea polyphenols.

We investigated the quorum sensing (QS) system of Shewanella baltica and the anti-QS related activities of green tea polyphenols (TP) against spoilage bacteria in refrigerated large yellow croaker. Autoinducer-2 (AI-2) and the diketopiperazines (DKPs) cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe) were detected in the culture extract of S. baltica XH2, however, no N-acylhomoserine lactones (AHLs) activity was observed. Green TP at sub-inhibitory concentrations interfered with AI-2 and DKPs activities of S. baltica without inhibiting cell growth and promoted degradation of AI-2. The green TP treatment inhibited biofilm development, exopolysaccharide production and swimming motility of S. baltica in a concentration- dependent manner. In addition, green TP decreased extracellular protease activities and trimethylamine production in S. baltica. A transcriptional analysis showed that green TP repressed the luxS and torA genes in S. baltica, which agreed with the observed reductions in QS activity and the spoilage phenotype. Epigallocatechin gallate (EGCG)-enriched in green TP significantly inhibited AI-2 activity of S. baltica. These findings strongly suggest that green TP could be developed as a new QS inhibitor for seafood preservation to enhance shelf life.

SigMol: repertoire of quorum sensing signaling molecules in prokaryotes.

Quorum sensing is a widespread phenomenon in prokaryotes that helps them to communicate among themselves and with eukaryotes. It is driven through quorum sensing signaling molecules (QSSMs) in a density dependent manner that assists in numerous biological functions like biofilm formation, virulence factors secretion, swarming motility, bioluminescence, etc. Despite immense implications, dedicated resources of QSSMs are lacking. Therefore, we have developed SigMol (http://bioinfo.imtech.res.in/manojk/sigmol), a specialized repository of these molecules in prokaryotes. SigMol harbors information on QSSMs pertaining to different quorum sensing signaling systems namely acylated homoserine lactones (AHLs), diketopiperazines (DKPs), 4-hydroxy-2-alkylquinolines (HAQs), diffusible signal factors (DSFs), autoinducer-2 (AI-2) and others. Database contains 1382 entries of 182 unique signaling molecules from 215 organisms. It encompasses biological as well as chemical aspects of signaling molecules. Biological information includes genes, preliminary bioassays, identification assays and applications, while chemical detail comprises of IUPAC name, SMILES and structure. We have provided user-friendly browsing and searching facilities for easy data retrieval and comparison. We have gleaned information of diverse QSSMs reported in literature at a single platform ‘SigMol’. This comprehensive resource will assist the scientific community in understanding intraspecies, interspecies or interkingdom networking and further help to unfold different facets of quorum sensing and related therapeutics.