Two 2[5H]-furanones as possible signaling molecules in Lactobacillus helveticus

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.     

Two 2[5H]-Furanones as Possible Signaling Molecules in Lactobacillus helveticus

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.     

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone reduces corrosion from Desulfotomaculum orientis

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming and biofilm formation of Gram-positive bacteria (Ren et al., 2002, Lett Appl Microbiol 34: 293-299). In the present study, the Gram-positive sulphate-reducing bacterium (SRB), Desulfotomaculum orientis, was used to study the inhibition of mild steel corrosion due to the addition of furanone. The weight loss from batch coupon experiments incubated with 40 microg x ml(-1) furanone was reduced fivefold compared with samples that lacked furanone. Analysis of the metal surface with environmental scanning electron microscopy further confirmed the protection afforded by the addition of furanone. In agreement with the corrosion inhibition, most probable number (MPN) analysis showed that 20 and 40 microg x ml(-1) furanone inhibited 58% and 96% of the D. orientis growth respectively. Hence, furanone has the potential to inhibit microbial-induced corrosion related to Gram-positive bacteria.     

A facile one-pot synthesis of 4,5-diaryl-2,2-dimethyl-3(2H)-furanones

An efficient and practical one-pot synthesis of 4,5-diaryl-2,2-dimethyl-3(2H)-furanones has been achieved from 1,2-diarylethanones and 2-bromoisobutyryl cyanide in the presence of excess base, by employing the 'hard soft acid base' principle. The reaction scope of 2-bromoisobutyryl cyanide could be expanded to prepare a variety of 2,2-dimethyl-3(2H)-furanone derivatives other than 4,5-diaryl-2,2-dimethyl-3(2H)-furanones.     

New sequential-assignment routes of nucleic acid NMR signals using a [5'-(13)C]-labeled DNA dodecamer

NMR signal assignments for DNA oligomers have been performed by the well-established sequential assignment procedures based on NOESY and COSY. The H4'/H5'/H5' resonance region is congested and difficult to analyze without the use of isotope-labeled DNA oligomers. Here a DNA dodecamer constructed with 2'-deoxy[5'-(13)C]ribonucleotides, 5'-d(*C*G*C*G*A*A*T*T*C*G*CG)-3' (*N = [5'-(13)C]Nucleotide), was prepared in an effort to analyze the H4'/H5'/H5' resonance region by 2D 1H-13C HMQC-NOESY. In the C5' and H1' resonance region, weak and strong cross peaks for C5'(i)-H1'(i) and C5'(i)-H1'(i-1), respectively, were found, thus enabling the sequential assignment within this region. A similar sequential assignment route was found between C5' and H2'. Proton pair distances evaluated from the canonical B-DNA as well as A-DNA indicated that these sequential-assignment routes on a 2D 1H-13C HMQC-NOESY spectrum work for most nucleic acid stem regions.     

Rhodium-catalyzed synthesis of 2(5H)-furanones from terminal alkynes and non-substituted alkynes under water-gas shift reaction conditions

Rhodium-catalyzed synthesis of 2(5H)-furanones from alkynes under water-gas shift reaction conditions was studied. By improving the reaction conditions for internal alkynes reported previously, the reaction could be extended to terminal alkynes. Terminal alkynes are selectively converted into 3- and 4-substituted 2(5H)-furanones (2 and 3). When acetylene itself is used, 2(5H)-furanone (2n) is obtained in a good yield. Examination of reaction solutions by IR spectroscopy and some other experimental findings suggest that the active species would be an alkyne-coordinated monomeric rhodium anion. A new reaction path is proposed.     

Antifungal 3,5-disubstituted furanones: From 5-acyloxymethyl to 5-alkylidene derivatives

5-Acetoxymethyl-3-(4-bromophenyl)-2,5-dihydrofuran-2-one previously described as highly antifungally active was found to provide the corresponding 5-methylene derivative via an unusual DMSO-promoted elimination of the ester group at C5 under antifungal assay conditions. Since the latter possessed nearly the same antifungal effect as that originally reported for the former, the 5-acetoxymethyl furanone just served as a precursor of the actual antifungally active species. A few series of compounds with alkyloxy, aryloxy and alkylidene substituents at C5 of the parent furanone structure were therefore prepared and evaluated. In line with the ease of elimination of the substituent from C5, low activities of the 5-alkoxy compounds were observed. On the other hand, their 5-aryloxymethyl congeners were found to be capable of liberating the antifungally active 5-methylene furanone into the testing medium. The antifungal effect of the 5-alkylidene derivatives was highly sensitive to substitution of the alkylidene moiety; a substituent in the allylic position was necessary for a compound to retain high activity. Parallel evaluation of cytostatic activity showed moderate activities of the antifungally active derivatives against HeLa S3 and CCRF-CEM lines. Cell cycle analysis of CCRF-CEM cells following the treatment with 5-methylene-3-(4-bromophenyl)-2,5-dihydrofuran-2-one revealed that this compound is a necrotic agent.     

Synthesis, structure, molecular docking, and structure-activity relationship analysis of enamines: 3-aryl-4-alkylaminofuran-2(5H)-ones as potential antibacterials

Thirty-one 3-aryl-4-alkylaminofuran-2(5H)-ones were designed, prepared and tested for their antibacterial activity. Some of them showed significant antibacterial activity against Gram-positive organisms, especially against Staphylococcus aureus ATCC 25923, but all were inactive against Gram-negative organisms. Out of these compounds, 3-(4-bromophenyl)-4-(2-(4-nitrophenyl)hydrazinyl)furan-2(5H)-one (4a11) showed the most potent antibacterial activity against S. aureus ATCC 25923 with MIC(50) of 0.42 μg/mL. The enzyme assay revealed that the possible antibacterial mechanism of the synthetic compounds might be due to their inhibitory activity against tyrosyl-tRNA synthetase. Molecular dockings of 4a11 into S. aureus tyrosyl-tRNA synthetase active site were also performed. This inhibitor snugly fitting the active site might well explain its excellent inhibitory activity. Meanwhile, this modeling disclosed that a more suitable optimization strategy might be to modify the benzene ring at 3-position of furanone with hydrophilic groups.     

The naturally occurring furanones: formation and function from pheromone to food

Three closely related 4-hydroxy-3(2H)-furanones have been found in a range of highly cooked foodstuffs where they are important flavour compounds with aroma threshold values as low as 20 micrograms kg-1 water (approximately 0.14 mumol l-1). The compounds are formed mainly as a result of the operation of the Maillard reactions between sugars and amino acids during heating but one compound, 5-(or 2)-ethyl-2-(or 5)-methyl-4-hydroxy-3(2H)-furanone, appears in practice to be produced by yeast, probably from a Maillard intermediate, during the fermentation stages in the production of soy sauce and beer. The compounds are also important in the flavour of strawberry, raspberry, pineapple and tomato but the route of biosynthesis is unknown. Two 3-hydroxy-2(5H)-furanones, emoxyfuranone and sotolon, which are produced spontaneously from amino acids such as threonine and 4-hydroxy-L-leucine are major contributors to meaty and spicy/nutty flavours in foods. The biosynthesis of 5-(1,2-dihydroxyethyl)-3,4-dihydroxy-2(5H)-furanone (ascorbic acid, vitamin C) and 5-hydroxymethyl-3,4-dihydroxy-2(5H)-furanone (erythroascorbic acid) from sugars in plants and yeast, respectively, has been characterized to the enzymic level. After treatment with chlorine, humic waters contain a range of chloro-furanones, some of which, particularly 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), are powerful mutagens. The furanones which occur in foods are also mutagenic to bacteria and cause DNA damage in laboratory tests. However, these compounds are, in practice, very effective anti-carcinogenic agents in the diets of animals which are being treated with known cancer-inducing compounds such as benzo[alpha]pyrene or azoxymethane. Two of the food-derived furanones have antioxidant activity comparable to that of ascorbic acid. A biological function has been discovered for some of the furanones besides vitamin C. 5-Methyl-4-hydroxy-3(2H)-furanone is a male pheromone in the cockroach Eurycolis florionda (Walker) and the 2,5-dimethyl derivative deters fungal growth on strawberries and is an important component of the attractive aroma of the fruit. The red seaweed Delisea pulchra (Greville) Montagne produces a range of brominated furanones which prevent colonisation of the plant by bacteria by interfering with the acylated homoserine lactone (AHL) signalling system used by the bacteria for quorum sensing. In addition, these compounds can deter grazing by marine herbivores. It is proposed here that the evolved biological function of a number of furanones is to act as inter-organism signal molecules in several different systems. This has resulted in two coincidental effects which are important for humans. Firstly, the easily oxidized nature of the furanones in general, which is likely to be an important property in their functioning as signal molecules, results in both mutagenic and anti-carcinogenic activity. The balance of these two effects from compounds in the diet has yet to be fully established. Secondly, and more specifically, the 4-hydroxy-3(2H)-furanones associated with fruit aromas act to attract animals to the fruit, which ensures seed dispersal. In the case of humans, the coincidental synthesis of some of these compounds in foods during preparation results in these foods appearing particularly attractive through the transferred operation of the original signalling mechanisms.     

Synthesis and biological evaluation of methanesulfonamide analogues of rofecoxib: Replacement of methanesulfonyl by methanesulfonamido decreases cyclooxygenase-2 selectivity

A new group of 3-(4-substituted-phenyl)-4-(4-methylsulfonamidophenyl)-2(5H)furanones in which the methylsulfonyl (MeSO(2)) COX-2 pharmacophore present in rofecoxib was replaced by a methanesulfonamido (MeSO(2)NH) moiety, and where the substituent at the para-position of the C-3 phenyl ring was simultaneously varied (H, F, Cl, Br, Me, OMe), were evaluated to determine the combined effects of steric and electronic substituent properties upon COX-1 and COX-2 inhibitory potency and COX isozyme selectivity. Structure-activity relationship (SAR) studies showed that compounds having a neutral (H), or electronegative halogen (F, Cl, Br), substituent at the para-position of the C-3 phenyl ring inhibited both COX-1 and COX-2 with COX-2 selectivity indexes in the 3.1-39.4 range. In contrast, compounds having an electron-donating Me or OMe substituent were selective inhibitors of COX-2 (COX-1 IC(50)>100 microM). These SAR data indicate the 3-aryl-4-(4-methylsulfonamidophenyl)-2(5H)furanone scaffold provides a suitable template to design COX inhibitors with variable COX-2 selectivity indexes.     

Differential gene expression to investigate the effect of (5Z)-4-bromo- 5-(bromomethylene)-3-butyl-2(5H)-furanone on Bacillus subtilis

(5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming, and biofilm formation of gram-positive bacteria. Using the gram-positive bacterium Bacillus subtilis as a test organism, we observed cell killing by 20 microg of furanone per ml, while 5 microg of furanone per ml inhibited growth approximately twofold without killing the cells. To discover the mechanism of this inhibition on a genetic level and to investigate furanone as a novel antibiotic, full-genome DNA microarrays were used to analyze the gene expression profiles of B. subtilis grown with and without 5 microg of furanone per ml. This agent induced 92 genes more than fivefold (P < 0.05) and repressed 15 genes more than fivefold (P < 0.05). The induced genes include genes involved in stress responses (such as the class III heat shock genes clpC, clpE, and ctsR and the class I heat shock genes groES, but no class II or IV heat shock genes), fatty acid biosynthesis, lichenan degradation, transport, and metabolism, as well as 59 genes with unknown functions. The microarray results for four genes were confirmed by RNA dot blotting. Mutation of a stress response gene, clpC, caused B. subtilis to be much more sensitive to 5 microg of furanone per ml (there was no growth in 8 h, while the wild-type strain grew to the stationary phase in 8 h) and confirmed the importance of the induction of this gene as identified by the microarray analysis.     

Synthesis and antibacterial activities of 5-hydroxy-4-amino-2(5H)-furanones

Starting from the mucohalogen acids 1a and b 5-hydroxy-2(5H)-furanones 2a-h have been prepared and tested. These novel 4-amino-5-hydroxy 2(5H)-furananones have shown a broad antibiotic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 in the micromolar range. A one step synthesis from mucohalogen acids towards the antibacterials 2a-h was developed, in which the target was obtained from 1a and b under reflux in toluene in presence of a catalytic amount of sulfuric acid. The derivatives 2b and c displayed a MIC and MBC of 4/8mug/ml, against Staphylococcus aureus with a selectivity towards the resistant strains.     

Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC50 = 1.4 microM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 microM; 15-LOX IC50 = 0.32 microM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC50 = 2.7 microM) and 5-LOX (IC50 = 0.30 microM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.     

3-Phenyl-5-methyl-2H,5H-furan-2-ones: tuning antifungal activity by varying substituents on the phenyl ring

A series of racemic 3-phenyl-5-methyl-2H,5H-furan-2-ones related to a natural product, (-)incrustoporine, was synthesized, and their antifungal activity evaluated. The key structural feature, furanone ring, was closed via H2SO4-mediated cyclization of 2-phenylpent-4-enoic acids. The compounds displayed antifungal activity, especially against filamentous fungi. Expressed as the minimum inhibition concentration (MIC) in micromol/L, the activity of the most promising derivative against Absidia corymbifera matched that of ketoconazole (31.25 micromol/L). In terms of microg/mL, the substance was more active (7.6 microg/mL) than this standard antifungal drug (16.6 microg/mL).     

Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

The quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was found to inhibit the swarming motility of Escherichia coli completely at 13 microg cm-2 (also at 20 microg ml-1) but did not inhibit its growth rate at 13-52 microg cm-2 or from 20 to 100 microg ml-1. Swimming was not inhibited by the furanone at 20-40 microg ml-1. In addition, confocal scanning laser microscopy revealed that this furanone at 60 microg ml-1 inhibited the biofilm formation of E. coli, as it decreased its thickness by 55%, reduced the number of water channels and decreased the percentage of live cells by 87%. This suggests that natural furanone may be used as a new method to control bacterial biofilms that does not involve toxicity. Furanone at 10 microg ml-1 also inhibited by 3300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1) and inhibited by 5500-fold that of V. harveyi via of autoinducer 2 (AI-2) as well as inhibited by 26-600-fold the quorum sensing of E. coli via AI-2; hence, this furanone is a non-specific intercellular signal antagonist.     

Novel 4,5-diaryl-3-hydroxy-2(5H)-furanones as anti-oxidants and anti-inflammatory agents

In order to study the effect of phenol moieties on biological activities of ascorbic acid derivatives, we synthesized 13 novel 4,5-diaryl-3-hydroxy-2(5H)-furanones 5a-m with various substitution patterns. Compound 5 g bearing a 2,3-dihydroxy phenyl ring on the 5-position of the heterocycle appeared to be the most powerful anti-oxidant furanone with reducing activity against DPPH (IC(50)=10.3 microM), superoxide anion quenching capacity (IC(50)=0.187 mM) and lipid peroxidation inhibitory effect (IC(50)=0.129 mM). To ascertain determinant molecular features for anti-oxidant activities, structure-activity relationships were studied. Lipophilicity and molecular parameters related to electron distribution and structure (difference in heats of formation between the compound and its radical or its cation radical, energy of the highest occupied molecular orbital, HOMO) were found to correlate with the anti-oxidant action of compounds 5 in the different tests used. Oxygen-derived free radicals are known to contribute to inflammatory disorders; therefore we have investigated effects of compounds 5 in two models of inflammation: phorbol ester-induced ear edema in mice (TPA-test) and carrageenan-induced paw edema in rat. At 100 mg/kg ip in the TPA-test, the anti-inflammatory activity of compounds 5 was potent compared with that of indomethacin and ketorolac and all the results suggested a cyclooxygenase inhibition in the emergence of such properties. The combined pharmacological actions of compounds 5 associated with a favorable therapeutic index prompt with interesting perspectives for their use in heart and brain disorders as well as in inflammatory diseases.     

Horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of VP16-213. Identification of a new metabolite

VP16 was submitted to oxidation catalyzed by horseradish peroxidase (HRP) and H2O2 in phosphate buffer (pH 7.0). The product of the reaction, which has a high performance liquid chromatographic (HPLC) retention time different from the previously known metabolites of VP16, was identified as 1,2,3,4-tetradehydro-VP16 by 1H-NMR and mass spectrometry (MS) analysis. It was found to result from the loss of four hydrogens and the formation of an aromatic ring (ring C of VP16). This new product retains, in the 4' position of the E ring of VP16, the hydroxy group which is crucial for the antitumoral activity of podophyllotoxin derivatives. The reaction was linear in a wide range of VP16 concentrations and was dependent on the concomitant presence of peroxidase and H2O2.     

Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

AIMS: (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(furanone) of the marine alga Delisea pulchra was synthesized, and its inhibition of swarming motility and biofilm formation of Bacillus subtilis was investigated. METHODS AND RESULTS: Furanone was found to inhibit both the growth of B. subtilis and its swarming motility in a concentration-dependent way. In addition, as shown by confocal scanning laser microscopy, furanone inhibited the biofilm formation of B. subtilis. At 40 microg ml(-1), furanone decreased the biofilm thickness by 25%, decreased the number of water channels, and reduced the percentage of live cells by 63%. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Natural furanone has potential for controlling the multicellular behaviour of Gram-positive bacteria.