Analysis of the isomeric composition of the proline peptide bond in an angiotensin-converting enzyme inhibitor using capillary electrophoresis

The cis and trans isomeric composition of a proline peptide bond can be determined by routine free-solution capillary electrophoresis measurements provided that one isomeric form is preferentially stabilized by a dissociable ionic group. This capability is illustrated using the angiotensin converting enzyme (ACE) inhibitor (S)-1-N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-ala-L-pro, which has the trade name enalapril. Electropherograms indicate that the two isomeric forms of enalapril can be separated with baseline resolution at 15 degrees C using capillary buffers having pH values in the dissociation ranges of the enalapril carboxyl group, pK(cis) and pK(trans) of 2.6 and 3.1, and of the enalapril amine group, pK(cis) and pK(trans) of 5.9 and 5.6. Such baseline resolution indicates that the isomeric composition does not change during analysis, facilitating measurement of the isomer composition of a sample prior to its injection into the capillary. Thus the effect of pH, ionic strength, or an aprotic solvent on the isomeric composition of enalapril can be measured under uniform analytical conditions. The trans isomer composition changes from 68% in the cationic form, pH <2, to 50% in the isoelectric form, pH approximately 4.5, to 60% in the anionic form, pH >7. Addition of salt to the isoelectric form or addition of an aprotic solvent to any form prior to analysis increases the trans isomer composition. Similar analyses can be made using the alternative ACE inhibitors captopril and enalaprilat.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Cyclohexanoid protoflavanones from the stem-bark and roots of Ongokea gore

Phytochemical investigation of root and stem-bark of the West African medicinal plant Ongokea gore resulted in the isolation of four novel flavonoids with an unusual cyclohexyl substituent instead of the common aromatic ring B. The structures of the isolated compounds were elucidated by spectroscopic methods, mainly 1D and 2D NMR, and subsequently, the structures were corroborated by chemical conversion to (-)-(S)-sakuranetin. The absolute configurations, and preferred conformations were determined by NOE experiments and CD measurements.     

Synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone from D-glucose

We have described the synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone by the reduction of a keto-conduritol derivative, the latter being prepared in five steps from (-)-(2S,3R,4S,5S)-2,3,4-tribenzyloxy-5-hydroxycyclohexanone, which is in turn readily synthesized from D-glucose.     

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.     

Evidence that the tertiary structure of 20(S)-ginsenoside Rg(3) with tight hydrophobic packing near the chiral center is important for Na(+) channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg(3) exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(S)-Rg(3)) and 20-R-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(R)-Rg(3)). Recently, we reported that 20(S)-Rg(3) regulates voltage-dependent Ca(2+) channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg(3) does not have this activity. In this study, we investigated the structure-activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na(+) channel (Na(v)1.5). We found that 20(S)-Rg(3) but not 20(R)-Rg(3) inhibited Na(+) channel current in a dose- and voltage-dependent manner. The difference between Rg(3) epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg(3) may be geometrically better aligned than that of 20(R)-Rg(3) for interaction with receptor regions in Na(+) channels. The (1)H and (13)C NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg(3) and 20(R)-Rg(3), were completely assigned, and their tertiary structures were determined. 20(S)-Rg(3) has more tight hydrophobic packing near the chiral center than 20(R)-Rg(3). Tertiary structures and activities of 20(S)-Rg(3) and 20(R)-Rg(3) indicate that 20(S)-Rg(3) may have stronger interactions with the receptor region in ion channels than 20(R)-Rg(3). This may result in different stereoselectivity of Rg(3) stereoisomers in the regulation of voltage-dependent Na(+) channel activity. This is the first structural approach to ginsenoside action on ion channel.     

Intramolecular hydrogen bonding in articaine can be related to superior bone tissue penetration: a molecular dynamics study

Local anesthetics (LAs) are drugs that cause reversible loss of nociception during surgical procedures. Articaine is a commonly used LA in dentistry that has proven to be exceptionally effective in penetrating bone tissue and induce anesthesia on posterior teeth in maxilla and mandibula. In the present study, our aim was to gain a deeper understanding of the penetration of articaine through biological membranes by studying the interactions of articaine with a phospholipid membrane. Our approach involves Langmuir monolayer experiments combined with molecular dynamics simulations. Membrane permeability of LAs can be modulated by pH due to a titratable amine group with a pKa value close to physiological pH. A change in protonation state is thus known to act as a lipophilicity switch in LAs. Our study shows that articaine has an additional unique lipophilicity switch in its ability to form an intramolecular hydrogen bond. We suggest this intramolecular hydrogen bond as a novel and additional solvent-dependent mechanism for modulation of lipophilicity of articaine which may enhance its diffusion through membranes and connective tissue.     

Kinetic mechanism and catalysis of a native-state prolyl isomerization reaction

Folding of tendamistat is a rapid two-state process for the majority of the unfolded molecules. In fluorescence-monitored refolding kinetics about 8% of the unfolded molecules fold slowly (lambda=0.083s(-1)), limited by peptidyl-prolyl cis-trans isomerization. This is significantly less than expected from the presence of three trans prolyl-peptide bonds in the native state. In interrupted refolding experiments we detected an additional very slow folding reaction (lambda=0.008s(-1) at pH 2) with an amplitude of about 12%. This reaction is caused by the interconversion of a highly structured intermediate to native tendamistat. The intermediate has essentially native spectroscopic properties and about 2% of it remain populated in equilibrium after folding is complete. Catalysis by human cyclophilin 18 identifies this very slow reaction as a prolyl isomerization reaction. This shows that prolyl-isomerases are able to efficiently catalyze native state isomerization reactions, which allows them to influence biologically important regulatory conformational transitions. Folding kinetics of the proline variants P7A, P9A, P50A and P7A/P9A show that the very slow reaction is due to isomerization of the Glu6-Pro7 and Ala8-Pro9 peptide bonds, which are located in a region that makes strong backbone and side-chain interactions to both beta-sheets. In the P50A variant the very slow isomerization reaction is still present but native state heterogeneity is not observed any more, indicating a long-range destabilizing effect on the alternative native state relative to N. These results enable us to include all prolyl and non-prolyl peptide bond isomerization reactions in the folding mechanism of tendamistat and to characterize the kinetic mechanism and the energetics of a native-state prolyl isomerization reaction.     

Aphid sex pheromones: from discovery to commercial production

This review charts the progress made with aphid sex pheromone chemistry, from initial identification of cyclopentanoid nepetalactones, for example (4aS,7S,7aR)-nepetalactone (1). and (1R,4aS,7S,7aR)-nepetalactol (2). to commercial production from a renewable non-food crop, the catmint, Nepeta cataria (Lamiaceae). The availability of aphid sex pheromone components is now facilitating the development of new aphid pest control strategies, incorporating the use of other semiochemicals, particularly in the manipulation of populations of aphid parasitoids and aphid predators such as lacewings, which can utilise the nepetalactones and closely related molecules to locate their hosts and prey. This is the first example of a plant resource being developed as a feedstock for the production of a commercially valuable insect pheromone. The development of a plant-based production route highlights the tremendous potential that higher plants offer as cheap and renewable resources for the production of insect semiochemicals, through the wide array of secondary metabolites that they can generate.     

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.     

Nuclear magnetic resonance characterization of the refolding intermediate of beta2-microglobulin trapped by non-native prolyl peptide bond

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex type I, is also found as a major component of amyloid fibrils formed in dialysis-related amyloidosis. Denaturation of beta2-m is considered to initiate the formation of fibrils. To clarify the mechanism of fibril formation, it is important to characterize the intermediate conformational states at the atomic level. Here, we investigated the refolding of beta2-m from the acid-unfolded state by heteronuclear magnetic resonance and circular dichroism spectroscopies. At low temperature, beta2-m refolded slowly, accumulating a rate-limiting intermediate with non-native chemical shift dispersions for several residues, but with compactness and secondary structures similar to those of the native protein. beta2-m has a cis proline residue at Pro32, located on the turn connecting the betaB and betaC strands. The slow refolding phase disappeared upon mutation of Pro32 to Val, indicating that Pro32 is responsible for the accumulation of the intermediate. The distribution of the perturbed residues in the intermediate suggests that the non-native prolyl peptide bond of Pro32 affects large areas of the molecule. A cis proline residue is common to various immunoglobulin domains involved in amyloidosis, implying that a non-native prolyl peptide bond that might occur under physiological conditions is related to the amyloidogenicity of these immunoglobulin domains.     

Activity-guided isolation of the chemical constituents of Muntingia calabura using a quinone reductase induction assay

Activity-guided fractionation of an EtOAc-soluble extract of the leaves of Muntingia calabura collected in Peru, using an in vitro quinone reductase induction assay with cultured Hepa 1c1c7 (mouse hepatoma) cells, resulted in the isolation of a flavanone with an unsubstituted B-ring, (2R,3R)-7-methoxy-3,5,8-trihydroxyflavanone (5), as well as 24 known compounds, which were mainly flavanones and flavones. The structure including absolute stereochemistry of compound 5 was determined by spectroscopic (HRMS, 1D and 2D NMR, and CD spectra) methods. Of the isolates obtained, in addition to 5, (2S)-5-hydroxy-7-methoxyflavanone, 2',4'-dihydroxychalcone, 4,2',4'-trihydroxychalcone, 7-hydroxyisoflavone and 7,3',4'-trimethoxyisoflavone were found to induce quinone reductase activity.     

Effect of the Phosphate Group with Different Negative Charges on the Conformation of Phosphorylated Ser/Thr-Pro Motif

Summary Ser/Thr-Pro motif is a widespread phosphorylated site in proteins, and its reversible phosphorylation is an important regulatory progress in many cell cycles and signal transduction. Recent research reveals that phosphorylation affects the local conformation of the peptide and its binding with the substrate through peptidyl--prolyl cis/trans isomerization. In order to further explore the effect of the phosphate group with different charges, four model peptides containing non- and phosphorylated Ser/Thr-Pro motif were synthesized using the classical solid-phase method. 1H-NMR, TOCSY, and ROESY were employed to characterize the conformation of the model peptides in solution with different pH value and analyze the peptidyl--prolyl isomerization at a molecular level. The results demonstrate that phosphorylation increases the cis conformation in the peptide and the maximum cis/trans ratio is obtained when the phosphate group has two negative charges. Furthermore, the experiments prove that the phosphorylation introduces a hydrogen bond between the phosphate and the NH of Ser/Thr residue, and the charges of the phosphate affect certain conformations of the phosphorylated Ser/Thr-Pro motif.     

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.     

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A

Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 A resolution and refined to an R-factor of 19.5% and an R(free) of 21.9%. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117-a 9.3 A shift of the Calpha atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

NMR solution structure and dynamics of the peptidyl-prolyl cis-trans isomerase domain of the trigger factor from Mycoplasma genitalium compared to FK506-binding protein

We have solved the solution structure of the peptidyl-prolyl cis-trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the beta sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central beta sheet. Our data indicate, however, that this strand may be partially structured as "nascent" beta strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.     

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO₂)LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.     

Constituents of the stems of Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2

As part of our program directed towards the discovery of new cancer chemopreventive agents from plants, the EtOAc-soluble extract of the stems of M. pomiferus was found to inhibit the enzyme cyclooxygenase-2 (COX-2). Bioassay-directed fractionation of this extract led to the isolation of two dibenzylbutyrolactone lignans, (8R,8'R)-3'-O-demethyl-5-hydroxymatairesinol (1) and (8R,8'R)-3'-O-demethyl-5-methoxymatairesinol (2), as well as seven known compounds, (-)-5'-methoxyyatein (3), blumenol A, (-)-deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, 2,6-dimethoxyhydroquinone, 4-hydroxybenzaldehyde, and beta-sitosterol glucoside. The structures of compounds 1 and 2 were determined using spectroscopic data (1D and 2D NMR, and HREIMS), and the 8R and 8'R absolute stereochemistry was established for both 1 and 2 on the basis of their CD spectra. All isolates obtained in the present study were evaluated for their inhibitory effects with both COX-1 and -2. Of these, only 5'-methoxyyatein (3) showed weak activity against COX-2, while all other compounds isolated were inactive. The COX-2 inhibitory activity of the EtOAc extract was also traced to the presence of several common fatty acids by LC-MS.     

Pyridinium-carbaldehyde: active Maillard reaction product from the reaction of hexoses with lysine residues

Besides the formation of the aminotriazine N6-[4-(3-amino-1,2,4-triazin-5-yl)-2,3-dihydroxybutyl]-L-lysine, the reaction of [1-13C]D-glucose with lysine and aminoguanidine leads to the generation of 6-[2-([[amino(imino)methyl]hydrazono]methyl)pyridinium-1-yl]-L-norleucine (14-13C1). The dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysine was shown to be a precursor in the formation of 14-13C1, which proceeds via the reactive carbonyl intermediate 6-(2-formylpyridinium-1-yl)-L-norleucine (13-13C1). In order to study the reactivity of 13-13C1, the model compound 1-butyl-2-formylpyridinium (18) was prepared in a two-step procedure starting from 2-pyridinemethanol. The reaction of the pyridinium-carbaldehyde 18 with L-lysine yielded the Strecker analogous degradation product 2-(aminomethyl)-1-butylpyridinium and another compound, which was shown to be as 1-butyl-2-[(2-oxopiperidin-3-ylidene)methyl]pyridinium. Reaction of 18 with the C-H acidic 4-hydroxy-5-methylfuran-3(2H)-one leads to the formation of the condensation product 1-butyl-2-[hydroxy-(4-hydroxy-5-methyl-3-oxofuran-2(3H)-ylidene)methyl]-pyridinium.