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Human lactate dehydrogenase (hLDH) is a tetrameric enzyme present in almost all tissues. Among its five different isoforms, hLDHA and hLDHB are the predominant ones. In the last few years, hLDHA has emerged as a therapeutic target for the treatment of several kinds of disorders, including cancer and primary hyperoxaluria. hLDHA inhibition has been clinically validated as a safe therapeutic method and clinical trials using biotechnological approaches are currently being evaluated. Despite the well-known advantages of pharmacological treatments based on small-molecule drugs, few compounds are currently in preclinical stage. We have recently reported the detection of some 2,8-dioxabicyclo[3.3.1]nonane core derivatives as new hLDHA inhibitors. Here, we extended our work synthesizing a large number of derivatives (42–70) by reaction between flavylium salts (27–35) and several nucleophiles (36–41). Nine 2,8-dioxabicyclo[3.3.1]nonane derivatives showed IC50 values lower than 10 µM against hLDHA and better activity than our previously reported compound 2. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC50 values against hLDHA (3.6–12.0 µM) and the highest selectivity rate (>25). Structure–activity relationships have been deduced. Kinetic studies using a Lineweaver–Burk double-reciprocal plot have indicated that both enantiomers of 68a and 68b behave as noncompetitive inhibitors on hLDHA enzyme.

Sweet cherry (Prunus avium L.) is a tree widely cultivated in temperate regions for its tasty and healing fruits. Pruning works on the tree give each year considerable amounts of woody wastes that hardly have any utility. The aims of this work were to detect the most active antioxidants present in a cherry pruning wood sample, to isolate and characterize them, and study the antimicrobial and antibiofilm activities of components found in the wood ethyl acetate extract against a selection of foodborne microorganisms. The online HPLC–DPPH technique allowed the detection of two active antioxidant peaks that, after being isolated by a combination of countercurrent chromatography (FCPC) and conventional preparative techniques, and subsequent structural characterization by NMR, MS and polarimetry, resulted to be (‒)-catechin (1) and (‒)-taxifolin (4). Other components of the cherry wood extract were also isolated, among which compounds 1, 4, and (+)-dihydrowogonin (12) have never been reported in P. avium. A selection of the isolated flavonoids was submitted to antimicrobial and antibiofilm activity evaluations against strains from type culture collections, as well as on multi-resistant strains previously isolated in our laboratory. Those compounds with antimicrobial activity detected in preliminary screenings by standard agar diffusion tests ‒the flavan-3-ol 1, the flavanonols 4 and (+)-aromadendrin (5), the flavanone (+)-pinocembrin (15), and the flavone tectochrysin (17)‒ were subjected to the minimal inhibitory concentration (MIC) test, showing all of them MIC values of 100 μg/mL. Compound 4 also induced a significative inhibition on the formation of biofilms by Enterobacter sp. UJA37p at a concentration of 1 μg/mL and a significative disruption of preformed biofilm by this strain at 0.1 µg/mL. Similar results on biofilm disruption were observed with compound 17.

Cinnamtannin B-1 (C-B1) is a commercially-available trimeric A-type procyanidin with remarkable cellular actions mainly derived from its high radical scavenging activity. C-B1 is the main phenolic compound of laurel wood, which has previously been isolated by a combination of conventional chromatographic techniques. The first aim of this work was to find laurel trees containing as much C-B1 as possible, and learn about the influence of variables, such as gender and harvest time, on the production of C-B1 by the tree. It was found that all studied trees tend to give higher C-B1 percentages in the May-July period, from 6 % to 18 %, and lower ones around March (spring) and November (fall), from 1 % to 8 %. In a general way, it also seems that the female trees tend to produce a bit more C-B1 (from 2.8 % to 17.3 %) than male ones (from 1.7 % to 13.4 %). In addition, eight minor phenolic compounds [(-)-ent-catechin (1), (-)-ent-epicatechin-(4α→8)-ent-epicatechin (2), (epi)catechin-(4→8)-(epi)afzelechin-(4→8)-(epi)catechin (3), (+)-epiafzelechin-(4β→8)-epicatechin (4), (-)-epicatechin (5), (-)-afzelechin-(4α→8)-epicatechin (6), (epi)afzelechin-(4→8)-(epi)afzelechin-(4→8)-(epi)catechin (7) and (+)-epicatechin-(4β→8,2β→O-7)-epicatechin-(4β→8)-catechin (C-D1)] were found and quantified in the ethyl acetate extract of the wood samples. The second aim of this work was to improve the recovery of C-B1 from laurel wood. The use of the fast centrifugal partition chromatography (FCPC) technique has allowed for a recovery of 96 % of a technical-grade C-B1 (64 % in a previous protocol using conventional column chromatography on silica gel and size-exclusion chromatography).

Human lactate dehydrogenase A (hLDHA) is one of the main enzymes involved in the pathway of oxalate synthesis in human liver and seems to contribute to the pathogenesis of disorders with endogenous oxalate overproduction, such as primary hyperoxaluria (PH), a rare life-threatening genetic disease. Recent published results on the knockdown of LDHA gene expression as a safe strategy to ameliorate oxalate build-up in PH patients are encouraging for an approach of hLDHA inhibition by small molecules as a potential pharmacological treatment. Thus, we now report on the synthesis and hLDHA inhibitory activity of a new family of compounds with 2,8-dioxabicyclo[ 3.3.1]nonane core (23–42), a series of twenty analogues to A-type proanthocyanidin natural products. Nine of them (25–27, 29–34) have shown IC50 values in the range of 8.7–26.7 μM, based on a UV spectrophotometric assay, where the hLDHA inhibition is measured according to the decrease in absorbance of the cofactor β-NADH (340 nm). Compounds 25, 29, and 31 were the most active hLDHA inhibitors. In addition, the inhibitory activities of those nine compounds against the hLDHB isoform were also evaluated, finding that all of them were more selective inhibitors of hLDHA versus hLDHB. Among them, compounds 32 and 34 showed the highest selectivity. Moreover, the most active hLDHA inhibitors (25, 29, 31) were evaluated for their ability to decrease the oxalate production by hyperoxaluric mouse hepatocytes (PH1, PH2 and PH3) in vitro, and the relative oxalate output at 24 h was 16% and 19 % for compounds 25 and 31, respectively, in Hoga1-/- mouse primary hepatocyte cells (a model for PH3). These values improve those of the reference compound used (stiripentol). Compounds 25 and 31 have in common the presence of two hydroxyl groups at rings B and D and an electron-withdrawing group (NO2 or Br) at ring A, pointing to the structural features to be taken into account in future structural optimization., Spanish Ministerio de Ciencia, Innovacion y Universidades - FEDER funds of the European Union RTI2018-098560-B-C21 RTI2018-098560-B-C22, Centro de Instrumentacion Cientifico-Tecnica (CICT) of the University of Jaen, Spain, University of Jaen, Andalusian Consejeria de Economia y Conocimiento (FEDER program 2014-2020) 1380682, European Youth Guarantee contract

Two polycyclic lumazine derivatives with different side chains (alkyl-acid and alkyl-ester chains) have been synthesized. Both novel bioinspired compounds exhibit interesting luminescent properties. Herein, the structural and optical properties of these compounds have been analyzed by single-crystal X-ray analysis, absorption, fluorescence and vibrational Raman spectroscopy together with DFT calculations. Their spectroscopic behavior depends on the five-fused core, which remains almost planar in the excited electronic state, and also on the nature of the side chain. In the solid phase, bathochromic shifts were observed with respect to solution for both molecules. These spectroscopic shifts were particularly notorious for the acid derivative and they might be related to the differences found in the molecular packing depending on the nature of the side chain. Two different theoretical strategies were employed to study the molecular packing in the crystalline state. Firstly, the energy of the S0→S1 transition was calculated for a molecule surrounded by a cluster of neighboring molecules extracted from the crystal structure. Secondly, the lowest-energy electronic transitions were computed for different-size molecular clusters extracted from the crystal structure. Thus, the J-aggregate photophysical behavior experimentally observed in the solid phase, and the spectroscopic differences between both compounds, were explained on the basis of short-range excitonic coupling. These results provide valuable insights for the rational design of new bioinspired compounds with luminescent properties.

The thermodynamic and kinetic behavior of 4-(2,4-dihydroxybenzylidene)-6-hydroxy-1,2,3,4-tetrahydroxanthylium chloride was studied in detail. Similarly to anthocyanins, this compound generates a multistate of species that are reversibly interconverted by pH stimuli. The multistate system was studied by NMR and UV–visible (conventional spectroscopy and stopped-flow techniques). A theoretical approach adapted from the one reported for anthocyanins and related compounds was used to rationalize the complex multistate. In particular, the thermodynamic diagram where all the species are positioned was extended to the ionized species. Cis and trans isomers of flavylium cation, quinoidal bases, and ionized chalcone at extremely high pH values were identified. The multistate is dominated in the range 5

Anthocyanins are the basis of the color of angiosperms, 3‐deoxyanthocyanins and sphagnorubin play the same role in mosses and ferns, and auronidins are responsible for the color in liverworts. In this study, the color system of cyanidin‐3‐O‐glucoside (kuromanin) as a representative compound of simpler anthocyanins was fully characterized by stopped flow. This type of anthocyanin cannot confer significant color to plants without intra‐ or intermolecular interactions, complexation with metals or supramolecular structures as in Commelina communis. The anthocyanin’s color system was compared with those of 3‐deoxyanthocyanins and riccionidin A, the aglycone of auronidins. The three systems follow the same sequence of chemical reactions, but the respective thermodynamics and kinetics are dramatically different. publishersversion published

Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism. PHs classification based on gene mutations parallel a variety of enzymatic defects, and all involve the harmful accumulation of calcium oxalate crystals that produce systemic damage. These geographically widespread rare diseases have a deep impact in the life quality of the patients. Until recently, treatments were limited to palliative measures and kidney/liver transplants in the most severe forms. Efforts made to develop pharmacological treatments succeeded with the biotechnological agent lumasiran, a siRNA product against glycolate oxidase, which has become the first effective therapy to treat PH1. However, small molecule drugs have classically been preferred since they benefit from experience and have better pharmacological properties. The development of small molecule inhibitors designed against key enzymes of glyoxylate metabolism is on the focus of research. Enzyme inhibitors are successful and widely used in several diseases and their pharmacokinetic advantages are well known. In PHs, effective enzymatic targets have been determined and characterized for drug design and interesting inhibitory activities have been achieved both in vitro and in vivo. This review describes the most recent advances towards the development of small molecule enzyme inhibitors in the treatment of PHs, introducing the multi-target approach as a more effective and safe therapeutic option.

Proanthocyanidins (PACs) are oligomers or polymers composed of units of flavanols. A-type PACs are a subclass of PACs characterized by the presence of at least a double linkage between two consecutive monomers of flavanol. These A-type PACs are found in some fruits and spices and possess potential health benefits as a result of their interesting biological activities, and consequently, their isolation and synthesis have given rise to great interest in the past. This review summarizes the synthetic efforts made to obtain both naturally occurring A-type PACs and their structurally simplified analogues. Most of the synthetic protocols reported involve the addition of a π-nucleophilic molecule over a molecule with two electrophilic carbons, such as a chalcone, a flavylium salt, or a flavanol derivative, among others. Synthesis of A-type PACs remains an issue at a very early stage of development compared to that of PACs with single linkages between monomers (B-type PACs), but the advances that are taking place in the last few years point to a significant development of the subject in the near future.

Porphyrins and derivatives form one of the most abundant classes of biochromes. They result from the breakdown of heme and have crucial physiological functions. Bilins are well-known representatives of this group that, besides significant antioxidant and anti-mutagenic properties, are also photosensitizers for photodynamic therapies. Recently, we demonstrated that the Polychaeta Eulalia viridis, common in the Portuguese rocky intertidal, holds a high variety of novel greenish and yellowish porphyrinoid pigments, stored as granules in the chromocytes of several organs. On the follow-up of this study, we chemically characterized pigment extracts from the worm&rsquo, s skin and proboscis using HPLC and evaluated their light and dark toxicity in vivo and ex vivo using Daphnia and mussel gill tissue as models, respectively. The findings showed that the skin and proboscis have distinct patterns of hydrophilic or even amphiphilic porphyrinoids, with some substances in common. The combination of the two bioassays demonstrated that the extracts from the skin exert higher dark toxicity, whereas those from the proboscis rapidly exert light toxicity, then becoming exhausted. One particular yellow pigment that is highly abundant in the proboscis shows highly promising properties as a natural photosensitizer, revealing that porphyrinoids from marine invertebrates are important sources of these high-prized bioproducts.

This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019). FCT/MCTES is also acknowledged through the National Portuguese NMR Network RECI/BBB-BQB/0230/2012. A.A.-A. is grateful for the postdoctoral fellowship from Fundacion Alfonso Marti0n Escudero. The multistate of chemical species generated by 4′-hydroxy-3,2′-furanoflavylium is similar to that of anthocyanins and related compounds. This furanoflavylium multistate system was fully characterized by UV-visible and NMR spectroscopy, allowing determination of the respective equilibrium and rate constants. In contrast to the multistate generated by flavylium cations derived from anthocyanins and related compounds, the furanoflavylium multistate is characterized by much slower hydration and tautomerization (pyran ring opening-closing). In addition, the cis-trans isomerization of the chalcones of this system (2′-hydroxyaurones) is extremely slow when compared with anthocyanins. The observed similar order of magnitude for tautomerization and isomerization rate constants leads to peculiar kinetics from the flavylium cation (pH = 1) to the stable trans-chalcone (higher pH values). The hemiketal appears and disappears during the first stages of the kinetics, which gives the intermediate cis-chalcone (pseudo-equilibrium). This last species disappears in a much slower process, as fully characterized by 1 H NMR, to give the final trans-chalcone. publishersversion published

The comparison of the ground-state reactivity of anthocyanins and aurone model compounds (i.e. with and without the furano bridge) has shown that the kinetic paradigm does not depend on the bridge but only on the hydroxyl substituent pattern, independently of the presence of the bridge: (i) bell shaped kinetics for those with two hydroxyl substituents in position 4' and 7, and (ii) four distinct kinetic steps for the mono substituted compounds with a hydroxyl in position 4'. The excited state proton transfer (ESPT) properties of these compounds were also investigated using steady-state and time-resolved spectroscopic techniques. It was found that the ESPT efficiency is significantly higher for the bridged compounds. Interestingly, pH-dependent steady-state fluorescence emission experiments show that in 4',7-dihydroxyfuranoflavylium the hydroxyl group in position 7 is the more acidic one in the excited state, while 1H NMR titration curves indicate a higher acidity constant in the ground state for the proton at the hydroxyl group in position 4'. Differently, the fluorescence emission spectrum of the quinoidal base deprotonated at position 7 is only observed upon excitation of the flavylium cation while the one from the base deprotonated at 4' is observed upon direct excitation.

As in supramolecular chemistry, complexity could also be achieved through a bottom-up approach. Anthocyanins and related compounds such as the compound (E)-6-(dimethylamino)-4-(4-(dimethylamino)-2-hydroxybenzylidene)-1,2,3,4-tetrahydroxanthylium chloride (1), here reported, exhibit this type of complexity. The thermodynamics and kinetics of the complex multistate of species of compound 1 were studied by conventional and stopped-flow UV–visible spectrophotometry as well as by NMR. The system follows the same multistate of species of anthocyanins, except for the presence at moderately basic pH values of a species possessing a spiro carbon. The introduction of two dimethylamino substituents in positions 4′ and 7, modulates deeply the thermodynamic and kinetics of the system. A beautiful pH-dependent palette of colors is obtained, including a blue flavylium cation at unusually high pH values. The protonation of the dimethylamino substituents is the key aspect for explaining the details of the spiro opening kinetics. The system was fully characterized by representing the mole fraction distribution and the relative energy level diagram of all multistate species as a function of pH.

A convenient method to synthesize A-type proanthocyanidin analogues from flavylium salts and π-nucleophiles has been developed. It was found that the thermodynamic stability of the starting flavylium salt, assessed by the measurement of the apparent acidity constant ( K'

Natural A-type procyanidins have shown very interesting biological activities, such as their proven antiadherence properties against pathogenic bacteria. In order to find the structural features responsible for their activities, we describe herein the design and synthesis of six A-type procyanidin analogues and the evaluation of their antimicrobial and antibiofilm properties against 12 resistant bacteria, both Gram positive and Gram negative, isolated from organic foods. The natural A-type procyanidin A-2, which had known antiadherence activity, was also tested as a reference compound for the comparative studies. Within the series, analogue 4, which had a NO2 group on ring A, showed the highest antimicrobial activity (MIC of 10 μg/mL) and was one of the best molecules at preventing biofilm formation (up to 40% decreases at 100 μg/mL) and disrupting preformed biofilms (up to 40% reductions at 0.1 μg/mL). Structure–activity relationships are also analyzed.

Summary This study aimed to evaluate the antimicrobial and antibiofilm activities of two procyanidins isolated from an ethyl acetate extract of laurel wood against a selection of foodborne pathogens. The analysis of the extract by HPLC–DAD/ESI–MS allowed us to detect the presence of two procyanidins, which were selectively isolated and identified by chromatographic and spectroscopic means as cinnamtannin B-1 (1) and procyanidin B-2 (2). Procyanidins 1 and 2 exhibited two biological activities: inhibition of bacterial growth at high concentrations and prevention of biofilm formation at lower concentrations. Synergistic effect was also detected when both compounds were tested in combination against Listeria monocytogenes. Significant effects were also detected on disruption of preformed biofilm. The ability of procyanidins to inhibit microbial growth and biofilm formation and to synergistically work with each other may stimulate a market as natural food preservatives, and/or natural sanitisers for processing equipment where foodborne pathogens reside.

Laurel tree (Laurus nobilis L.) wood is a byproduct of the pruning of this tree widely cultivated for its aromatic leaves. The phenolic composition of laurel wood has not been reported yet. The aim of this work was to study the phenolic profile of an ethyl acetate extract of laurel wood in order to know its potential as a source of antioxidants and valuable compounds. Three flavan-3-ols, 4 dimeric B-type proanthocyanidins, and 2 trimeric A-type procyanidins were isolated by preparative column chromatography and semi-preparative HPLC, using a fast online post-column radical scavenging detection technique as guidance. All isolated compounds were fully characterized by spectroscopic (NMR, IR, UV) and spectrometric (MS) techniques. In addition, 17 minor proanthocyanidins were tentatively identified by liquid chromatography coupled to mass spectrometry (HPLC–ESI–MS). The extract was dominated by the presence of cinnamtannin B-1, a bioactive trimeric A-type procyanidin reported as a strong antioxidant of commercial value. According to the chemical composition found, it can be concluded that laurel wood is a renewable source of potential bioactive phenolic compounds.

The similarity between 2,6-Bis(2-hydroxyarylidene)cyclohexanones and 2′-hydroxychalcones in generating respectively styrylflavylium and flavylium cations was explored. The complex pH and light dependent multistate of species of the asymmetric of 2,6-Bis(2-hydroxyarylidene)cyclohexanone bearing a diethyl amine in position 7 of the flavylium cation was studied by pH jumps, monitored by UV–vis spectrophotometry, stopped flow, and NMR. The possible structural isomers corresponding to the migration of the amine substituent at the benzopyrylium to ring B of the flavylium cation via chalcone, or spiro species were not detected. However, cis and trans isomers from the pendent arm take place and a photochromic system involving flavylium cation switching from red to blue was characterized. In acidic medium the flavylium cation and the hemiketal protonated at the amine substituent are the equilibrium species. In moderately acidic solutions the spiro is the most stable species and in very basic medium the ionized trans-chalcone.

The effect of methyl, hydroxyl, and chloride substituents in position 3 of the 3′,4′,7-trihydroxyflavylium core structure was studied. The stability, relative energy of each of chemical species (thermodynamics), and their rates of interconversion (kinetics) are very dependent on these substituents. By comparing the mole fraction distribution at equilibrium of the three multistate systems with the parent 3′,4′,7-trihydroxyflavylium, introduction of a methyl substituent in position 3 increases the mole fraction of hemiketal at the expense of the trans-chalcone and increases the hydration rate very significantly; a hydroxyl substituent in position 3 gives rise to a degradation process, as observed in anthocyanidins. In the case of 3-chloro-3′,4′,7-trihydroxyflavylium, a dramatic increase of the flavylium cation acidity was observed and a photochromic system can be operated upon irradiation of the respective trans-chalcone in 1 m HCl. According to the photochromic response of 3,3′,4′,7-tetrahydroxyflavylium and 3′,4′,7-trihydroxyflavylium, some requirements for a good photochromic performance are discussed.

This review summarizes the chemical composition reported up to date on Laurus nobilis L. (Lauraceae), an evergreen shrub or tree cultivated for its aromatic leaves and ornamental interest. It has been focused on non-volatile phytochemicals such as sesquiterpene lactones, flavonoids and proanthocyanidins, among others. Moreover, biological activities of laurel extracts and pure compounds have also been reviewed.