A Plausible Prebiotic One-Pot Synthesis of Orotate and Pyruvate Suggestive of Common Protometabolic Pathways.

A reaction between two prebiotically plausible building blocks, hydantoin and glyoxylate, generates both the nucleobase orotate, a precursor of biological pyrimidines, and pyruvate, a core metabolite in the citric acid cycle and amino acid biosynthesis. The reaction proceeds in water to provide significant yields of the two widely divergent chemical motifs. Additionally, the reaction of thiohydantoin and glyoxylate produces thioorotate in high yield under neutral aqueous conditions. The use of an open-chain thiohydantoin derivative also enables the potential pre-positioning of a nucleosidic bond prior to the synthesis of an orotate nucleoside. The observation that diverse building blocks of modern metabolism can be produced in a single reaction pot, from common reactants under mild conditions, supports the plausibility of orthogonal chemistries operating at the origins of chemical evolution.

Protein-Ribofuranosyl Interactions Activate Orotidine 5'-Monophosphate Decarboxylase for Catalysis.

The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5'-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5'-monophosphate (OMP) is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of OMP. The D37 and T100' side chains of OMPDC interact, respectively, with the C-3' and C-2' hydroxyl groups of enzyme-bound OMP. D37G and T100'A substitutions result in 1.4 kcal/mol increases in the activation barrier ΔG⧧ for catalysis of decarboxylation of the phosphodianion-truncated substrate 1-(ß-d-erythrofuranosyl)orotic acid (EO) but result in larger 2.1-2.9 kcal/mol increases in ΔG⧧ for decarboxylation of OMP and for phosphite dianion-activated decarboxylation of EO. This shows that these substitutions reduce transition-state stabilization by the Q215, Y217, and R235 side chains at the dianion binding site. The D37G and T100'A substitutions result in <1.0 kcal/mol increases in ΔG⧧ for activation of OMPDC-catalyzed decarboxylation of the phosphoribofuranosyl-truncated substrate FO by phosphite dianions. Experiments to probe the effect of D37 and T100' substitutions on the kinetic parameters for d-glycerol 3-phosphate and d-erythritol 4-phosphate activators of OMPDC-catalyzed decarboxylation of FO show that ΔG⧧ for sugar phosphate-activated reactions is increased by ca. 2.5 kcal/mol for each -OH interaction eliminated by D37G or T100'A substitutions. We conclude that the interactions between the D37 and T100' side chains and ribosyl or ribosyl-like hydroxyl groups are utilized to activate OMPDC for catalysis of decarboxylation of OMP, EO, and FO.

Synthesis, Structure and Impact of 5-Aminoorotic Acid and Its Complexes with Lanthanum(III) and Gallium(III) on the Activity of Xanthine Oxidase.

The superoxide radical ion is involved in numerous physiological processes, associated with both health and pathology. Its participation in cancer onset and progression is well documented. Lanthanum(III) and gallium(III) are cations that are known to possess anticancer properties. Their coordination complexes are being investigated by the scientific community in the search for novel oncological disease remedies. Their complexes with 5-aminoorotic acid suppress superoxide, derived enzymatically from xanthine/xanthine oxidase (X/XO). It seems that they, to differing extents, impact the enzyme, or the substrate, or both. The present study closely examines their chemical structure by way of modern methods-IR, Raman, and 1H NMR spectroscopy. Their superoxide-scavenging behavior in the presence of a non-enzymatic source (potassium superoxide) is compared to that in the presence of an enzymatic source (X/XO). Enzymatic activity of XO, defined in terms of the production of uric acid, seems to be impacted by both complexes and the pure ligand in a concentration-dependent manner. In order to better relate the compounds' chemical characteristics to XO inhibition, they were docked in silico to XO. A molecular docking assay provided further proof that 5-aminoorotic acid and its complexes with lanthanum(III) and gallium(III) very probably suppress superoxide production via XO inhibition.

Theoretical and Experimental Vibrational Characterization of Biologically Active Nd(III) Complex.

The neodymium(III) complex of orotic acid (HOA) was synthesized and its structure determined by means of analytical and spectral analyses. Detailed vibrational analysis of HOA, sodium salt of HOA, and Nd(III)-OA systems based on both the calculated and experimental spectra confirmed the suggested metal-ligand binding mode. Significant differences in the IR and Raman spectra of the complex were observed as compared to the spectra of the ligand. The calculated vibrational wavenumbers, including IR intensities and Raman scattering activities, for the ligand and its Nd(III) complex were in good agreement with the experimental data. The vibrational analysis performed for the studied species, orotic acid, sodium salt of orotic acid, and its Nd(III) complex helped to explain the vibrational behaviour of the ligand's vibrational modes, sensitive to interaction with Nd(III). In this paper we also report preliminary results about the cytotoxicity of the investigated compounds. The cytotoxic effects of the ligand and its Nd(III) complex were determined using the MTT method on different tumour cell lines. The screening performed revealed that the tested compounds exerted cytotoxic activity upon the evaluated cell lines.

A Procedure to Design One-Pot Multi-enzyme System for Industrial CDP-Choline Production.

Fermentation and chemical methods for industrial cytidine diphosphate choline (CDP-choline) catalytic production both suffer from several disadvantages such as relatively low efficiency and productivity. To overcome these problems, we applied the concept of synthetic biology to develop a new one-pot multi-enzyme system to produce CDP-choline from orotic acid. Enzymes from different sources were selected and optimized as building blocks of the system, and parameters such as oxygen supply were also optimized. This system shows a titer of 37.6 ± 1.1 mM and a reaction rate of 1.6 mM L-1 h-1, both increase 66 % from traditional processes. It also has an efficiency of energy of 25.4%, improves 2-folds. This new one-pot CDP-choline-producing system has a potential for industrial use, and the procedure to design one-pot multi-enzyme system can be applied to build other one-pot system producing energy-rich compounds.

Hypoxia Active Platinum(IV) Prodrugs of Orotic Acid Selective to Liver Cancer Cells.

Platinum(IV) complexes of orotic acid selectively target liver cancer cells displaying enhanced activity and higher uptake in Hep G2. The comparatively higher expression of Organic Anion Transporter 2 (OAT2) in Hep G2 and decrease in toxicity in the presence of OAT2 inhibitor suggest its involvement in the uptake of the complexes. They are resistant to sequestration by the copper transporter ATP7B, unlike cisplatin and oxaliplatin.

In Vitro Interaction of 5-Aminoorotic Acid and Its Gallium(III) Complex with Superoxide Radical, Generated by Two Model Systems.

Increased levels of the superoxide radical are associated with oxidative damage to healthy tissues and with elimination of malignant cells in a living body. It is desirable that a chemotherapeutic combines pro-oxidant behavior around and inside tumors with antioxidant action near healthy cells. A complex consisting of a pro-oxidant cation and antioxidant ligands could be a potential anticancer agent. Ga(III) salts are known anticancer substances, and 5-aminoorotic acid (HAOA) is a ligand with antioxidant properties. The in vitro effects of HAOA and its complex with Ga(III) (gallium(III) 5-aminoorotate (GaAOA)) on the in vitro accumulation of superoxide and other free radicals were estimated. Model systems such as potassium superoxide (KO2), xanthine/xanthine oxidase (X/XO), and rat blood serum were utilized. Data suggested better antioxidant effect of GaAOA compared to HAOA. Evidently, all three ligands of GaAOA participated in the scavenging of superoxide. The effects in rat blood serum were more nuanced, considering the chemical and biochemical complexity of this model system. It was observed that the free-radical-scavenging action of both compounds investigated may be manifested via both hydrogen donation and electron transfer pathways. It was proposed that the radical-scavenging activities (RSAs) of HAOA and its complex with Ga(III) may be due to a complex process, depending on the concentration, and on the environment, nature, and size of the free radical. The electron transfer pathway was considered as more probable in comparison to hydrogen donation in the scavenging of superoxide by 5-aminoorotic acid and its gallium(III) complex.

Supramolecular Interactions in Hybrid Polylactide Blends-The Structures, Mechanisms and Properties.

The conformation of polylactide (PLA) chains can be adjusted by supramolecular interactions (the formation of hydrogen bonds or host-guest complexes) with appropriate organic molecules. The structures formed due to those intermolecular interactions may act as crystal nuclei in the PLA matrix ("soft templating"). In this review, the properties of several supramolecular nucleating systems based on synthetic organic nucleators (arylamides, hydrazides, and 1,3:2,4-dibenzylidene-d-sorbitol) are compared to those achieved with biobased nucleating agents (orotic acid, humic acids, fulvic acids, nanocellulose, and cyclodextrins) that can also improve the mechanical properties of PLA. The PLA nanocomposites containing both types of nucleating agents/additives are discussed and evaluated in the context of their biomedical applicability.

Multicomponent Approach to Libraries of Substituted Dihydroorotic Acid Amides.

A process featuring a sequential multicomponent reaction followed by a regioselective postcyclization strategy was implemented for the facile synthesis of N,N'-disubstituted dihydroorotic acid amides under mild conditions. We obtained, for the first time, a library of 29 derivatives, encompassing 19 Nα-substituted-N4-dihydroorotyl-4-aminophenylalanine derivatives, a key residue of gonadotropin-releasing hormone antagonist Degarelix. The corresponding products were prepared from easily accessible starting materials in good to excellent yields with broad substrate scope.

Enhancement of the properties of biosourced poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by the incorporation of natural orotic acid.

The aim of this study is to use natural orotic acid (OA) as a sustainable, environmentally friendly additive to improve the crystallization, rheological, thermal, mechanical, and biodegradation properties of bacterially synthesized poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). OA was found to be an efficient nucleating agent for P34HB, and dramatically enhanced both non-isothermal and isothermal crystallization rates. The incorporation of OA increased nucleation density and decreased spherulite size, but had little effect on the crystalline structure. The rheological properties of the P34HB were greatly improved by the solid filler OA, particularly when a percolation network structure was formed in the blends. The thermal stability of P34HB was strongly enhanced, as exemplified by the ~23 °C increase in the onset thermal decomposition temperature (To) for the blend loaded with 5 wt% OA compared to that of pure P34HB. Moreover, the yield strength and elongation at break of P34HB containing 0.5 wt% OA increased by 25% and 119%, respectively. The most intriguing result was the clear enhancement in the enzymatic hydrolysis rates of the P34HB/OA blends compared to that of neat P34HB. The synergetic improvement in these properties may be of significant importance for the wider practical application of biosourced P34HB.

Highly Water-Soluble Orotic Acid Nanocrystals Produced by High-Energy Milling.

Orotic acid (OA), a heterocyclic compound also known as vitamin B13, has shown potent antimalarial and cardiac protection activities; however, its limited water solubility has posed a barrier to its use in therapeutic approaches. Aiming to overcome this drawback, OA freeze-dried nanocrystal formulations (FA, FB, and FC) were developed by using the high-energy milling method. Polysorbate 80 (FA) and povacoat® (FC) were used alone and combined (FB) as stabilizers. Nanocrystals were fully characterized by dynamic light scattering, laser diffraction, transmission electron microscopy, thermal analysis (thermogravimetry and derivative thermogravimetry, and differential scanning calorimetry), and X-ray powder diffraction revealing an acceptable polydispersity index, changes in the crystalline state with hydrate formation and z-average of 100-200 nm, a remarkable 200-time reduction compared to the OA raw material (44.3 µm). Furthermore, saturation solubility study showed an improvement of 13 times higher than the micronized powder. In addition, cytotoxicity assay revealed mild toxicity for the FB and FC formulations prepared with povacoat®. OA nanocrystal platform can deliver innovative products allowing untapped the versatile potential of this drug substance candidate.

Antioxidant Potential and Angiotensin-Converting Enzyme (ACE) Inhibitory Activity of Orotic Acid-Loaded Gum Arabic Nanoparticles.

Orotic acid (OA) nanoparticles were prepared using the freeze-drying method. The antihypertensive activity and antioxidant capacity of OA and orotic acid-loaded gum arabic nanoparticles (OAGANPs) were examined using the angiotensin-converting enzyme (ACE), 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and ß-carotene assays, as well as the quantification of total phenolic content (TPC). The DPPH and NO scavenging activities of OAGANPs were significantly higher than those of the OA solution. The ß-carotene bleaching assay of OAGANPs showed a dose-dependent trend, while 500 µg/ml was significantly more effective than the other concentrations, which exerted 63.4% of the antioxidant activity. The in vitro antihypertensive assay revealed that the OAGANPs exhibited the most potent ACE inhibition activity, when compared to the OA solution. Hence, results revealed the potential of preparing the OA as a nanoparticle formulation in enhancing the antioxidant and antihypertensive properties compared to the OA solution.

The crystallization behavior of poly(lactic acid) with different types of nucleating agents.

The effects of six nucleating agents (NAs), i.e., orotic acid (OA), potassium salt of 3,5-bis(methoxycarbonyl)benzenesulfonate (LAK-301), substituted-aryl phosphate salts (TMP-5), talc (TALC), N'1,N'6-dibenzoyladipohydrazide (TMC-306) and N1,N1'-(ethane-1,2-diyl)bis(N2-phenyloxalamide) (OXA), on the crystallization behavior of poly(lactic acid) (PLA) were compared by DSC. Under the same dosing of 0.5wt%, the nucleation effect of the NAs for PLA declines in the order of TMC-306≈OXA>TALC≫TMP-5≈LAK-301≈OA. The nucleation efficiency (NE) of TMC-306 and OXA is around 50%, which is almost 2 times of the NE of TALC. In the best case of the PLA/TMC-0.5% sample, the half-time of crystallization decreases from 30s to 9s with decreasing the crystallization temperature from 120°C to 100°C, which is of great significance to the fast production of highly crystallized PLA materials. As high-efficient NAs, TMC-306 and OXA are able to accelerate the crystallization rate of PLA even upon fast cooling at 50°C/min, while make no difference on PLA crystal form, as identified by WAXD. DMA analysis shows that the storage modulus of PLA is significantly improved by TMC-306 and OXA.

Enzyme Architecture: Erection of Active Orotidine 5'-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes.

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with kcat/Km = 1.4 × 10-7 M-1 s-1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.

Three Pyrimidine Decarboxylations in the Absence of a Catalyst.

The epigenetic modification of DNA by 5-methylation of cytosine residues can be reversed by the action of the TET family of dioxygenases that oxidize the methyl group to produce 5-carboxycytosine (5caC), which can be converted to cytosine in a final decarboxylation step. Likewise, 5-carboxyuracil (5caU) is decarboxylated to uracil in the last step in pyrimidine salvage. In view of the extreme difficulty of decarboxylating derivatives of orotic acid (6caU), it seemed desirable to establish the rates of decarboxylation of 5caC and 5caU in the absence of a catalyst. Arrhenius analysis of experiments performed at elevated temperatures indicates that 5caU decomposes with a rate constant of 1.1 × 10-9 s-1 (ΔH⧧ = 25 kcal/mol) in a neutral solution at 25 °C. The decomposition of 5caC is somewhat slower (k25 = 5.0 × 10-11 s-1; ΔH⧧ = 27 kcal/mol) and leads to the initial accumulation of cytosine as an intermediate, followed by the relatively rapid deamination of cytosine (k25 = 1.9 × 10-10 s-1; ΔH⧧ = 23.4 kcal/mol). Both 5caC and 5caU are decarboxylated many orders of magnitude more rapidly than 6caU is (k25 = 1.3 × 10-17 s-1). Ab initio simulations indicate that in all three cases, the favored route of spontaneous decarboxylation in water involves direct elimination of CO2 with the assistance of an explicit water molecule.

Design and synthesis of potent substrate-based inhibitors of the Trypanosoma cruzi dihydroorotate dehydrogenase.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, is the leading cause of heart disease in Latin America. T. cruzi dihydroorotate dehydrogenase (DHODH), which catalyzes the production of orotate, was demonstrated to be essential for T. cruzi survival, and thus has been considered as a potential drug target to combat Chagas disease. Here we report the design and synthesis of 75 compounds based on the orotate structure. A comprehensive structure-activity relationship (SAR) study revealed two 5-substituted orotate analogues (5u and 5v) that exhibit Kiapp values of several ten nanomolar level and a selectivity of more than 30,000-fold over human DHODH. The information presented here will be invaluable in the search for next-generation drug leads for Chagas disease.

Biochemical characterization of dihydroorotase of Leishmania donovani: Understanding pyrimidine metabolism through its inhibition.

De novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is third enzyme of the pathway. The enzyme was cloned, expressed in E. coli BL21 (DE3), purified to homogeneity and biochemically characterized. The estimated kcat for the forward reaction and reverse reactions were 2.1 ± 0.1 s-1 and 1.1 ± 0.15 s-1, respectively. Homology modeling and docking studies were done to find out potential inhibitors for LdDHOase. Biotin sulfone and Kaempferol were found to be potential inhibitors of LdDHOase based on docking studies. These inhibitors were verified using recombinant LdDHOase and their anti-leishmanial effect was evaluated. Moreover, alterations in expressions of de novo as well as salvage pathways enzymes, after treatment of L. donovani with dihydroorotase inhibitor(s) were evaluated and discussed as survival mechanism of the pathogen. Further, effect of inhibition of cytidine deaminase, a key enzyme of salvage pathway of pyrimidine biosynthesis, was also evaluated on parasitic survival and alteration in gene expression of enzymes of both pathways. Further, effect of both pathways inhibition was also evaluated. The data suggests that the inhibition of single pathway can be overcome by increased expression of enzyme(s) of alternate pathway and both pathways seem to be equally important in the pathogen. When both pathways are simultaneously inhibited, parasite shows significant DNA damage and parasitic death.

Investigation of vital pathogenic target orotate phosphoribosyltransferases (OPRTase) from Thermus thermophilus HB8: Phylogenetic and molecular modeling approach.

Biosynthesis pathways of pyrimidine and purine are shown to play an important role in regular cellular activities. The biosynthesis can occur either through de novo or salvage pathways based on the requirement of the cell. The pyrimidine biosynthesis pathway has been linked to several disorders and various autoimmune diseases. Orotate phosphoribosyl transferase (OPRTase) is an important enzyme which catalyzes the conversion of orotate to orotate monophosphate in the fifth step of pyrimidine biosynthesis. Phylogenetic analysis of 228 OPRTase sequences shows the distribution of proteins across different living forms of life. High structural similarities between Thermusthermophilus and other organisms kindled us to concentrate on OPRTase as an anti-pathogenic target. In this study, a homology model of OPRTase was constructed using 2P1Z as a template. About 100 ns molecular dynamics simulation was performed to investigate the conformational stability and dynamic patterns of the protein. The amino acid residues (Met1, Asp2, Glu43, Ala44, Glu47, Lys51, Ala157 and Leu158) lining in the binding site were predicted using SiteMap. Further, structure based virtual screening was performed on the predicted binding site using ChemBridge, Asinex, Binding, NCI, TosLab and Zinc databases. Compounds retrieved from the screening collections were manually clustered. The resultant protein-ligand complexes were subjected to molecular dynamics simulations, which further validates the binding modes of the hits. The study may provide better insight for designing potent anti-pathogenic agent.

Structural Properties, Order-Disorder Phenomena, and Phase Stability of Orotic Acid Crystal Forms.

Orotic acid (OTA) is reported to exist in the anhydrous (AH), monohydrate (Hy1), and dimethyl sulfoxide monosolvate (SDMSO) forms. In this study we investigate the (de)hydration/desolvation behavior, aiming at an understanding of the elusive structural features of anhydrous OTA by a combination of experimental and computational techniques, namely, thermal analytical methods, gravimetric moisture (de)sorption studies, water activity measurements, X-ray powder diffraction, spectroscopy (vibrational, solid-state NMR), crystal energy landscape, and chemical shift calculations. The Hy1 is a highly stable hydrate, which dissociates above 135 °C and loses only a small part of the water when stored over desiccants (25 °C) for more than one year. In Hy1, orotic acid and water molecules are linked by strong hydrogen bonds in nearly perfectly planar arranged stacked layers. The layers are spaced by 3.1 Å and not linked via hydrogen bonds. Upon dehydration the X-ray powder diffraction and solid-state NMR peaks become broader, indicating some disorder in the anhydrous form. The Hy1 stacking reflection (122) is maintained, suggesting that the OTA molecules are still arranged in stacked layers in the dehydration product. Desolvation of SDMSO, a nonlayer structure, results in the same AH phase as observed upon dehydrating Hy1. Depending on the desolvation conditions, different levels of order-disorder of layers present in anhydrous OTA are observed, which is also suggested by the computed low energy crystal structures. These structures provide models for stacking faults as intergrowth of different layers is possible. The variability in anhydrate crystals is of practical concern as it affects the moisture dependent stability of AH with respect to hydration.

Lanthanide(III) complexes are more active inhibitors of the Fenton reaction than pure ligands.

OBJECTIVES: This study is an extension to our finding of direct anti-oxidant activities of lanthanide(III) complexes with the heterocyclic compound, 5-aminoorotic acid (AOA). In this experiment, we used AOA and coumarin-3-carboxylic acid as the two heterocyclic compounds with anti-oxidant potential, to produce the complexes with different lanthanides. METHODS: Lanthanide(III) complexes were tested on the iron-driven Fenton reaction. The product of this reaction, the hydroxyl radical, was detected by HPLC. RESULTS: All complexes as well as their ligands had positive or neutral effect on the Fenton reaction but their behavior was different. Both pure ligands in low concentration ratio to iron were inefficient in contrast to some of their complexes. Complexes of neodymium, samarium, gadolinium, and partly of cerium blocked the Fenton reaction at very low ratios (in relation to iron) but the effect disappeared at higher ratios. In contrast, lanthanum complexes appeared to be the most promising. Both blocked the Fenton reaction in a dose-dependent manner. CONCLUSION: Lanthanide(III) complexes were proven to block the iron-driven production of the hydroxyl radical. Second, the lanthanide(III) element appears to be crucial for the anti-oxidant effect. Overall, lanthanum complexes may be promising direct anti-oxidants for future testing.