6-methylnicotinic Acid Research Articles

Effector Or Substrate: Characterizing Molecular Features of 6‐HNA That Govern Enhanced Rates of NADH Oxidation and Hydroxylation by 6‐Hydroxynicotinate‐3‐Monooxygenase (NicC)

Group A flavin‐dependent monooxygenases are involved in the degradation of aromatic compounds and the biosynthesis of natural products. These enzymes catalyze the regioselective incorporation of one oxygen atom from dioxygen into their substrates. Comparison of the mechanisms through which the enzymes in this class work could assist in future bioremediation efforts and aid in lowering the costs of synthesis of hydroxylated aromatic compounds. 6‐Hydroxynicotinate 3‐monooxygenase (NicC), a member of this class of enzymes, catalyzes the decarboxylative‐hydroxylation of 6‐hydroxynicotinic acid (6‐HNA) to 2,5‐dihydroxypyridine with concomitant oxidation of NADH. Like other enzymes in its class, the binding of 6‐HNA stimulates an increase in the rate of oxidation of NADH, however the structural determinants of 6‐HNA responsible for this are unknown. Here we show by kinetic analyses of NicC variants that NicC uses Arg184 to form an ion pair with the carboxylate of 6‐HNA to stabilize the ES complex. Once 6‐HNA is bound, an electronegative atom para to the carboxylate (the 6‐oxo group in 6‐HNA) strongly increases the rate of NADH oxidation, possibly by changing the hydrogen bonding network in the active site and allowing the FAD cofactor to swing to the “out orientation”, allowing for closer contact with NADH and therefore more efficient hydride transfer. Previous work showed that NicC is able to bind its substrates (6‐HNA and 5‐chloro‐6‐HNA) via a 2‐step mechanism, where deprotonation of the pyrindolic group of the substrate governs the second step. Molecules that bind and increase the rate of NADH oxidation without turning over into products are called effectors. Thus, effectors share a common molecular characteristic that prevents them from undergoing the coupled hydroxylative pathway in NicC. Through this we sought to determine the 6‐HNA analogues that act as effectors of NicC in attempt to determine how NicC distinguishes between substrates and effectors. 6‐Chloro‐nicotinic acid, 6‐amino‐nicotinic acid and 6‐methyl nicotinic acid were found to be effectors in NicC (Fig 1). These three molecules are not likely to be ionized by deprotonation, signifying that deprotonation of the substrate could govern the coupled and uncoupled hydroxylation pathways in NicC (Scheme 1). Thus, NicC’s mechanism of controlling NADH reduction of FAD appears to be distinct from the mechanism utilized by p‐hydroxybenzoic acid hydroxylase, the quintessential group A flavin dependent monooxygenase.

Study on growth, spectral, optical and thermal characterization of an NLO crystal: 6-Methyl nicotinic acid (6MNA)

6-methyl-nictinic acid (6MNA), organic non linear optical (NLO) single crystals were grown by slow evaporation solution technique. The cell parameters of 6MNA were confirmed from single crystal X-ray diffraction analysis. The Fourier transforms infrared and proton Nuclear magnetic resonance spectral analysis confirm the presence of various functional groups and the placement of protons respectively in 6MNA compound. UV–vis–NIR spectral studies revealed that the grown 6MNA has good optical transmission in the range of entire visible region. The thermal properties of crystals were evaluated from thermogravimetric (TG) and differential thermal analysis (DTA). It has shown that the grown crystals were stable up to 213°C. The second harmonic generation (SHG) measurements indicated that the efficiency of 6MNA is two times greater than that of the KDP crystals and is suitable for frequency conversion applications.

Two new complexes with 6-methylnicotinic acid ligand: Synthesis, crystal structure and Hirshfeld surfaces

Two new complexes derived from 6-methylnicotinic acid: 1(Mn(C7H6NO2)2(H2O)2) and 2 ([Ni(C7H7NO2)2Cl2(H2O)2]·4H2O) have been synthesized and characterized. In complex 1, the MnII ion exhibits a distorted octahedral geometry and featured with 1D chain, while the NiII ion in complex 2 exhibits a regular octahedral geometry and featured with intricate 3D motif. We further compared the molecular Hirshfeld surface and fingerprint plot analysis of ligand 6-methylnicotinic acid in complexes 1 and 2 with the individual 6-methylnicotinic acid, for the purpose of investigating the influence of different metals on the intermolecular interaction around 6-methylnicotinic acid. Which revealed that the closest contacts of these two complexes were dominated by H⋯H, H⋯O, N⋯H, C–H⋯π and π⋯π (C–C) interactions, and the complexation with transition metals lead to the increase of C–H⋯π interactions while the decrease of H–H, N–H, and π⋯π interactions.

Pulse radiolysis studies on reactions of α-hydroxyalkyl radicals with nicotinamide and 6-methyl nicotinic acid

Reactions of α-hydroxyalkyl radicals derived from 2-propanol, ethanol and methanol with nicotinamide (NICAM) and 6-methyl nicotinic acid (6-MNA) were studied at various pHs using pulse radiolysis technique. It is found that α-hydroxyalkyl radicals react with NICAM and 6-MNA at pHs when nitrogen is in the protonated state. In these reactions, radical adducts of NICAM/6-MNA with α-hydroxyalkyl radicals are formed which have absorption maxima at about 340–350nm which subsequently decay to give pyridinyl type of radicals of NICAM and 6-MNA having λmax at 410nm. Rate constants for the reactions of (CH3)2COH, CH3CHOH and CH2OH radicals with NICAM and 6-MNA were found to have linear dependence on reduction potentials of corresponding α-hydroxyalkyl radicals. Adducts formed in the reactions of CH3CHOH and CH2OH radicals with both NICAM and 6-MNA decayed slowly compared to the decay of adduct formed in reactions with (CH3)2COH radicals.

6-Methylnicotinic acid

All non-H atoms of the title compound, C7H7NO2, are nearly coplaner, the r.m.s. deviation being 0.0087 Å. In the crystal, the partially overlapped arrangement and the face-to-face distance of 3.466 (17) Å between parallel pyridine rings of neighboring mol­ecules indicates the existence of π–π stacking. Inter­molecular O—H⋯N hydrogen bonding and weak C—H⋯O hydrogen bonding are present in the crystal structure.

Preparation of deuterated methyl and dimethyl substituted nicotinoylating agents for derivatization of the N-terminal of protein.

Methyl groups of 6-methylnicotinic acid and 2,6-dimethylnicotinic acid were deuterated by an H-D exchange reaction under conditions of 1% NaOD/D(2)O on heating. With a condensation reaction between the D-labeled nicotinic acid derivative and N-hydroxysuccinimide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the nicotinoylating agents, 1-(6-methyl[D(3)]nicotinoyloxy)succinimide (2c) and 1-(2,6-dimethyl[D(6)]nicotinoyloxy)succinimide (2f) were prepared. Both D-labeled nicotinoylating agents and their unlabeled counterparts quantitatively modified the N-terminal of protein.

A molybdenum-containing dehydrogenase catalyzing an unusual 2-hydroxylation of nicotinic acid.

An enzyme of Ralstonia/ Burkholderia strain DSM 6920 catalyzing the initial hydroxylation of 6-methylnicotinic acid at position 2 was purified to apparent homogeneity. It also catalyzed the unusual conversion of nicotinic acid to 2-hydroxynicotinic acid and was therefore designated as nicotinic acid dehydrogenase (NDH). Native NDH had a molecular mass of 280 kDa and was composed of subunits of 75, 30 and 16 kDa. It contained molybdenum, iron, acid-labile sulfur and FAD in a ratio of 1.6:7.3:8.0:0.6 mol(-1) of native enzyme. The molybdenum cofactor was characterized as molybdopterin cytosine dinucleotide. Zinc was identified as an additional metal ion in a molar ratio of 1.8 mol mol(-1) of native enzyme. Purified NDH exhibited a maximal specific activity of 22.6 micromol nitro blue tetrazoliumchloride reduced min(-1) mg(-1) of protein, using nicotinic acid as electron donor. The apparent K(m) value for nicotinic acid was determined to be 154 microM. Pyridine-3,5-dicarboxylic acid and quinoline-3-carboxylic acid were further substrates, but exhibited significantly different activity pH optima. Several artificial electron acceptors were reduced by NDH, but no activity was detected with NAD or O(2). NDH was inactivated upon incubation with cyanide, but no loss of activity was obtained in the presence of arsenite.

Pitfalls in the supramolecular assembly of silver(I) coordination compounds

The syntheses and crystal structures of (6-methylnicotinic acid) (6- methylnicotinato)silver(I), di (6-methylnicotinic acid) silver(I) nitrate, (2-chloro-6-methylnicotinato) silver(I), di (2-chloro-6-methylnicotinic acid) silver(I) nitrate, and (2-methylni-cotinic acid) (2-methylnicotinato) silver(I) are described. These coordination complexes were synthesized under the same conditions and with very similar ligands but their resulting structures are quite different, in part due to the degree of protonation of the nicotinic acid ligand(s). The structures vary from coordination polymers to hydrogen-bonded coordination dimers, to linear structures linked by anion-carboxylic acid hydrogen bonds. This study illustrates some of the difficulties involved in predicting hydrogen-bonded networks in transition-metal systems which contain carboxylic acid moieties and coordinatively unsaturated metal ions.

Microbial production of 2-hydroxynicotinic acid from nicotinic acid by intact cells of MCI3289

We investigated the microbial production of 2-hydroxynicotinic acid (2-HNA) from nicotinic acid (NA) by intact cells of MCI3289, which was isolated from soil by enrichment culture with 6-methylnicotinic acid as the sole carbon source. The strain which showed the 2-HNA-producing activity was revealed to be a member of the β-subgroup of Proteobacteria. The 2-HNA-producing activity was induced by the addition of 6-methylnicotinic acid and 2-hydroxy-6-methylnicotinic acid to the culture medium. Intact cells of this strain incubated at 30°C in an optimal reaction mixture containing 15 g/l of NA produced 6.40 g/l of 2-HNA for 29 h.

Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2.

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3-3c, Mena 25/4-1, and Mena 25/ 4-3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3-3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3-3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia.

A method for the preparation of nicotinic acid and its amide from 2-methyl-5-ethylpyridine

Potassium permanganate is a suitable agent for the preparative-scale synthesis of carbonic acids with a variety of structures. Such agents are also quite frequently used in technology, for example, for the preparation of nicotinic acid from 13picoline. We have shown that the permanganate method of oxidation can successfully be used for preparing nicotinic acid from such widely available raw materials as 2-methyl-5-ethylpyridine (MEP), although additional stages must be included, to extract and decarboxylate isocinchomeronic acid (ICA). The first stage of the reaction is carried out using 6 moles of KMnO 4 for each mole of MEP. Addition of the correct quantity of oxidizing agent to the reaction vessel in a single aliquot or as 2-3 portions results in the reaction occurring too violently and with excessively low selectivity, and ICA is then formed with yields close to those described in the literature, i.e., not greater than 45% [1]. During experiments to identify the optimal reaction conditions it was found that the KMn04 dosing should be changed, reducing the amount of oxidizing agent to 0.5-1 moles per mole of pyridine base, and each portion should only be added after complete utilization of the preceding portion, which is easily detected because of the decolorization of the reaction solution. MEP itself has an extensive tendency to oxidize: even at 80~ the first 2-3 portions of KMnO 4 are entirely utilized within 10-15 min. As shown in Table 1, the reaction product at this time point consists of three pyridinecarboxylic acids, namely, 6-methylnicotinic, 5-ethylpicolinic, and isocinchomeronic acids, though the ftrst of these is predominant. As more oxidizing agent is added, the total rate of the process decreases, and the content of 6-methylnicotinic acid in the reaction mix decreases, and the proportion of isocinchomeronic acid increases. Side reactions occur simultaneously, accompanied by breakage of the pyrimidine ring, profound oxidation of the organic material, and correspondingly unexpected degradation of the oxidizing agent. Thus, both 6-methylnicotinic acid and the initial MEP are present in the decolorized oxidation reaction after addition of 6 moles of KMnO4. Increasing the temperaatre of the reaction and addition of excess oxidizing agent promote ICA accumulation. At 90~ and at boiling temperature, addition of 7 moles of KMnO 4 results in completion of the oxidation process within 3-3.5 h. By this time, MEP and pyrimidine monocarboxylic acids are completely consumed, and the ICA yield is 68-72% of the theoretical maximum. Excess utilization of the oxidizing agent is no more than 15% of the amount required for the quantity of MEP used. Experiments with greater excess quantities of KMnO 4 did not give positive results: oxidation of the ICA product started, though all sources of ICA were exhausted before this started. Experiments with increased quantities of oxidizable material and oxidizing agent present risks of overheating, especially in the initial phases of the reaction. In order to avoid complications, the first portions of KMnO 4 should be made smaller, the intervals between addition of KMn04 portions should be increased, the mixing of the reaction should be improved, removal of heat from the reaction area should be efficient, and the oxidizing agent should be added as an aqueous solution. It should also be noted that losses during extraction of ICA from the reaction are small, because its water solubility is low, i.e., 0.05-0.1 at pH 1.3-2.8 [2]. The stage producing nicotinic acid consists of decarboxylation of ICA, which is carried out by heating ICA in a steel autoclave at 230-250~ for 2 h in the presence of a minimal quantity of water: up to 15 mmoles per mole of initial substance [3]. In these conditions, the desired product is obtained with a yield of 98-99%.

An Improved Method for the Determination of Nicotinic Acid in Human Plasma by High-Performance Liquid Chromatography

Abstract A simple, specific, and sensitive HPLC method was developed for the determination of nicotinic acid in human plasma. Plasma is deproteinized with acetonitrile and centrifuged. The supernatant solution is evaporated and reconstituted in mobile phase. Separation is achieved using an IB-SIL CN column with a mobile phase composed of acetonitrile:methanol:water:acetic acid (700:150:150:1, v/v/v/v). Detection is by ultraviolet absorbance at 263 nm. The retention times of nicotinic acid and 6-methyl nicotinic acid (internal standard) are approximately 3.8 and 8.8 minutes, respectively. The assay is linear in concentration ranges of 100 to 10,000 ng/ml (original method) and 20 to 2,000 ng/mL (high-sensitivity method). The analysis of pooled quality controls (150, 1,250, and 7,530 ng/mL) demonstrates excellent precision with relative standard deviations (RSD) (n = 18) of 3.4%, 2.7%, and 2.8%, respectively. For the high sensitivity method, quality control pools prepared at 60, 150, and 1,250 ng/mL had RSDs...