5-Methyl-2-pyrazinecarboxylic acid is a pyrazine derivative. Simultaneous membrane based solvent extraction (MBSE) and membrane based solvent stripping (MBSS) of 5-methyl-2-pyrazine carboxylic acid (MPCA) has been studied. Mechanism of pertraction of MPCA through layered bulk liquid membranes has been studied. MPCA reacts with Ln(NO3)3·6H2O and K4[W(CN)8]·2H2O to form unique three-dimensional network structure which contains open channels. 5-Methyl-2-pyrazinecarboxylic acid can be used in the preparation of novel europium(III) complexes, and in the synthesis of Ln-metal organic frameworks (MOFs).

Mixing Properties of 5-Methyl-2-pyrazinecarboxylic Acid in Different Solvents

5-Methyl-2-pyrazinecarboxylic acid (CAS no. 5521-55-1) as a pyrazine derivative is an important pharmaceutical intermediate. It is mainly used to synthesize the second generation sulfonylurea hypoglycemic drug glipizide, a new generation of long-acting lipid-lowering drug acimox, and an effective drug for tuberculosis, methyl 5-methylpyrazine-2-carboxylate. However, butanone is controlled by the Drug Enforcement Administration, which brings inconvenience to industrial production. In order to obtain the satisfying yield and purity of the product, it is of great significance to study the dissolution and purification process of MPCA. Up to now, the dissolution process of 5-Methyl-2-pyrazinecarboxylic acid in different solvents has not been reported. Hence, we studied the dissolution behavior of MPCA in methanol, ethanol, n-propanol, isopropanol, 1-butanol, acetone, 2-butanone, toluene, ethyl acetate, acetonitrile, 1,4-dioxane, and water at temperatures ranging from 273.15 to 313.15 K by using the isothermal saturation method, and the results were correlated by the modified Apelblat equation, λh equation, and Wilson model. Moreover, the solvent effect and solute–solvent intermolecular interaction were analyzed during the dissolution process of 5-Methyl-2-pyrazinecarboxylic acid in pure organic solvents. Furthermore, the mixing Gibbs energy, mixing enthalpy, and mixing entropy were also evaluated by the Wilson model.[1]

A reverse-phase column LP-C18 (250 mm × 4.6 mm) at the temperature of 303.15 K and a UV detector with the wavelength of 276 nm was applied to determine the concentration of 5-Methyl-2-pyrazinecarboxylic acid in different solvents. The nonlinear regression method was used to correlate the solubility results of MPCA in selected pure solvents by software Mathcad. Usually, the resultant correlation of thermodynamic models is evaluated by the relative average deviation (RAD) and root-mean-square deviation (RMSD). The solubility of 5-Methyl-2-pyrazinecarboxylic acid in 12 pure solvents within the temperature range from 273.15 to 313.15 K under atmospheric pressure (101.3 kPa) was determined. It increased with increasing temperature, and at a certain temperature, it decreased with the following sequence: 1,4-dioxane > ethanol > n-propanol > 1-butanol > methanol > isopropanol > acetone > 2-butanone > ethyl acetate > water > acetonitrile > toluene. The results were correlated by the modified Apelblat equation, λh equation, and Wilson model, and the maximum values of RAD and RMSD were no more than 1.67% and 3.43 × 10–4, respectively. The values of RAD in the modified Apelblat equation are all less than 0.67%, and the largest RMSD is 1.08 × 10–4; the results show that the modified Apelblat equation could be used preferentially to correlate solubility data. Furthermore, the results of the mixing Gibbs energy, mixing enthalpy, and mixing entropy show that the mixing process is not only exothermic but also entropy-driven. Understanding the dissolution process of 5-Methyl-2-pyrazinecarboxylic acid is of great industrial significance.

Bioretrosynthesis of Functionalized N‐Heterocycles

Based on a retrosynthetic analysis, eleven genes are selected, systematically modulated, and overexpressed in three Escherichia coli strains to construct an artificial pathway to produce 5‐methyl‐2‐pyrazinecarboxylic acid, a key intermediate in the production of the important pharmaceuticals Glipizide and Acipimox. A microbial system with three engineered Escherichia coli strains is developed for the production of N‐heterocycles directly from glucose. Via one‐pot tandem collaborations, it realizes high‐level production of 5-Methyl-2-pyrazinecarboxylic acid, a key intermediate for the important pharmaceuticals Glipizide and Acipimox. This work provides new insight into integrating bioretrosynthetic principles with synthetic biology to perform complex syntheses. DMP is the precursor for the synthesis of 5-Methyl-2-pyrazinecarboxylic acid (MPCA), which is a key component of widely used pharmaceuticals, including Glipizide (an antidiabetic medication with annual prescriptions of 17 million in the United States and 30 million in China. Unlike chemical processes, biotransformation processes are environmentally friendly: for example, reactions occur under mild conditions in the absence of toxic chemicals and metal catalysts, and there is a sustainable supply of raw materials. These evident benefits make the production of DMP and 5-Methyl-2-pyrazinecarboxylic acid from inexpensive bio‐based carbon sources highly desirable.[2]

Towards developing an optimal biosynthetic pathway for 5-Methyl-2-pyrazinecarboxylic acid, we first analyzed different potential routesfrom a retrosynthetic perspective. Interconversion of functional groups can be applied to produce the MPCA carboxylate group via the serial oxidation of a methyl group. The artificial pathway for 5-Methyl-2-pyrazinecarboxylic acid was constructed in reverse. Initially, we used an Escherichia coli strain for the N‐heterocycle functionalization module. Pseudomonas putida is a well‐known degrader of aromatic compounds and has been reported to convert DMP to MPCA, although the respective gene cluster has not been elucidated. In summary, we used a retrosynthetic principle to develop a facile microbial system for the synthesis of industrially important N‐heterocyclic compounds. The modular nature of our microbial system enabled the plug‐and‐play application of the biocatalysts, resulting in high (>5 g L?1) production of the pharmaceutical intermediate 5-Methyl-2-pyrazinecarboxylic acid and the odorant DMP. This study highlights the potential of the environmentally benign production of artificial compounds from renewable biomass via biocatalysis and broadens the perspective of designing complex microbial systems for synthetic applications.

Synthetic pathways for microbial biosynthesis

Due to its aromatic properties, 2,5-DMP is an attractive natural flavor additive for the food industry. For instance, 5-Methyl-2-pyrazinecarboxylic acid (MPCA) can be produced from 2,5-DMP via oxidation of one of the methyl groups. MPCA plays a crucial role in synthesizing commercial drugs such as acipimox (a lipid-lowering agent), glipizide (an anti-diabetic medication), pyrazinamide compounds (medications used to treat tuberculosis), and many more. Currently, 2,5-DMP and its derivatives are primarily produced through chemical synthesis. The Maillard reaction and Strecker degradation are the main methods to obtain 2,5-DMP, while oxidation routes facilitate the production MPCA, 2,5-DMP-N-OX, and 2,5-DMP-di-OX. Our findings have shown that (1) 2,5-DMP biosynthesis is achievable in the KT2440 strain, (2) the biosynthesis of 5-Methyl-2-pyrazinecarboxylic acid from glucose is feasible in shake flask cultivation using a single microbial host, (3) 2,5-DMP-N-OX and 2,5-DMP-di-OX can be produced via de novo biosynthesis.[3]

We studied the 2,5-DMP functionalization pathway leading to 5-Methyl-2-pyrazinecarboxylic acid production via the utilization of XylMABC monooxygenase system (XMO for short). XMO comprises three key enzymes: XylMA hydroxylase (NADH-consuming), XylB benzyl alcohol dehydrogenase (NADH-consuming), and XylC benzaldehyde dehydrogenase (NAD+-consuming). In this instance, both plasmid variants facilitated the formation of MPCA, yielding similar amounts of product after 120 h of cultivation. Specifically, the pBAD2_XylMABC construct produced an 5-Methyl-2-pyrazinecarboxylic acid titer of 204 ± 24 mg L?1, while the pBNT_XylMABC construct achieved a titer of 198 ± 21 mg L?1. Pseudomonas putida KT2440 has evolved from a simple soil-dwelling, root-colonizing bacterium with a versatile metabolism into one of the most popular and standardized chassis for developing effective whole-cell catalysts. This can be perfectly illustrated by the study on 5-Methyl-2-pyrazinecarboxylic acid biosynthesis in E. coli strains.  One strategy involves using XMO-produced MPCA as a substrate for Pml, which oxidizes it to acipimox.

References

[1]Yang, Z., Shao, D., & Zhou, G. (2019, August 21). Solubility Determination and Thermodynamic Mixing Properties of 5-Methyl-2-pyrazinecarboxylic Acid in Different Solvents. *Journal of Chemical & Engineering Data*, 64(9), 4046-4053.

[2]Feng J, Li R, Zhang S, Bu Y, Chen Y, Cui Y, Lin B, Chen Y, Tao Y, Wu B. Bioretrosynthesis of Functionalized N-Heterocycles from Glucose via One-Pot Tandem Collaborations of Designed Microbes. Adv Sci (Weinh). 2020 Jul 21;7(17):2001188. doi: 10.1002/advs.202001188. PMID: 32995125; PMCID: PMC7507072.

[3]Petkevi?ius V, Juknevi?iūt? J, Ma?onis D, Me?kys R. Synthetic pathways for microbial biosynthesis of valuable pyrazine derivatives using genetically modified Pseudomonas putida KT2440. Metab Eng Commun. 2025 Mar 30;20:e00258. doi: 10.1016/j.mec.2025.e00258. PMID: 40236303; PMCID: PMC11999294.