2-Chloronicotinic Acid: Applications in Pesticides and its Biosynthesis by Amidase-Catalyzed Hydrolysis

General Description

2-Chloronicotinic acid is a key compound used in pesticides, particularly neonicotinoid insecticides, due to its effectiveness in pest control. It serves as an important intermediate in their synthesis. Through amidase-catalyzed hydrolysis of 2-chloronicotinamide, biosynthesis of 2-chloronicotinic acid is facilitated. Engineering efforts on the amidase enzyme from Pantoea sp. resulted in significantly improved catalytic properties, leading to enhanced production of 2-chloronicotinic acid. This biocatalytic approach offers a promising and environmentally friendly method for producing this essential chemical intermediate, addressing the demand for 2-chloronicotinic acid in pharmaceuticals and pesticides effectively.

Figure 1. 2-Chloronicotinic acid

Applications in Pesticides

2-Chloronicotinic acid is a chemical compound that finds extensive applications in the field of pesticides. This organic acid has proven to be highly effective in controlling and eliminating pests, making it a valuable component in the development of various pesticide formulations. One of the primary applications of 2-Chloronicotinic acid is its role as a key intermediate in the synthesis of neonicotinoid insecticides. Neonicotinoids are a class of pesticides that specifically target insects, disrupting their central nervous system and causing paralysis and death. By acting on specific receptors in the insect's nerve synapses, neonicotinoids provide highly targeted and efficient pest control. 2-CNA serves as a crucial building block in the synthesis of many widely used neonicotinoid insecticides, such as imidacloprid and clothianidin. Additionally, 2-Chloronicotinic acid exhibits broad-spectrum activity against a wide range of pests, including aphids, thrips, whiteflies, and beetles. It can be formulated into various pesticide products, including sprays, granules, and seed treatments. The versatility of 2-CNA allows for its incorporation into different pesticide formulations, enabling effective pest management in various agricultural settings. Moreover, the use of 2-Chloronicotinic acid in pesticides offers several advantages. It shows excellent systemic properties, meaning that once applied, it can be absorbed by plants and distributed throughout their tissues, providing prolonged protection against pests. Furthermore, 2-CNA demonstrates low toxicity to mammals, making it a safer option for pest control. In conclusion, 2-Chloronicotinic acid plays a vital role in the development of neonicotinoid insecticides and offers broad-spectrum pest control. Its effectiveness, versatility, and relatively low toxicity make it a valuable component in the formulation of pesticides for agricultural applications. ¹

Biosynthesis by Amidase-Catalyzed Hydrolysis

2-Chloronicotinic acid serves as a crucial intermediate in the production of pharmaceuticals and pesticides. Its biosynthesis from 2-chloronicotinamide via amidase-catalyzed hydrolysis presents a promising enzymatic approach, although the strong steric and electronic effects caused by the 2-position chlorine substituent of the pyridine ring make this process challenging. In response to this, an amidase from Pantoea sp. (Pa-Ami) was subjected to design and engineering efforts to enhance its catalytic properties. Through the introduction of single mutant G175A and double mutant G175A/A305T strains, significant improvements in specific activity for 2-chloronicotinamide were achieved, with 3.2- and 3.7-fold enhancements observed, respectively. Moreover, the catalytic efficiency saw substantial increases, with kcat/Km values 3.1 and 10.0 times higher than those of the wild type, demonstrating the effectiveness of the engineered strains. Structural analysis revealed that the G175A mutation resulted in a shortened distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide, thereby facilitating the nucleophilic attack on the Oγ of Ser177 due to improved orientation. Additionally, the A305T mutation played a role in the formation of a suitable tunnel, aiding in substrate entry and product release, ultimately contributing to enhanced catalytic efficiency. The use of the G175A/A305T double mutant as a biocatalyst led to the production of a maximum of 1,220 mM 2-chloronicotinic acid with a 94% conversion rate, achieving a remarkable space-time yield of 575 gproduct liter-1 day-1. These findings not only present a novel robust biocatalyst for 2-chloronicotinic acid production but also offer valuable insights into the structure-function relationships of amidase. In light of the increasing demand for 2-chloronicotinic acid and the limitations of current chemical synthesis methods, the engineered Pa-Ami holds promise as an efficient and environmentally friendly route for the biosynthesis of 2-chloronicotinic acid from 2-chloronicotinamide. This study highlights the potential of structure-guided saturation mutagenesis in enhancing enzymatic activity and paves the way for sustainable production of important chemical intermediates. ²

Reference

1. Dai AD, Wu ZM, Zheng RC, Zheng YG. Constitutive expression of nitrilase from Rhodococcus zopfii for efficient biosynthesis of 2-chloronicotinic acid. 3 Biotech. 2022; 12(2): 50.

2. Tang XL, Jin JQ, Wu ZM, Jin LQ, Zheng RC, Zheng YG. Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis. Appl Environ Microbiol. 2019; 85(5): e02471-18.