L-alanyl-L-glutamine (Ala-Gln) is a white crystalline powder, odorless and tasteless, with the molecular formula of C8H15N3O4, relative molecular mass of 217.22, and the structure as shown in Fig. 1.The solubility of Ala-Gln is up to 586 g/L (20 ℃), which is 20-fold higher than that of free L-Gln, and it is slightly soluble in glacial acetic acid and almost insoluble in methanol. It was slightly soluble in glacial acetic acid and almost insoluble in methanol. Free L-Gln is easy to decompose and generate toxic pyroglutamic acid and ammonia under acidic or high-temperature conditions, while Ala-Gln has good thermal stability and does not decompose at 120°C. Ala-Gln can decompose and generate free L-Gln rapidly in the body, and increase the content of free L-Gln in the body without side effects. At present, it is believed that Ala-Gln is the best substitute for L-Gln, which can overcome the shortcomings of L-Gln such as not easy to store, poor water solubility, and easy to produce toxic substances at high temperature, etc. As a novel nutritional supplement, Ala-Gln has been applied in clinical treatment.

1.Application of Ala-Gln

Ala-Gln can be rapidly decomposed in the body to produce L-Gln, which can regulate the L-Gln content in organisms, maintain cellular homeostasis, improve the activity of intestinal cells, and participate in the synthesis of proteins and lipids, etc. Moreover, the latest study found that there is a close relationship between L-Gln deficiency and the susceptibility of COVID-19. In addition, Ala-Gln can be mixed with other amino acids or substances with active ingredients, such as fish oil, propolis and collagen, to synthesize nutraceuticals with anti-fatigue and anti-stress functions. With the deepening of Ala-Gln related research, its applications in medical and agricultural fields have been gradually explored.

Ala-Gln has great potential in medical treatment. In intestinal diseases, L-Gln is the main source of energy for rapidly dividing cells in the intestine, such as intestinal epithelial cells, macrophages and lymphocytes. Supplementation of Ala-Gln can increase the content of L-Gln in the relevant cells, increase the speed of cell division, maintain the integrity of the intestinal tract, activate the immune system, improve the cellular structure of the gastrointestinal mucosa, and maintain the normal function and structure of the mucosa of the small intestine. Some studies have reported that Ala-Gln supplementation in mice can significantly alleviate the symptoms of colitis in mice more than L-Gln supplementation, reduce the concentration of interleukin (1L)-1β, 1L-6, etc., significantly increase the concentration of immunoglobulin and superoxide dismutase, enhance the intestinal barrier effect, and regulate the intestinal flora and metabolites of the flora. Researchers found that patients suffering from severe diarrhea disease or sepsis can reduce the symptoms of severe diarrhea or sepsis by injecting Ala-Gln, and greatly reduce the recurrence rate and mortality. Supplementation with Ala-Gln reduces endothelial cell damage and restores disordered essential proteins, thereby reducing peritoneal dialysis-related vascular lesions, restoring intestinal integrity, ameliorating inflammation, and decreasing relapse rates. In corneal diseases, Jin et al. found that Ala-Gln effectively maintained the corneal endothelial pump function and barrier function by protecting the mitochondrial function and preventing cytoskeletal rearrangement in mice with acute high intraocular pressure (IOP), which provided a new method for the treatment of corneal endothelial diseases, and enlarged the potential clinical application scope of Ala-Gln. Elsewhere, Ala-Gln supplementation increased L-Gln levels in plasma, liver, and skeletal muscle, while decreasing cellular redox status, enhancing hepatic oxidative stress, improving liver injury and lipid metabolism, and thus improving glucose homeostasis in obese and diabetic mice. For traumatic coagulopathy, Ala-Gln supplementation can significantly reduce its incidence and improve patients' postoperative survival. Some reports have shown that Ala-Gln has a protective effect against liver ischemia/reperfusion injury by improving microcirculation, reducing inflammation and increasing glutathione (GSH) levels in the liver. The injection of Ala-Gln can safely and effectively reduce or prevent the formation of postoperative cellular adhesions and provide a new direction for the treatment of related diseases. In the field of agriculture, adding Ala-Gln to fish feed can treat gastrointestinal inflammation and injury caused by feeding soybean meal, increase the length of intestinal villi and the expression of genes related to nutrient absorption, increase the surface area of the villi, so that the intestinal tract is more likely to absorb nutrients, and increase the activity of lipase and trypsin in the liver of the fish, so as to improve the quality of the fish meat and the economic efficiency. Xu et al. found that the addition of Ala-Gln in feed could significantly promote the growth performance and antioxidant capacity of juvenile fish in cold fresh water, thus accelerating the growth rate of fish, reducing the feeding cost, and preventing the metabolic disorders and death of fish in low-temperature environments.The application of Ala-Gln has been carried out in many fields, and the high efficiency of its preparation has attracted much attention. Currently, the methods of Ala-Gln preparation are mainly divided into chemical synthesis and biosynthesis.

2.Chemical synthesis of Ala-Gln

The chemical synthesis of Ala-Gln has realized the large-scale production of Ala-Gln at a relatively early stage, and before 2004, most of Ala-Gln was produced by chemical synthesis, which was accomplished by using L-Ala and L-Glu as raw materials through different synthesizing steps, and was mainly classified into the following types.

2.1 Protective group method

Protective group method can be divided into benzyloxycarbonyl (Cbz) protective group method and tert-butoxycarbonyl (Boc) protective group method.

2.1.1 Cbz protecting group method

Cbz protects the amino group of L-Ala, p-nitrophenol ester activates the carboxyl group of L-Ala, the amide group of L-Gln is protected by anthracene (-xanthyl) and the carboxyl group is protected by methyl ester. After the reaction of the protected L-Gln with L-Ala was complete, the ester group was removed by saponification reaction, and the -xanthyl group and Cbz were removed by HBr gas to produce Ala-Gln. Shimonishi et al. synthesized dipeptides by dicyclohexylcarbodiimide (DCC) condensation reaction using methyl glutamate and Cbz-protected Ala (Cbz-Ala) as substrates, and the amide group was removed by HBr gas. The Cbz-protecting group of the amino group was removed by HBr to produce alanine glutamic acid y-carboxymethyl ester, which could be converted to Ala-Gln after amination with concentrated ammonia.Wang Wanfu found that the active ester of Cbz-protected L-Ala was condensed with L-Gln to produce Cbz-Ala-Gln under the condition of DCC, and the Cbz-protecting group was removed by hydrogenation to obtain Ala- Gln. Gln .

References

[1]

Stehle P, Zander J, Mertes N, et al. Effect of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance after major surgery . Lancet, 1989, 1(8632): 231-233.

[2]

Stehle P, Pfaendler P, Fürst P. Isotachophoretic analysis of a synthetic dipeptide l-alanyl-l-glutamine. Journal of Chromatography A, 1984, 294: 507- 512.

[3]

Ferrantelli E, Liappas G, Vila Cuenca M, et al. The dipeptide alanyl-glutamine ameliorates peritoneal fibrosis and attenuates IL-17 dependent pathways during peritoneal dialysis. Kidney International, 2016, 89(3): 625-635.

[4]

El Hamamsy M, Bondok R, Shaheen S, et al. Safety and efficacy of adding intravenous N-acetylcysteine to parenteral L-alanyl-L-glutamine in hospitalized patients undergoing surgery of the colon: a randomized controlled trial. Annals of Saudi Medicine, 2019, 39(4): 251-257.