The mechanism of action of o-phthalaldehyde_Kain Industrial Additives

Background and overview^[1]

O-phthalaldehyde is an important pharmaceutical and chemical intermediate. It was previously mainly used in amine alkaloids, fluorometer histamine determination reagents and medical testing. Since it was used for endoscope disinfection in 1994 and was found to have good disinfection effects, many studies have been conducted abroad on the disinfection of o-phthalaldehyde, and it has been developed into a new type of highly efficient disinfectant and passed the U.S. FDA Certification. Compared with the disinfectant glutaraldehyde (GTA), o-phthalaldehyde has the advantages of glutaraldehyde's broad spectrum, high efficiency, and low corrosion. It also has its own characteristics such as low irritation and low concentration. In addition, o-phthalaldehyde also has a good killing effect on glutaraldehyde-resistant mycobacteria isolated in recent years. Therefore, o-phthalaldehyde has the potential to be a substitute for glutaraldehyde.

Biological activity

O-phthalaldehyde belongs to the dialdehyde compound. Like glutaraldehyde, it mainly cross-links through the aldehyde group with the amino acid groups of amino acids, proteins and some other components of microorganisms. Many experiments have shown that the cross-linking effect of o-phthalaldehyde is weaker than that of glutaraldehyde. This may be because o-phthalaldehyde can only react with the primary amino group (NH2), so it can only react with lysine, arginine and the amino acids at the end of the peptide chain. Some studies have proposed a hypothesis to explain the reason for the weak cross-linking effect of o-phthalaldehyde: First, the addition reaction of aromatic aldehydes (such as o-phthalaldehyde) is weaker than that of aliphatic aldehydes (such as glutaraldehyde). Second, the structure of o-phthalaldehyde contains a benzene ring. This knot makes the spatial structure of o-phthalaldehyde lack of stretchability, so it is difficult to fully react with amino-containing substances by adjusting the spatial conformation. On the contrary, glutaraldehyde has greater stretchability and is easy to react with the amino groups in the substance.

Stability and corrosiveness

O-phthalaldehyde has good stability in the pH range of 3-9, and its solution can be used continuously for 14 days. The American company iCdxe has developed test strips for accurately monitoring concentration. Some data show that if an automatic endoscope cleaning program is used, it can be used repeatedly 82 times before reaching the minimum effective concentration, while glutaraldehyde can only be used 40 times. Phthalaldehyde is not harmful to many metals, plastics and other substances.

Mechanism of action

1) Mechanism of action on bacterial propagules

EDTA, sodium laurate and staphylococcal lysins (lysostaphin) are three substances that can damage the cell wall or cell membrane structure, resulting in increased cell permeability and leakage of contents. EDTA, sodium laurate and staphylococcal lysin were used to induce leakage of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus after the action of o-phthalaldehyde, respectively. It was found that the bacteria after the action of o-phthalaldehyde The degree of cell leakage was much lower than that in the control group. This shows that o-phthalaldehyde forms a strong cross-linked bond with the cell walls or membranes of these bacteria, forming a barrier that prevents these substances from damaging the cell wall/membrane structure. However, the sealing and anti-leakage effect of o-phthalaldehyde also causes dysfunction in the exchange of substances inside and outside the bacteria, so that the normal physiological functions of the bacteria cannot be carried out, thereby promoting cell death. Ethambutol, D-cycloserine and fluorescent phenylalanine respectively act on Mycobacterium tuberculosis cells to increase their permeability, affect different stages of glycolipid C synthesis and inhibit its synthesis. The above three substances were added to the culture medium to cultivate the Mycobacterium tuberculosis under study, and then the bacteria were exposed to a certain concentration of o-phthalaldehyde solution. It was found that the Mycobacterium tuberculosis group in the culture treatment group became very sensitive to o-phthalaldehyde. This may be due to the permeability of the cell wall and the destruction of the glycolipid C and carboglycan structures in the cell wall, making it easier for o-phthalaldehyde to act on target sites in Mycobacterium tuberculosis cells. Analytical studies have shown that the molecular basis of the cross-linking reaction of o-phthalaldehyde is weaker than that of glutaraldehyde, and the resistance of bacteria to permeation inducers after the action of o-phthalaldehyde is also lower than that of glutaraldehyde. However, o-phthalaldehyde has a stronger killing effect on Mycobacterium tuberculosis and Gram-negative bacteria than glutaraldehyde. This may be because o-phthalaldehyde is an aromatic aldehyde with good lipid solubility and can more easily penetrate the cell membranes of Mycobacterium tuberculosis and Gram-negative bacteria with more lipids, thus acting on the target site inside the bacteria to trigger cells. of rapid death.

2) Mechanism of action against bacterial spores

The outer layer of the spore is an important barrier to its resistance to phthalaldehyde. The outer layer of Bacillus subtilis spores was removed using different methods and then exposed to o-phthalaldehyde solution. It was found that the removed outer layer of spores became very sensitive to the killing effect of o-phthalaldehyde. Bacillus subtilis spores containing cotE (the cotE gene will cause serious defects in the outer layer of the bud) were used to perform sporicidal experiments on o-phthalaldehyde and found that they were more sensitive to o-phthalaldehyde than Bacillus subtilis spores without this gene. sensitive. However, when comparing these outer spores or cotE-containing spores with Bacillus subtilis propagules, Bacillus subtilis propagules are more sensitive to o-phthalaldehyde. It can be seen that whether the outer spores are removed or the cotE-containing spores are removed, the integrity of the outer layer is not completely destroyed, and the remaining remaining parts may still be highly resistant to o-phthalaldehyde. The saturated outer layer of the spores is a structure formed in the later stages of spore maturation and contains a large amount of disulfide-diclosolic acid (DPA), which enhances the spores' resistance to the outside world. The study found that spores treated with o-phthalaldehyde did not release DPA. This may be because the spores' sensors for nutrients in the external environment were affected by the effects of the o-phthalaldehyde.The destruction of diformaldehyde prevents cells from receiving signals for nutrient uptake, thus weakening the release of DPA. Spores treated with o-phthalaldehyde will not germinate.

Toxicity

Although the concentration of o-phthalaldehyde is reduced compared with glutaraldehyde, it does not have a pungent smell. However, direct contact with o-phthalaldehyde can still cause damage to the eyes, skin, digestive tract, and respiratory mucosa. Disinfectant liquid should avoid direct contact with eyes, skin, and clothing. Direct contact with skin may cause temporary skin coloring, and repeated contact may cause skin allergies. Avoid contaminating food. Drinking or swallowing disinfectant by mistake can cause irritation and burns to the mouth, throat, esophagus, stomach and other digestive tracts. Avoid exposure to phthalaldehyde vapors, as they can irritate the respiratory tract and eyes, cause tingling of the nose, throat, and bronchial tubes, cough, and cause chest discomfort, tension, difficulty breathing, and headaches. There are certain residues of medical equipment after disinfection with o-phthalaldehyde, which must be carefully cleaned, otherwise it will cause discoloration of the patient's skin and mucous membranes. At the same time, the content of o-phthalaldehyde in hospital sewage should be limited, which can be neutralized with glycine to make drainage safer.

Applied Research

As a new high-efficiency disinfectant, although o-phthalaldehyde is more expensive than glutaraldehyde, it has been used in many countries because of its many advantages. At present, its main application value is as an endoscope disinfectant. Clinical studies have shown that after 100 endoscopes are disinfected with 500 mg/L o-phthalaldehyde for 5 minutes, the bacterial load is reduced by more than 5109, and the aqueous solution soaked for more than 30 minutes reduces the Gram-positive bacteria on the bronchoscope, gastroscope, and colonoscope after use by the patient. , negative bacteria, fungi and enterovirus all turned negative. However, to use phthalaldehyde for sterilization, 400-500mg/L of phthalaldehyde is required at 20°C. Each country has its own regulations. In many countries in Europe, Asia, and Latin America, high-efficiency disinfection can be achieved in 5 minutes; in Canada and Australia, it is 10 meters; in the United States, it is 12 minutes.

Preparation^[2]

The green preparation process of o-phthalaldehyde includes the following preparation steps:

1) Add 0.1 to 0.3 mol of 1,3-dihydroisobenzofuran, 0.2 to 0.6 mol of oxidant, 0.3 to 0.5 g of catalyst heteropolyacid, and 10 to 30 g of water in a three-necked flask;

2) Add an electromagnetic stirrer to the above three-necked flask, install a reflux condenser and a temperature probe, and perform an ultrasonic stirring reaction; set the reaction temperature to 30 to 60°C, the ultrasonic power to 200 to 500W, and the reaction running time 30min～180min;

3) After the reaction is completed, lower to room temperature, use an equal volume of ethyl acetate to extract three times, combine the extracts, and remove the ethyl acetate by distillation to obtain crude o-phthalaldehyde;

4) Mix the crude o-phthalaldehyde and the recrystallization solvent petroleum ether at a mass ratio of 1:3 and then recrystallize. Filter to obtain o-phthalaldehyde crystals. Place the o-phthalaldehyde crystals in a vacuum with a degree of Dry in a vacuum drying oven at 60Pa and 50℃ for 4 hours to obtain o-phthalaldehyde product;

5) Use a rotary evaporator to recover the petroleum ether obtained in step 4. The recovered petroleum ether can be reused.

Main reference materials

[1] Research progress on o-phthalaldehyde disinfectants

[2] CN201310242491.6 Green preparation process of o-phthalaldehyde