N-Hydroxysuccinimide CAS 6066-82-6

N-Hydroxysuccinimide (NHS) is a versatile organic compound with the chemical formula C4H5NO3. It exists primarily as a white crystalline solid and is widely recognized for its role as an active ester in biochemical reactions. NHS serves as a key intermediate in the synthesis of activated esters, which are crucial for conjugating biomolecules such as proteins, peptides, and oligonucleotides.

The structure of NHS features a succinimide ring substituted with a hydroxyl group at the nitrogen atom. This unique structure imparts NHS with specific reactivity properties, enabling it to form stable amide bonds with amines under mild conditions. This reactivity makes NHS derivatives highly valuable in bioconjugation techniques, where they are used to link various biomolecules together while preserving their biological activity.

One of the most common applications of NHS is in the preparation of NHS esters. These esters can react with primary amines to form stable amide linkages, a process often employed in labeling experiments with fluorescent dyes, radioisotopes, or other reporter molecules. Furthermore, NHS-activated compounds are utilized in the immobilization of biomolecules onto solid supports, such as beads or slides, for use in assays and affinity chromatography.

The mild reaction conditions required for NHS-mediated conjugation, coupled with its high yield and specificity, make it a preferred choice in biotechnological and pharmaceutical applications. However, it's important to note that NHS compounds should be handled with care due to their potential irritancy and reactivity. In summary, it stands as a cornerstone in the field of biochemical conjugation, facilitating the attachment of various moieties to biomolecules with precision and efficiency.

There are two manufacturing processes for HOSU. One is the aqueous solution method, which dissolves hydroxylamine salt in water, adds inorganic base to make hydroxylamine free, and then adds succinic anhydride to react to produce H0SU. This method is reported in U.S. Patent 5.426.190. In this method, hydroxylamine sulfate with high price is used as raw material to obtain HOSU product through three steps of alkalization, reaction and separation. The process is complex and the reported yield is less than 72%.

• Suspend hydroxylamine hydrochloride in ether organic solvent, control the temperature range within - 10~30 ° C, drop methanol solution of inorganic base with the concentration within 5~30% under the protection of nitrogen, then filter the NaCl solid salt produced under the protection of nitrogen, add succinic anhydride and composite acid catalyst to the filtrate at room temperature, and conduct temperature rise reaction. During the temperature rise reaction, methanol is first evaporated, and then rises to 105~130 ° C, The reaction time shall be controlled within the range of I-20hr, and the ether organic solvent shall be evaporated under reduced pressure to obtain the HOSU crude product; The weight ratio of hydroxylamine hydrochloride and ether organic solvent is 1:5-1:20; The ether organic solvent is one of dioxane, butyl ether, diethylene glycol methyl ether or tetrahydrofuran; Either NaOH or KOH is selected as the inorganic base, and the molar ratio of the inorganic base and hydroxylamine hydrochloride is 1:1; The molar ratio of succinic anhydride to hydroxylamine hydrochloride is 0.6-1.2: 1.0; The composite acid catalyst is at least one of sulfuric acid, phosphoric acid, acetic acid and propionic acid, and the added amount is I-10% of the weight of succinic anhydride;
• Add acetic acid acetic acid into the crude HOSU obtained in step a, heat it up and return it to the crude HOSU for dissolution, then filter it while hot to remove a small amount of solid insoluble matter, cool it to 15 ° C~10 ° C, and product crystallizes out, and filter it to obtain the required HOSU product; The ratio of the added amount of ethyl acetate to the weight of the HOSU crude product is I-10: I.

Another method is to use organic solvent system to produce H0SU. This method is reported in Fudan University patent CN2008.1020.1278. In this patent, pyridine, triethylamine and other organic bases are used to neutralize (lower price) hydroxylamine hydrochloride after being added to succinic anhydride for reaction. It is difficult to separate the generated organic base hydrochloride from the product HOSU, which requires a large amount of ethyl acetate for multiple extraction (90 times of the product amount), so it is not suitable for industrial production.

N-Hydroxysuccinimide, commonly known as NHS, holds a significant position in the realm of peptide chemistry and organic synthesis. Its research history dates back more than half a century, with pivotal contributions from the Anderson group, who first proposed its use for preparing active esters in the 1960s.

NHS is an intermediate widely employed in the preparation of various activated esters of acylamino acids. These esters are crucial for promoting coupling reactions with nucleophiles, particularly in the conjugation of fluorophores to amine residues of proteins. The favorable reactivity, relative stability towards hydrolysis, good crystallizability, and low toxicity of NHS esters have made them indispensable in biochemical applications.

The synthesis and application of NHS have undergone extensive research over the years. The formation of NHS esters involves the reaction of carboxylic acid groups with NHS, often facilitated by dicyclohexylcarbodiimide. This reaction is frequently utilized in preparing conjugates for contaminant immunoassays, owing to the stability of the active ester under acidic conditions, which facilitates purification and storage.

NHS esters have found diverse applications, ranging from conjugating proteins to peptides and labeling them with fluorescent tags, to serving as key reagents in the synthesis of peptides, antibiotics, amino acids, and proteins. Their ability to react with amines under mild alkaline pH conditions without carbodiimide activation has further expanded their utility.

In summary, the research history of N-Hydroxysuccinimide showcases a journey from its initial proposal as an active ester precursor to its establishment as a versatile reagent in peptide chemistry, biochemical conjugations, and beyond. With continuous advancements in its synthesis and application, NHS continues to be a cornerstone in the field of organic and biochemical research.

N-Hydroxysuccinimide (NHS) has cemented its position as a versatile and indispensable reagent in chemistry and biotechnology. Its ability to form stable amide bonds through reaction with primary amines has enabled breakthroughs in bioconjugation, peptide synthesis, and material science. From labeling proteins with fluorescent dyes to crosslinking biomaterials for tissue engineering, NHS continues to drive innovation across diverse scientific domains. As research progresses, the development of novel NHS derivatives and hybrid methodologies promises to expand its applications even further, solidifying its role as a cornerstone of modern chemistry.

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