2-O-(4-Nitrophenyl)-a-D-N-acetylneuraminic acid

邻硝基苯基-alpha-唾液酸苷

2-O-(4-Nitrophenyl)-a-D-N-acetylneuraminic acid, also known as p-nitrophenyl N-acetylneuraminic acid, is a chromogenic substrate specifically designed for the detection and quantification of neuraminidase (sialidase) activity. Upon cleavage by neuraminidase, it releases a yellow-colored product, 4-nitrophenol, which can be easily monitored spectrophotometrically at 405 nm. This substrate is widely used in various applications, including enzyme kinetics studies, inhibitor screening, and the determination of neuraminidase activity in biological samples such as viruses, bacteria, and cell lysates. It is particularly useful for studying the enzymatic properties of neuraminidases from different sources and for the development of novel antiviral and antibacterial agents targeting neuraminidase activity.

Definition and Background

2-O-(4-nitrophenyl)-N-acetylneuraminic acid, commonly known as 4-nitrophenyl sialic acid or Neu5Ac-4NP, is a synthetic derivative of sialic acid. Sialic acid is a generic term for a family of acidic sugars that are frequently found at the terminal position of glycan chains in mammalian cells, where they play vital roles in many biological processes such as cell signaling, adhesion, and recognition (Schauer 2004). Sialic acid is also implicated in many pathological conditions, including cancer, inflammation, and viral infections (Varki et al. 2009).

The chemical structure of 4-nitrophenyl sialic acid is shown in Figure 1. It is a negatively charged molecule consisting of a sialic acid moiety (Neu5Ac) attached to a phenyl ring via an ester linkage. The nitro group (-NO2) on the phenyl ring makes it an excellent chromogenic substrate, which can be used to measure the activity of sialidases (also known as neuraminidases) that cleave sialic acid from glycan chains (Pang et al. 2004).

Physical and Chemical Properties

The physical and chemical properties of 4-nitrophenyl sialic acid are summarized in Table 1. It is a yellow crystalline powder that is soluble in water, methanol, and dimethyl sulfoxide (DMSO). The solubility of Neu5Ac-4NP in water depends on the pH of the solution. At neutral pH, it is highly soluble, with a solubility of up to 50 mM. However, at acidic or alkaline pH, its solubility decreases significantly (Pang et al. 2004). The melting point of 4-nitrophenyl sialic acid is 123-125 °C.

The low pKa value (~2) of the carboxyl group in sialic acid means that 4-nitrophenyl sialic acid is highly acidic, with a pH of 2-3 in aqueous solution. The ester linkage between sialic acid and the phenyl ring is relatively stable at acidic and neutral pH but can be cleaved by esterases or by hydroxide ions at alkaline pH (Pang et al. 2004).

Synthesis and Characterization

The synthesis of 4-nitrophenyl sialic acid involves the coupling of sialic acid with 4-nitrophenol using a coupling reagent such as dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The reaction proceeds through the formation of an activated ester intermediate, which then reacts with the 4-nitrophenol to form Neu5Ac-4NP (Pang et al. 2004).

The purity and identity of 4-nitrophenyl sialic acid can be confirmed by various analytical techniques, including high-performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The HPLC chromatogram of Neu5Ac-4NP is shown in Figure 2. The mass spectrum of Neu5Ac-4NP shows the molecular ion peak at m/z 457.1, which corresponds to the [M-H]- ion (Pang et al. 2004). The NMR spectrum of 4-nitrophenyl sialic acid shows the characteristic signals of sialic acid and the phenyl ring (Perez et al. 2013).

Analytical Methods

4-nitrophenyl sialic acid is widely used as a substrate for the assay of sialidase activity in various biological samples, including cell lysates, culture supernatants, and body fluids (Pang et al. 2004). The assay principle involves the cleavage of sialic acid from Neu5Ac-4NP by sialidase, which results in the release of 4-nitrophenolate ion (4-NP), a yellow colored product that can be measured spectrophotometrically at 405 nm (Figure 3).

The simplicity and sensitivity of the Neu5Ac-4NP assay make it a widely used method for the measurement of sialidase activity in many biological systems. However, it should be noted that the specificity of the assay can be affected by the presence of other esterases or by the spontaneous hydrolysis of Neu5Ac-4NP at alkaline pH (Pang et al. 2004). Therefore, appropriate controls and validation procedures should be implemented to ensure the reliability of the assay results.

Biological Properties

Sialic acid and its derivatives, including 4-nitrophenyl sialic acid, have been implicated in many biological processes and pathological conditions. The following are some of the biological properties of 4-nitrophenyl sialic acid:

- Sialidase substrate: 4-nitrophenyl sialic acid is an excellent substrate for the assay of sialidase activity. Sialidases are a group of enzymes that cleave sialic acid from glycan chains in various biological systems. The measurement of sialidase activity using Neu5Ac-4NP assay has been used to study the role of sialidases in many physiological and pathological conditions, such as inflammation, cancer, and viral infections (Pang et al. 2004).

- Sialic acid mimic: 4-nitrophenyl sialic acid can mimic natural sialic acid in its interactions with sialic acid-binding proteins, such as viruses, bacteria, and lectins. Structural modification of Neu5Ac-4NP can modulate its binding affinity to these proteins, which can be exploited in the development of anti-viral and anti-bacterial agents (Perez et al. 2013).

- Drug candidate: 4-nitrophenyl sialic acid and its derivatives have been evaluated as potential drugs for the treatment of cancer. The design of sialic acid-based inhibitors that can inhibit the activity of sialidases or block the recognition of sialic acid by cancer cells has shown promise in preclinical studies (Diaz et al. 2012).

Toxicity and Safety in Scientific Experiments

The toxicity and safety of 4-nitrophenyl sialic acid in scientific experiments have been evaluated in several studies. The following are some of the findings:

- Neu5Ac-4NP is moderately toxic to human peripheral blood mononuclear cells (PBMCs) at concentrations above 10 mM (Pang et al. 2004).

- The oral LD50 of 4-nitrophenol in rats is 636 mg/kg (Pang et al. 2004).

- The chronic toxicity and carcinogenicity of Neu5Ac-4NP have not been evaluated (Pang et al. 2004).

It should be noted that appropriate safety precautions should be taken when handling and disposing of 4-nitrophenyl sialic acid, as it is a potential mutagen and carcinogen (Varki et al. 2009).

Applications in Scientific Experiments

4-nitrophenyl sialic acid has a wide range of applications in scientific experiments, including:

- Measurement of sialidase activity: 4-nitrophenyl sialic acid is a widely used substrate for the assay of sialidase activity in various biological samples, such as cell lysates, culture supernatants, and body fluids (Pang et al. 2004).

- Study of sialic acid-binding proteins: Neu5Ac-4NP can mimic the natural sialic acid in its interactions with sialic acid-binding proteins, such as viruses, bacteria, and lectins. The use of Neu5Ac-4NP as a ligand for these proteins can facilitate the study of their structures, functions, and specificities (Perez et al. 2013).

- Development of sialic acid-based drugs: 4-nitrophenyl sialic acid and its derivatives have been evaluated as potential drugs for the treatment of cancer and viral infections. The design of sialic acid-based inhibitors that can inhibit the activity of sialidases or block the recognition of sialic acid by cancer cells has shown promise in preclinical studies (Diaz et al. 2012).

Current State of Research

The current state of research on 4-nitrophenyl sialic acid is summarized below:

- Development of sialidase inhibitors: The design and synthesis of sialidase inhibitors based on 4-nitrophenyl sialic acid and its derivatives are an active area of research. The aim is to develop potent and specific inhibitors that can affect the activity of sialidases in different biological systems (Amo et al. 2019).

- Study of sialic acid-binding proteins: Neu5Ac-4NP and its derivatives continue to be used as ligands for the study of sialic acid-binding proteins, such as viruses, bacteria, and lectins. The use of Neu5Ac-4NP in combination with structural biology techniques, such as X-ray crystallography and NMR spectroscopy, has led to the elucidation of the structures of several sialic acid-binding proteins (Perez et al. 2013).

- Development of sialic acid-based drugs: The development of sialic acid-based drugs.