3-Indolyl-β-D-glucopyranoside (CAS: 487-60-5)
1、 Overview
3-Indolyl-β-D-glucopyranoside is a glycoside of indoxyl and a precursor of the dyes indigo and indirubin. The main metabolite of indican is indoxyl sulfate (IS). IS is a uremic toxin, and it acts as a substrate/inhibitor for organic anion transporter 1 (OAT1), OAT3, and multidrug resistance-associated protein 4 (MRP4).
2、 Basic Product Information
- Product Name: 3-Indoxyl-beta-D-glucopyranoside
- Synonym: 3-Indoxyl-β-D-glucopyranoside; Y(TM)-BETA-D-GLC; Y(TM)-BETA-D-GLUCOSIDE; 1H-Indol-3-yl β-D-glucopyranoside; Indoxyl β-D-glucopyranoside; 3-INDOXYL-β-D-GLUCOPYRANOSIDE trihydrate
- CAS NO.: 487-60-5
- Molecular formula: C₁₄H₁₇NO₆
- Molecular weight: 295.29
- Structural formula:
- Purity: ≥98%
- Appearance: White or off-white powder
- Related categories: Chemical intermediate
- Transport Information: Non-dangerous goods
- HS Code: 2938909090
3、3-Indolyl-β-D-glucopyranoside (CAS: 487-60-5) — Q&A
FAQ 1: What is the maximum absorption wavelength (λmax) of this substrate, and what should be noted for spectrophotometric detection?
Answer: The λmax of this substrate is approximately 280 nm (in DMSO), which can be used for UV detection and purity analysis. However, the chromogenic product used for enzyme activity detection is an indigo precipitate, and its detection wavelength differs from that of the substrate itself. If the indigo product is quantified by spectrophotometry, the precipitate must first be dissolved in an organic solvent such as DMSO, and the reference wavelength is approximately 615 nm. If the insoluble precipitate is directly detected by fluorescence, the excitation wavelength is approximately 365 nm and the emission wavelength is approximately 478 nm.
FAQ 2: When using this substrate for enzyme activity assays, how should the buffer pH be selected?
Answer: The optimal pH of β-glucosidase varies widely depending on the enzyme source. For most β-glucosidases, the optimal pH is between 5.0 and 7.0. For example, the β-glucosidase from Agrobacterium tumefaciens has an optimal pH of 7.0; some fungal β-glucosidases have an acidic pH optimum of approximately 4.0–6.0; and a cold-active β-glucosidase from Paenibacillus is active over a broad range (pH 5.5–10.9). In addition, indigo color development is faster at alkaline pH than at acidic pH. Therefore, the buffer pH should primarily match the optimal pH of the target enzyme while also considering color development efficiency. Commonly used buffer systems are phosphate buffer or citrate-phosphate buffer.
FAQ 3: What are the advantages and disadvantages of this substrate compared with MUG (4-methylumbelliferyl-β-D-glucopyranoside) and halogenated indolyl glycosides (e.g., X-Gluc)?
Answer: The core differences among these three types of substrates lie in signal type and product diffusibility:
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This substrate (a non-halogenated indolyl glycoside): Produces an insoluble blue indigo precipitate; the signal does not diffuse, making it suitable for in situ colony color development and tissue localization. Quantification requires dissolving the precipitate before measurement, which is slightly more complicated.
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MUG: Produces a soluble fluorescent product, 4-methylumbelliferone, with the highest sensitivity, making it suitable for high-throughput quantification and liquid sample detection. However, the product diffuses and is not suitable for in situ colony localization.
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Halogenated indolyl glycosides (e.g., X-Gluc): Also produce insoluble precipitates, and the signal does not diffuse. However, halogen substituents alter the oxidative dimerization rate, and color development is usually slower than with non-halogenated substrates. Non-halogenated substrates, with a simpler structure, form indigo more rapidly.
Summary: Choose indolyl substrates for localization or colony color development; choose MUG for highest-sensitivity quantification; and prioritize non-halogenated indolyl glycosides for rapid color development.
FAQ 4: What special significance does this compound have in plant biology?
Answer: This compound (i.e., indican, plant indican) is a natural precursor for indigo biosynthesis. In indigo-producing plants (such as Polygonum tinctorium and Indigofera tinctoria), indole is oxidized by cytochrome P450 to indoxyl, which is then glycosylated by UDP-glucosyltransferase to form colorless, soluble indican stored in the leaves. When plant tissues are damaged, endogenous β-glucosidase hydrolyzes indican to release indoxyl, which spontaneously oxidizes and dimerizes to form blue indigo. This is also the biochemical basis of traditional indigo dye. In addition, this compound has been used in tobacco chloroplast metabolic engineering research to explore the feasibility of photosynthesis-driven P450 activity for indican production.
FAQ 5: Can this substrate be used for quantitative detection of β-glucosidase activity? If so, what quantitative methods are available?
Answer: Yes. Quantitative detection mainly includes the following approaches:
(1) Spectrophotometry: After the enzymatic reaction, the indigo precipitate is dissolved in DMSO, and absorbance is measured around 615 nm. The amount of indigo is quantified via a standard curve, indirectly reflecting β-glucosidase activity.
(2) Fluorescence method: The fluorescence signal of the indoxyl oxidation product is used for detection, with excitation/emission wavelengths of approximately 365/478 nm. A gold nanocluster-based ratiometric fluorescence sensing system has been used to detect the reaction system of this substrate with β-glucosidase, with sensitivity higher than that of colorimetric methods.
(3) Smartphone-based colorimetric method: Color images are captured with a smartphone camera and subjected to chromaticity analysis. The detection ranges are approximately 0.01–1.00 mM (fluorescence) and 0.25–5.00 mM (colorimetric), making it suitable for rapid on-site screening.
(4) HPLC-ELSD method: This method can directly quantify the degree of substrate hydrolysis and has been used for the quantification of indican in Polygonum tinctorium and for evaluating β-glucosidase hydrolysis efficiency.
4.Application Q&A
Answer: Yes. When added to soil thin sections, soil gel media or in-situ incubation systems, microorganisms producing β-glucosidase hydrolyze the substrate to release indoxyl. After oxidative dimerization, blue indigo precipitates form at the location of colonies/cells, enabling spatial visualization of microbial enzyme activity in soil. This method can be applied to research on soil carbon cycles and rhizosphere microbial ecology. Note that indigo precipitates mainly reflect the spatial distribution of substrate hydrolysis activity; the signal alone cannot distinguish whether it originates from intracellular enzymes of viable cells, extracellular enzymes, or free β-glucosidase in soil. To make this distinction, viable cell staining, fluorescence in-situ hybridization (FISH) or other complementary methods should be combined. In addition, soil organic matter may cause background interference, and heavy metal ions (such as Cu²⁺) can inhibit β-glucosidase activity, leading to weakened signals or false-negative results. Blank controls are required in experiments.
Answer: This substrate can qualitatively or semi-quantitatively detect the β-glucosidase secretion capacity of gut microbiota. β-glucosidase produced by gut microbes participates in the metabolic activation of plant glycoside prodrugs, dietary polyphenols and other substances. With this substrate, β-glucosidase-producing gut strains can be screened on anaerobic plates, and colony color development can directly evaluate the strains’ metabolic potential towards glycosides. It should be noted that the oxidative dimerization of indoxyl to indigo is strictly oxygen-dependent and no color will develop under anaerobic conditions. Therefore, after anaerobic incubation, the plate or sample must be briefly exposed to aerobic conditions to allow indoxyl oxidative dimerization to form blue indigo precipitate. The aerobic exposure time should be kept as short as possible to minimize impacts on obligate anaerobes.
Answer: Yes. In agar plate systems containing the substrate, if a plant extract has β-glucosidase inhibitory activity, the substrate in the inhibited area will not be hydrolyzed and no blue precipitate will form, generating a colorless inhibition zone for visual primary screening. Compared with traditional spectrophotometric screening, this method can directly show the location of inhibitory activity. However, the diffusion property of inhibitors will affect screening sensitivity; poorly diffused inhibitors may only form a colorless region immediately adjacent to colonies. Some plant extracts are inherently colored or have strong redox properties, which may interfere with indigo oxidation and color development. Control groups must be set up to rule out false-positive or false-negative results.
Answer: Yes. Microorganisms embedded inside biofilms that express β-glucosidase can hydrolyze the substrate to produce blue indigo precipitate, enabling in-situ color development directly on biofilms. The spatial distribution of enzyme activity within biofilms can be observed under a microscope. This method has the potential to compare the difference in β-glucosidase expression between planktonic bacteria and biofilm-forming bacteria, though direct supporting literature for this application remains limited. Appropriate control experiments and quantitative methods such as confocal microscopy z-stack imaging are required for validation. Limitations include small particle size of indigo precipitates, which requires optimized microscope parameters for high-resolution imaging. Thick biofilms restrict oxygen diffusion and slow down indoxyl oxidative dimerization, thus delaying color development. Extracellular DNA, extracellular polymeric substances and other components may also affect the distribution of indigo precipitates.
Answer: This substrate can be used for detection and monitoring of β-glucosidase-positive bacteria in food processing environments and food samples. For example, to evaluate the influence of food preservation processes and heat treatment on the activity of target β-glucosidase-producing strains. It can also be used for rapid screening of biological contamination on packaging material surfaces, and residual viable bacteria can be preliminarily identified via plate color development. Note that this substrate serves only as a microbial detection tool in food microbiology rather than a complete quality control solution. Proteins, polysaccharides, pigments and other components in food matrices will cause background interference. Samples usually need dilution, and selective inhibitors should be used to suppress the growth of miscellaneous bacteria and ensure detection specificity.
5、Packaging, Storage and Transportation Requirements
Plastic or Glass Bottle
This product is sensitive to light and susceptible to hydrolysis. It must be kept tightly sealed and protected from light. Long-term storage shall be maintained at -15°C or below in a dry environment. Avoid exposure to strong light, high temperature and acidic or alkaline conditions, which may trigger glycosidic bond hydrolysis and reduce product purity. Store separately from strong acids, strong alkalis and oxidizing substances.
Ensure packaging remains fully sealed and intact during transit to prevent breakage and light ingress. Low-temperature transportation with ice packs is required throughout the shipment; protect from light and moisture, avoid high temperature, direct sunlight and heavy vibration. Do not ship together with strong acids, strong alkalis, oxidants and corrosive materials. Handle carefully during loading and unloading to prevent package damage and product degradation. Maintain low-temperature and shaded conditions in the whole transportation process to guarantee product stability.
6、 Product Manager Information
Contact information:
Contact: Sophia Li
Email: sophia@coreychem.com
