What is Biotinylation and Biotinylated PEG?

Biotin, also known as vitamin H or B7, is an essential nutrient for various biological processes in the human body. It plays a vital role in cellular metabolism, particularly in the synthesis of fatty acids, glucose metabolism, and the breakdown of amino acids. Biotin is commonly found in food sources such as eggs, nuts, dairy products, and green leafy vegetables. However, in some cases, supplementation may be necessary to meet the body's needs. Although biotin is mainly recognized for its importance in metabolism and energy production, its application in drug discovery has received increasing attention in recent years. This multifunctional molecule shows promise in different aspects of drug development, from target identification and validation to drug delivery systems and treatment.

What is Biotin?

Biotin, also known as vitamin B7 or vitamin H, is a water-soluble B vitamin that plays a vital role in a variety of important body functions. Biotin was first discovered in the early 20th century as a growth factor for yeast and later in rats. It was originally known as vitamin H before being officially named in 1940. This vitamin is essential for the metabolism of fats, carbohydrates, and proteins in the body. It acts as a coenzyme in several carboxylation reactions, helping to transfer carbon dioxide. Biotin is naturally found in a variety of foods and can also be taken as a dietary supplement.

Fig. 1. Structure of biotin.

Biotin Structure

The molecular weight of biotin is 244.31. There are two ring structures in the molecule, of which ring I is an imidazoline ring, which is the main site for binding to avidin; ring II is a thiazole ring with a valeric acid side chain, and its terminal carboxyl group is the only structure for binding to antibodies and other biological macromolecules. The carboxyl group of biotin can be chemically modified to produce derivatives with various active groups, called activated biotin. At the same time, the chemical properties of these reagents (solubility, arm length, shearability) can be optimized according to special uses to meet the needs of binding to various biological macromolecules.

Biotin Streptavidin

Streptavidin is a protein with four identical binding sites that binds to biotin in a very tight complex and one of the strongest known non-covalent interactions (dissociation constant (Kd) of about 10x^-15 mol/L). The bond between biotin and streptavidin forms very rapidly and once formed, it is not affected by extremes of pH, temperature, organic solvents and other modifiers. The binding between biotin and streptavidin is very specific because of the high affinity between them. This affinity makes the biotin-streptavidin complex a very useful tool for the detection, separation and purification of many biomolecules such as proteins, DNA or RNA. The interaction between biotin and streptavidin is commonly used in labeling and purification techniques in many biological experiments. This interaction is also widely used in the binding of biomolecules (such as proteins, lipids and nucleic acids), synthetic molecules (such as fluorescent labels), biochip technology, immunohistochemistry, Western blot analysis and other molecular biology experiments.

Biotinylation

Biotinylation is the process of binding a chemical substance or molecule to biotin. Biotin is a water-soluble vitamin, also known as vitamin B7 or vitamin H, which plays an important role in the biochemical processes of many organisms. Biotin can be obtained through chemical synthesis or from natural sources such as food. Biotinylation is often used in biological laboratories to label and detect specific molecules or proteins, where biotin is bound to the molecule or protein to be detected, and the target molecule can then be detected or purified through the specific interaction between biotin and other molecules.

Biotinylated PEG

Biotinylated PEG molecules consist of a PEG polymer chain linked to a biotin molecule. The biotin molecule acts as a functional group that can specifically bind to avidin or streptavidin proteins with high affinity. This biotin-avidin interaction is one of the strongest non-covalent interactions known in nature, making biotinylated PEG an important tool for a variety of bioconjugation applications. Researchers can attach biotinylated PEG to surfaces, nanoparticles, antibodies, or other biomolecules to create targeted and stable bioconjugates for imaging, drug delivery, diagnostics, and other biomedical applications. A key advantage of using biotinylated PEG is its ability to enhance the stability and biocompatibility of bioconjugates. The hydrophilicity of PEG helps protect the bioconjugate from nonspecific interactions with proteins, cells, and other biological components. This stealth effect, often referred to as the "PEGylation" effect, can improve the circulation time of bioconjugates in the body, reduce immunogenicity, and enhance their therapeutic efficacy. Based on this, we at BOC Sciences offer a variety of functional group-modified PEGylated biotin products:

Biotinylated Antibody

Biotinylated antibodies are antibodies that have biotin attached to them. Biotin can bind to avidin with high affinity, and the affinity is 10,000 times that of antigen-antibody binding. Moreover, one avidin can bind to four biotins, which plays a role of cascade amplification. Therefore, it can more sensitively display trace antigens. This method is also called affinity immunohistochemistry. At present, antibody protein labeling technology has been widely used in analytical research and technical determination in the fields of medical pathology, immunohistochemistry, molecular biology, biopharmaceuticals, etc. Common antibody labeling techniques include biotinylation labeling, enzyme labeling, fluorescein labeling and colloidal gold labeling. Samples labeled with biotinylated antibodies can be detected by avidin or biotin-avidin complex labeled with enzyme or fluorescent dye. Biotin-SP is a 6-atom spacer located between biotin and its conjugated protein. When biotin-SP conjugated antibodies are used in enzyme immunoassays, the sensitivity is improved compared with biotin-conjugated antibodies without a spacer. This is particularly evident when biotin-SP conjugated antibodies are used together with alkaline phosphatase conjugated chain avidin. Apparently, the long spacer extends the biotin moiety away from the antibody surface, making it more accessible to the binding site of streptavidin.

Biotinylated Protein

The valerate side chain of the biotin molecule can be derivatized to incorporate a variety of reactive groups that are used to link biotin to other molecules. Using these reactive groups, biotin can be easily attached to most proteins and peptides. Biotinylation reagents can be used to target a variety of functional groups, including primary amines, thiols, carbohydrates, and carboxyl groups. There are also options for photosensitive biotin compounds that respond nonspecifically to photoactivation. This functional group-specific variety is useful in selecting biotinylation reagents that do not inactivate the target macromolecule.

Fig. 2. Biotinylated Protein and DNA (ACS Chem. Biol. 2022, 17(12): 3270-3275).

The solubility of biotinylation reagents will largely affect the labeling ability of proteins or other macromolecules. Proteins have hydrophilic and hydrophobic regions depending on the side chains of the amino acids and the protein configuration, and these regions can promote or hinder the biotinylation process depending on the solubility of the biotinylation reagent. Some studies require biotinylated proteins that are highly hydrophilic. For example, when studying the expression of molecules on the surface of cells or the internalization process of tracer surface molecules, it is necessary for the biochemical protein to be hydrophilic to limit the biotinylated-labeled protein from passing through the hydrophobic cell membrane and thus remaining on the surface of the cell. Biotin with PEG chains has higher solubility, which can improve the biotinylation process of proteins and prevent the aggregation of biotinylated proteins.

Biotinylated Nucleotides

Biotin-modified nucleoside triphosphate analogs, such as dUTP and dCTP, can be enzymatically incorporated into DNA or RNA fragments for use in fluorescence in situ hybridization (FISH), DNA arrays, microarrays, and other hybridization techniques. Standard enzymatic non-radioactive DNA labeling reactions, including 3' end labeling, cDNA labeling, nick translation, PCR, and random primer labeling. Each biotinylated nucleotide contains a spacer of 11, 14, 16, or 20 atoms between the biotin and its attachment point on the nucleotide. For example, the common Biotin-dATP or Biotin-dCTP is used to add to the enzyme nick in HiC experiments; biotin-labeled linker (21bp dsDNA) can be used to connect two chromatin DNA fragments in ChIAPET experiments; RNA is labeled with biotin in RNA pull down experiments.

Biotin Sources

The sources of biotin can be natural or synthetic. Natural food sources such as eggs, liver, and nuts are rich in biotin. These sources provide the body with the necessary amounts of biotin to perform basic functions. In addition, as biotin is used in the field of pharmaceutical research, methods for chemically synthesizing biotin have also been developed.

Biosynthesis of Biotin

Both plants and bacteria in nature can synthesize biotin to meet normal physiological functions. In addition, plants and bacteria use the same starting materials and reaction pathways in the synthesis of biotin. That is, L-alanine and pimelic acid are used as starting materials. First, 7-carbonyl-8-aminononanoic acid is formed through a condensation process; then, 7,8-diaminononanoic acid is formed through the transamination process of the carbonyl group; then, dethiobiotin is obtained through further condensation under the action of dethiobiotin synthase (DBS); finally, the final product is obtained under the action of biotin synthase.

Chemical Synthesis of Biotin

As mentioned above, biotin plays an important role in various biochemical reactions such as fatty acid biosynthesis and glycolysis. Biotin deficiency in the human body can cause a variety of nutritional diseases. Not only that, biotin deficiency in poultry can also lead to growth retardation, stunted development and even death, especially with the continuous development of poultry and animal husbandry, the demand for biotin as a feed additive has surged. Therefore, the study of the chemical synthesis of biotin is necessary and urgent. So far, the total synthesis strategies of biotin reported in the literature are basically divided into two types, namely enantioselective synthesis and stereospecific synthesis. The former uses fumaric acid as a raw material, and constructs three chiral carbon centers in the molecule through asymmetric synthesis or other chiral techniques, and undergoes multi-step reactions to finally obtain the target product with sufficient optical purity. The other strategy is to use chiral compounds such as L-cysteine as starting materials, and use the inherent chiral carbon atoms in the raw materials to transform the structure to construct the biotin molecule.

Biotin Benefits

Biotin is a cofactor in the enzyme system related to the carboxylation and decarboxylation reactions in the body's metabolism, and can participate in carboxylation reactions, gluconeogenesis, and protein synthesis. Therefore, biotin is a substance necessary for life, epithelial tissue growth and maintenance, and reproduction. In addition, biotin plays an important role in the metabolism of carbohydrates, fatty acids, proteins, and nucleic acids in the body. In most cases, people pay more attention to its nutritional and health functions. It is generally believed that biotin has a positive effect on maintaining the normal functions of skin, nails, and hair. Lack of biotin leads to thinning and loss of hair, gray hair and hair loss, dermatitis, eczema and other skin symptoms. Nevertheless, biotin has a very wide source (present in a variety of foods including egg yolks, nuts, beans, fish, and fruits), so daily diet can meet the needs without additional supplementation. However, studies have also shown that diabetes, pregnancy, and malnutrition can lead to biotin deficiency, and appropriate exogenous supplementation is needed at this time.

What Does Biotin Do?

Water-soluble vitamin biotin functions as a cofactor for multiple carboxylase enzymes involved in important metabolic processes, especially those involving the production of fatty acids, gluconeogenesis, and amino acid metabolism. The primary role of biotin is to transport reactive carbon dioxide, which is necessary for a number of carboxylation processes that are vital to the body's synthesis of vital macromolecules and energy production. Consequently, a lack of biotin can result in a variety of metabolic diseases and clinical presentations, underscoring the significance of this vitamin for human well-being.

Biotin for Diagnostics

Biotin has proven to be an effective tool for a range of detection and analytical approaches in the field of biomedical diagnostics. Enzyme-linked immunosorbent assays (ELISAs) and fluorescence-based assays are two sensitive detection techniques that can be made possible by conjugating biotin to target molecules like antibodies or nucleic acids. Diagnostic kits frequently employ this biotin-streptavidin conjugation method to find infections, biomarkers, and other analytes in clinical samples. Furthermore, biotin can be applied to imaging research, acting as a flexible tag for molecules that can be observed by methods like magnetic resonance imaging (MRI) or fluorescence microscopy.

Biotin for Therapeutic Applications

Biotin has demonstrated potential as a therapeutic agent for the treatment of a range of disorders, in addition to its diagnostic uses. Treating biotinidase deficiency, a rare genetic condition marked by reduced biotin circulation and usage, is one of the most well-established uses of biotin. To avoid severe metabolic disruptions and neurological problems, patients with this syndrome need to take therapeutic amounts of biotin supplements for the rest of their lives. Furthermore, biotin has become known as a possible supplementary treatment for a number of skin disorders, including alopecia and brittle nail syndrome. Research indicates that administering biotin supplements to those suffering from some forms of alopecia can enhance their nail strength and look as well as encourage hair growth and thickness.

Biotin for Target Identification

The usefulness of biotin in target identification and validation is a significant application in drug research. Researchers can selectively label and capture proteins or other biomolecules engaged in particular biological pathways by conjugating biotin to target molecules. Target proteins can be isolated and characterized using a technique known as biotinylation, which aids in the clarification of their connections and roles. Because biotin-based affinity purification methods, including biotin tags and streptavidin beads, make it possible to quickly and effectively isolate target proteins from complicated biological materials, they have completely changed the area of proteomics. The identification of possible therapeutic targets and the molecular knowledge of disease causes have both benefited greatly from this technique.

Biotin for Drug Delivery

The creation of tailored drug delivery systems is another area in which biotin is used in drug discovery. On the surface of several cell types, such cancer and inflammatory cells, biotin receptors are extensively expressed. Researchers can build tailored delivery vehicles that specifically bind to biotin receptors on target cells, hence improving drug absorption and therapeutic benefits, by conjugating pharmaceuticals or nanoparticles to biotin. Drug delivery methods mediated by biotin provide several benefits, such as enhanced drug solubility, stability, and bioavailability. Furthermore, biotin-conjugated carriers are excellent candidates for precision medicine approaches due to their targeting capabilities.

Reference

1. Seiple, I.B. et al. Biotin as a Reactive Handle to Selectively Label Proteins and DNA with Small Molecules. ACS Chem. Biol. 2022, 17(12): 3270-3275.