Protein-carbohydrate interactions are essential in numerous biological processes. For the study of protein-carbohydrate interactions, Grün et al., in 2006, reported a simple and fast protocol for the biotinylation of carbohydrates (Figure 3). The labeling process utilizes reductive amination. Reductive amination is also part of the Maillard reaction. The Maillard reaction is a non-enzymatic browning reaction involving reduced sugars with compounds containing free amino groups such as amino acids.
The research group demonstrated that biotinylated glycans enable the production of glycan arrays to determine specific interactions of lectins. Furthermore, the study showed that fluorescent beads coated with selected biotinylated glycans bind to dendritic cell-specific ICAM-3-grabbing nonintegrin (DC–SIGN)-expressing dendritic cells in vitro. Finally, using biotinylated high-mannose N-glycans, Grün et al. visualized DC–SIGN-expressing cells in lymph node tissue.
Carbohydrates with the chemical formulas Cn(H2O)n or Cn(H2O)n-1 are commonly known as sugars or saccharides. Simple carbohydrates are monosaccharides. Simple monosaccharides are classified according to the number of their carbon atoms. The larger polysaccharides compromise chemically linked monosaccharides.
Monosaccharides with four or more carbons are usually cyclic molecules. Five-membered monosaccharides are known as furanoses, and six-membered monosaccharides are known as pyranoses. Furanoses and pyranoses are cyclic hemiacetals. The formation of a hemiacetal is illustrated in figure 2.
Since monosaccharides contain chiral carbon atoms, they occur in stereoisomers. Stereoisomers differ in the spatial arrangement of their atoms. The monosaccharide hexose D-glucose primarily exists in the pyranose ring form. Glucose can exist in an open-chain form and a cyclic structure. The two cyclic glucose enantiomers are known as D-glucose and L-glucose. D and L define the configuration of a monosaccharide's highest-numbered chiral carbon atom.
The human body uses the D-enantiomer for energy production. It has n = 4 stereocenters. Therefore there are 2n = 24 = 16 possible enantomers.
Alpha and beta anomers of monosaccharides usually have different specific optical rotations. The two anomers slowly interconvert in an aqueous solution. This interconversion is known as mutarotation (Figure 1).
Mutarotation of glucose in aqueous solution
Freshly prepared solutions of glucose in water gradually change in optical rotary power. The cause is a mutarotation reaction in which the dissolved glucose transforms from one form to another. Figure 1 shows the mutarotation between the α-anomer and the β-anomer of glucose.
Figure 1: Mutarotation of glucose. Mutarotation is characteristic of the cyclic forms of glucose. Aldehydes cannot undergo mutarotations. Mutarotation occurs by opening the pyranose ring to the free aldehyde form. This reaction is a reversal of a hemiacetal formation reaction. A rotation of 180° of the carbon-carbon bond to the carbonyl group allows reclosure of the hemiacetal ring via the reaction of the hydroxy group at the opposite site of the carbonyl carbon. In the glucose molecule, the two pyranose forms interconvert. However, other carbohydrates can undergo more complex mutarotations. For example, D-fructose can mutarotate into pyranose and furanose forms.
Figure 2: Formation of carbohydrate semiacetals containing N-acetyl-galactose moieties.
One-step Biotinylation of Carbohydrates
The hemiacetal form of monosaccharides allows the conjugation of commercially available biotin-hydrazides to the reducing ends of the carbohydrates. Examples are biotinylated glucose, galactose, and mannose.
Figure 3: Biotinylation of carbohydrate reducing ends via a biotin-hydrazide. The hemiacetal formed in aqueous solution reacts with the hydrazide group of a biotin-hydrazide to form a hydrazone linkage. The reductant NaCNBH3 reduces the hydrazone linkage to the stable product.
Bioconjugation [Bioconjugation at Biosynthesis]
Biotin Oligonucleotide Modification [Biotin-oligonucleotide-modification]
Biotinylated Oligonucleotides [Biotinylated-Oligonucleotide-Synthesis-Services]
Biotinylated RNA affinity probes [RNA-network-analysis-using-biotinylated-RNA-affinity-probes]
Classification of carbohydrates [IUPAC] [Nomenclature-of-carbohydrates-the-fundamentals]
Grün CH, van Vliet SJ, Schiphorst WE, Bank CM, Meyer S, van Die I, van Kooyk Y. One-step biotinylation procedure for carbohydrates to study carbohydrate-protein interactions. Anal Biochem. 2006 Jul 1;354(1):54-63. [Pubmed]
Why use peptide biotinylation? [Peptide-biotinylation]