TFP-dPEG®₄-biotinidase resistant biotin

TFP-dPEG®4-biotinidase-resistant biotin is a water-soluble biotinylation reagent that has been modified to resist hydrolysis by biotinidase (EC 3.5.1.12). This product combines Biotinoyl-2-aminobutyric acid, with the 2,3,5,6-tetrafluorophenyl (TFP) ester of a single molecular weight, discrete polyethylene glycol (dPEG®) linker. TFP-dPEG®4-biotinidase-resistant biotin has biotin's typical high affinity for avidin and streptavidin. However, the modified biotin prevents recycling by biotinidase. This product is useful in applications such as ELISAs of tissue samples and antibody pretargeting, where endogenous biotinidase potentially can recycle biotin.

Development and Uses of TFP-dPEG®4-biotinidase-resistant biotin

Biotinidase is an amidohydrolase that breaks the covalent bond between biotin and amino acids such as lysine (i.e., biocytin). In vivo, biotinidase recycles biotin. Although necessary for healthy bodily function, biotinidase potentially creates problems when biotinylated proteins are used in serological assays, since endogenous biotinidase removes the biotin from these proteins.

Dr. D. Scott Wilbur and colleagues at the University of Washington in Seattle developed biotinidase-resistant biotin in 2006. Development of biotinidase-resistant biotin arose from a need for biotinylation reagents that could facilitate in vivo antibody pretargeting. However, to improve the product's water solubility, Quanta BioDesign conjugated a short amino-dPEG®4-propionic acid linker to biotinidase-resistant biotin. For practical use in bioconjugation, the TFP ester then was synthesized.

Like our other dPEG® biotinylation products, TFP-dPEG®4-biotinidase-resistant biotin will not cause aggregation and precipitation of biomolecules to which it is conjugated. Moreover, the dPEG® spacer in this product reduces non-specific binding. Aggregation, precipitation, and non-specific binding are common problems with the widely used LC-biotin reagents.

TFP-dPEG®4-biotinidase-resistant biotin resists amide hydrolysis because the non-natural amino acid to which it is conjugated prevents the active site of biotinidase from hydrolyzing the amide bond connecting biotin to the dPEG®4 spacer. This product potentially is beneficial for applications that use serological fluids containing endogenous biotinidase. Useful roles for TFP-dPEG®4-biotinidase-resistant biotin include the following:

therapeutic applications such as antibody pretargeting with biotin-streptavidin systems;
supramolecular constructs (g., nanoparticles) used in vivo that depend on biotin-(strept)avidin affinity to maintain structural integrity; and,
ELISAs of serological fluids and tissue samples using biotin-(strept)avidin affinity.

Products Similar to TFP-dPEG®4-biotinidase-resistant biotin
In addition to this TFP ester-activated construct, Quanta BioDesign offers a more extended version of this product and an equivalent-length product functionalized as the N-hydroxysuccinimidyl (NHS) ester. TFP esters possess greater hydrolytic stability and amine reactivity compared to NHS esters.

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