A reagent for covalently attaching biotin to proteins via a cleavable connector arm

Abstract

The preparation of a reagent which can be used to attach biotin to proteins via a cleavable connector arm is described. The reagent, 3-(4-(N-biotinoyl-6-aminocaproyloxy)phenyl)propionic acid N-hydroxysuccinimide ester (BPE), can acylate amino groups of proteins and other compounds via reaction of the N-hydroxysuccinimide ester function. Compounds which have been covalently labeled with BPE can be selectively retrieved (at physiological pH values) from complex mixtures by adsorbing the target compounds on immobilized avidin. These compounds can be released from the avidin by cleavage of the phenyl ester linkage of the connector arm under conditions which don't denature proteins. Insulin was labeled with BPE. The binding of the labeled insulin to immobilized avidin, and its subsequent release by treatment at room temperature with 1 M hydroxylamine at pH 7 was demonstrated.The highly specific and very strong binding of biotin to avidin (KD -lo-l5 M; (7)) could provide us with a basis for the design of elegant protocols for the retrieval of compounds from cells and physiological fluids even when the compounds targeted for retrieval are present at very low concentrations.For example, a polypeptide hormone that had been covalently labeled with biotin could be incubated with cells and later retrieved by adsorption on immobilized avidin for studies of hormone catabolism and for isolation of hormonereceptor complexes. Unfortunately, the direct application of this approach is usually precluded by the very low rate constant for the dissociation of biotin from

Gas‐phase fragmentation study of biotin reagents using electrospray ionization tandem mass spectrometry on a quadrupole orthogonal time‐of‐flight hybrid instrument

“…During the past few decades, biotin reagents have been developed specifically to be attached covalently to proteins via the intermediacy of a cleavable connector 1–4. Biotin, which is also known as either vitamin H or B 7 , is a water‐soluble B‐complex vitamin, which has a nominal mass of 244 Da and an empirical formula of [C 10 H 16 N 2 O 3 S].…”

“…[1][2][3][4] Biotin, which is also known as either vitamin H or B 7 , is a watersoluble B-complex vitamin, which has a nominal mass of 244 Da and an empirical formula of [C 10 H 16 N 2 O 3 S]. It is composed of a tetrahydroimidizalone ring fused to a tetrahydrothiophene ring substituted at the C-2 position with valeric acid (Scheme 1).…”

“…5 The high affinity of biotin for avidin provides the basis for many established procedures for the detection and isolation of biotinassociated proteins. [1][2][3][4][5] It has also become possible to apply this affinity system to the detection of specific DNA sequences by using biotinylated DNA compounds as hybridization probes. 6,7 There have been a multitude of reports dealing with the identification of intact protein-biotin complexes by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).…”

Synthetic Strategies for the Biotinylation of Bioactive Small Molecules

“…The N-hydroxysuccinimide ester 4 has the advantage of being soluble in a range of organic solvents. [17] The application of biotinylation in in vivo systems is an expanding area, especially with regard to imaging and chemotherapy. However, both N-hydroxysuccinimide moieties are prone to base hydrolysis to an extent that depends on the exact conditions used, but as much as a threefold decrease in reaction rate of the desired biotinylated compound.…”

“…Before analysis by SDS-PAGE, the small molecule can be released from the biotin, and the majority of nonspecifically bound proteins, by cleavage of the phenyl ester bond under mild, non-denaturing conditions (1 m hydroxylamine at pH 7), subsequent centrifugation allows separation of the heavier beaded biotin and associated nonspecific interacting proteins from the lighter probe and protein target complex. [17] The application of biotinylation in in vivo systems is an expanding area, especially with regard to imaging and chemotherapy. [18] A significant issue in this setting is the cleavage of the amide-linked biotin residue by the enzyme biotinidase.…”