Biotin amine reactive derivatives

The pH-dependent binding of 2-imino-biotin (Fig. 3g) has been used in affinity chromatography to purify strept(avidin) because binding is easily reversible [85]. Specifically,bin ding occurs under alkaline conditions (pH 10) and release occurs under acidic conditions (pH 4). An amine-reactive derivative of desthiobiotin (Fig. 3h), a non-sulfur containing metabolic precursor to biotin that is bound to strept(avidin) with lower affinity than biotin, has been used to form reversible complexes with strept(avidin) [74]. Release is achieved by incubation in the presence of free desthiobiotin or biotin. Recently developed lower affinity derivatives include 9-methyl biotins [86]. Another approach covalently attaches a photocleavable biotin derivative (Fig. 3h) to molecules that are released following exposure to 200 nm light [75]. 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

NHS-LC-biotin is an extended spacer arm derivative of biotin containing an amine-reactive NHS ester (Chapter 11, Section 1). The compound is a popular choice for biotinylating a wide... 

Perhaps the most common method of DNA biotinylation is through enzymatic incorporation with the use of a biotin-labeled deoxynucleoside triphosphate. First reported by Langer et al. and Leary et al. in 1981, the procedure is probably the most popular nonradioactive labeling technique reported for oligonucleotide probes. Although biotinylated derivatives of dCTP and dATP are reported in the literature, by far the most frequently employed derivative is biotin—dUTP prepared from the reaction of an amine-modified dUTP with an amine-reactive biotinylation reagent, such as NHS-LC—biotin (Chapter 8, Section 3.1). 

Biotinylation Using Amine-Reactive Biotin Derivatives... 

Reaction buffer 100 mM bicarbonate buffer (pH 8.0-8.3) is preferred for labeling antibody amines (see Note 3). The optimum reaction pH values for labeling antibodies with other reactive biotin derivatives are summarized in Table 3. 

These derivatives have been prepared according to equation 62 and their reactivity was investigated140. [2-18F]Fluoropropionic acid derivatives have been used for NCA fluo-roacetylation of a variety of amines, R3NH2, including homoveratrylamine, benzoyl hydrazide, biotin hydrazide, -butylamine, 2-butylamine and phenylalanine ethyl ester1. 

The first class of luminescent biotinylation reagents, derived from rhenium(I) polypyridine complexes [Re(NAN)(CO)3(py-biotin-NCS)]+ (24), have been reported by Lo and co-workers [62], To investigate the amine-specific reactivity... 

Fig. 3 d Photocleavable biotin derivative made up of a biotin moiety linked through a spacer arm (6-aminocaprioic acid) to allow binding to streptavidin and an alpha-substituted 2-nitrobenzyl nucleus with a iV-hydroxysuccinimidyl reactive group (NHS carbonate) that reacts with primary amines under mild conditions (pH 8) (Taken from [75])... 

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