Azido-biotin

Once the protein is modified to contain an alkynyl group at its C-terminal it can be used to covalently link to its click chemistry reactant partner, an azide on the surface of an array. Other azido molecules also can be conjugated with an alkyne-protein to facilitate the detection or capture of the protein using affinity techniques. For instance, an azido-fluorescein reagent can be used to detect fluorescently the expressed protein in complex samples or an azido-biotin... 

Figure 11.22 Azido-sialic acid-containing glycans can be labeled in vivo with biotin-PEG-phosphine using the Staudinger ligation reaction, which forms an amide bond.

Figure 17.3 Maleimide-modified glass slides (1) can be derivatized using two chemoselective ligation reactions to create biotin modifications. In the first step, alkyne-PEG4-cyclopentadiene linkers (2) are added to the maleimide groups using a Diels-Alder reaction. In the second reaction, an azido-PEG4-biotin compound (3) is reacted with the terminal alkyne on the slide using click chemistry to result in another cycloaddition product, a triazole ring.

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

Add to the washed cells 60 pi of a 5 mM concentration of the phosphine derivative to couple to the azido-sugar groups on the cell surface (e.g., biotin-PEG-phosphine). 

Figure 17.19 An azido-sialic acid derivative that gets incorporated into glycans in cells can be labeled specifically with a biotin-phosphine tag using the Staudinger ligation process. The result is an amide bond linkage with the glycan.

Figure 17.20 An azido-palmitic acid derivative can be added to cells to obtain palmitoylated proteins that contain an azide group able to participate in the Staudinger ligation reaction. Biotinylation of these post-translationally modified sites then can be done in vivo using a biotin-phosphine reagent.

Methyl 4,6-0-benzylidene-3-deoxy-a-D-ribo-hexopyranoside (56) was benzoylated, debenzylidenated, and partially p-toluenesulfon-ylated to 57 this was converted into 58 by reaction with sodium iodide, followed by catalytic reduction. The methanesulfonate of 58 was converted into 59 by reaction with sodium azide in N,N-dimethylformamide, and 59 was converted into 4-azido-3,4,6-trideoxy-a-D-xylo-hexose (60) by acetolysis followed by alkaline hydrolysis. Reduction of 60 with borohydride in methanol afforded 61, which was converted into 62 by successive condensation with acetone, meth-anesulfonylation, and azide exchange. The 4,5-diazido-3,4,5,6-tetra-deoxy-l,2-0-isopropylidene-L-ara/uno-hexitol (62) was reduced with hydrogen in the presence of Raney nickel, the resultant diamine was treated with phosgene in the presence of sodium carbonate, and the product was hydrolyzed under acidic conditions to give 63. The overall yield of 63 from 56 was 4%. The next three reactions (with sodium periodate, the Wittig reaction, and catalytic reduction) were performed without characterization of the intermediate products, and gave (+)-dethiobiotin methyl ester indistinguishable from an authentic sample thereof prepared from (+)-biotin methyl ester. 

A biotin containing the cleavable aryl azido moiety, sulfosuccinimidyl-2[6-biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-l,3 -dithiopropionate, has been prepared (1). 

Af-(D-Biotinyl)-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl)-L-threonine te/t-Butyl Ester 23 [34], A mixture of D-biotin (150 mg, 0.6 mmol), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC 580 mg, 3 mmol), and 1-hydroxy-benzotriazole (HOBt 540 mg, 4 mmol) in dimethylformamide (DMF 2 mL) is stirred under exclusion of moisture at 22 C. After 45 min, the biotin is dissolved, and a solution of freshly prepared glycosyl threonine ester 22 (0.3 mmol, preceding procedure) in dichloro-methane (2 mL) is added at 0°C. After stirring for 16 h at room temperature, the solvent is evaporated in vacuo, the remainder dissolved in dichloromethane (50 mL), extracted with ice-cold 0.2 N HCl (3 x 25 mL), water (25 mL), and saturated NaHCOj solution (2 x 25 mL), dried with MgSO, and concentrated in vacuo. Purification by flash chromatography on silica gel (20 g) in dichloromethane-ethanol (25 1) yields 23 200 mg (93%) [a] 96.5° (c 1, CHClj) Rf 0.29 (toluene-acetone 4 1). 

A chemoenzymatic synthesis of the P-a-methyl 2 -deoxynucleoside triphosphates 122 has been described which involves reaction of the 5 -0-(methylpho-sphonyl)-N-protected nucleosides with pyrophosphate in the presence of CDI. Removal of the base protection by treatment with penicillin amidase gave compounds 122 leaving the labile a-methylphosphonate intact. A number of 2 -deoxythymidine 5 -triphosphate and 3 -azido-2, 3 -dideoxythymidine 5 -tripho-sphate analogues (123) containing a hydrophobic phosphonate group have also been synthesised and evaluated as substrates for several viral and mammalian polymerases. Some y-ester (124) and y-amide (125) derivatives of dTTP and 3 -azido-2, 3 -dideoxythymidine 5 -triphosphate (AZTTP) were also synthesized and studied. The y-phenylphosphonate triphosphate 126 and its conjugation to biotin and fluorescein labels has also been described. 

Forster et al. (1985) synthesized a derivative of biotin containing a photoactivable azido group. This product is commercially available... 

The RAFT polymerization of a -azido- u-dithiopyridine affords azide-terminated het-erotelechelic polymers which were reacted with biotin/avidine glutathion and bovin serum albumine via click and thiol-disulflde exchange reactions... 

The pseudodipeptide 55 (see Scheme 10.16) was regarded as a conformationally restricted Xaa-Pro dipeptide and Scolastico et al. functionalized the azido-group by copper(l)-catalyzed dipolar cycloaddition with an alkynylated sugar, biotin or fluorescein to afford substituted triazolyl-Xaa-Pro-dipeptide mimics 56 in good to excellent yields. ... 

PEG-methacrylate was selected as the monomer to ensure biocompatibility, as PEGylated surfaces are known to inhibit protein binding. To demonstrate the accessibility of the surface azide groups, an azido-derivative of biotin was clicked to the polymer-brush-modified surface subsequently, surface plasmon resonance (SPR) was used to confirm the selective binding with streptavidin in preference to other proteins. 

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