Azide group amines

Figure 4.21 BASED can react with molecules after photoactivation to form crosslinks with nucleophilic groups, primarily amines. Exposure of its phenyl azide groups to UV light causes nitrene formation and ring expansion to the dehydroazepine intermediate. This group is highly reactive with amines. The cross-bridge of BASED is cleavable using a disulfide reducing agent.

Figure 5.16 Photoactivation of a phenyl azide group with UV light results in the formation of a short-lived nitrene. Nitrenes may undergo a number of reactions, including insertion into active carbon-hydrogen or nitrogen-hydrogen bonds and addition to points of unsaturation in carbon chains. The most likely route of reaction, however, is to ring-expand to a dehydroazepine intermediate. This group is highly reactive toward nucleophiles, especially amines.

Of the following amine-reactive and photoreactive crosslinkers, the overwhelming majority use an aryl azide group as the photosensitive functional group. Only a few use alternative photoreactive chemistries, particularly perfluorinated aryl azide, benzophenone, or diazo compounds. For general background information on photoreactive crosslinkers, see Das and Fox (1979), Kiehm and Ji (1977), Vanin and Ji (1981), and Brunner (1993). 

HSAB (N-hydroxysuccinimidyl-4-azidobenzoate) is a heterobifunctional reagent containing an amine-reactive NHS ester on one end and a photoreactive phenyl azide group on the other end... 

Figure 5.20 Sulfo-HSAB is a short photoreactive crosslinker that can be used to modify amine-containing molecules through its NHS ester end to form amide linkages. After photoactivation, the phenyl azide group can react with amines to create a covalent bond.

SANPAH (N-succinimidyl-6-(4 -azido-2 -nitrophenylamino)hexanoate) is a heterobifunctional crosslinking agent containing an NHS ester and a photoreactive phenyl azide group (Thermo Fisher). The NHS ester end can react with amine groups in proteins and other molecules, forming... 

SADP, N-succinimidyl-(4-azidophenyl)l,3 -dithiopropionate, is a photoreactive heterobifunctional crosslinker that is cleavable by treatment with a disulfide reducing agent (Thermo Fisher). The crosslinker contains an amine-reactive NHS ester and a photoactivatable phenyl azide group, providing specific, directed coupling at one end and nonselective insertion capability at the other end. 

Figure 5.25 The reaction of sulfo-SAPB with an amine group is done first to form an amide bond derivative through its NHS ester end. Subsequent exposure to UV light causes the phenyl azide group to ring-expand to a highly reactive dehydroazepine, which can couple to nucleophiles, such as amines.

Sulfo-SAMCA, sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate, is a heterobifunctional reagent similar in design to SAED (Section 3.9, this chapter) (Thermo Fisher). One end of the crosslinker contains an amine-reactive sulfo-NHS ester, while the other end is an AMCA derivative (Chapter 9, Section 3) that contains a photosensitive phenyl azide group. Unlike... 

Figure 5.37 APG can be used to label specifically arginine residues in proteins, producing stable, cyclic Schiff base-like bonds with the side-chain guanidino groups. Photoactivation with UV light then causes ring expansion of the phenyl azide group, initiating covalent bond formation with amines.

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20 minutes in phosphate buffer at room temperature conditions), it should be coupled to an amine-containing protein or other molecule before the photolysis reaction is done. During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide group. The degree of derivatization should be limited to no more than a 5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible precipitation of the modified molecules. For a particular protein, studies may have to be done to determine the optimal level of modification. 

Figure 6.1 The Wedekind trifunctional crosslinker can react with amine groups via its p-nitrophenyl ester to form amide bond linkages. The phenyl azide group then can be photoactivated with UV light to generate covalent bond formation with a second molecule. The biotin side chain provides binding capability with avidin or streptavidin probes.

Figure 9.38 The acetyl azide group of this Cascade Blue derivative has dual functions. It can react with amine groups to form amide bonds, or it can be converted to an isocyanate at high temperatures to couple with hydroxyl functional groups, creating a carbamate linkage.

Cascade Blue acetyl azide is soluble in aqueous solution, but the reactive azide group will hydrolyze and should be used immediately in a conjugation reaction. A concentrated stock solution may be prepared in water, dissolved quickly, and an aliquot quickly added to a buffered reaction medium. For aqueous reactions, a pH range of 7-9 is optimal. Avoid amine-containing buffers. 

Figure 28.12 Sulfo-SBED first is used to label a bait protein through reaction of the sulfo-NHS ester with available amine groups on the protein, yielding an amide bond linkage. This labeled bait protein then is added to a sample containing proteins that potentially could interact with the bait. After an incubation period, the sample is exposed to UV light to photoactivate the phenyl azide group. This reaction causes any interacting prey proteins to be crosslinked with the bait protein, forming a complex containing a biotin affinity tag.

The authors have shown that changing the hydroxyl group in the 8-position to an azide group causes transformation of the azocine ring into azocinoindole 75 under UV light. This leads to the exocyclic amine 76 and the product of azocine fragment enlargement, diazonino[4,3,2-cd]indole 77 (Scheme 23). 

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