Photoreactive crosslinkers

Psoralen, or derivatives of 9-methoxy-7H-furo[3,2-g]chromen-7-one tricyclic ring structures, are used as photoreactive groups in crosslinkers, biotinylation compounds, and nucleic acid probes. Psoralens have been used for many years as photochemotherapy agents for treatment of psoriasis and vitiligo (Smith and Barker, 2006). Psoralens react when exposed to UV light... 

Psoralens can react by two different routes upon photoactivation (Parsons, 1980 Pathak, 1984). The first route is through the well-known photoreaction mechanism that principally involves intercalation within double-stranded DNA or RNA with the formation of adducts with adjacent thymine bases. The furan-side and pyrone-side rings in psoralen both can form cycloaddition products with the 5,6-double bond of thymine to create a crosslink between two DNA strands (Reaction 57) or to a lesser extent, within double-strand regions of RNA. 

Reported structures for homobifunctional aryl azides include a biphenyl derivative and a naphthalene derivative (Mikkelsen and Wallach, 1976), a biphenyl derivative containing a central, cleavable disulfide group (Guire, 1976), and a compound containing a central l,3-diamino-2-propanol bridge between phenyl azide rings that are nitrated (Guire, 1976). The only commercially available homobifunctional photoreactive crosslinker is BASED. 

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). 

Figure 5.18 SASD is a photoreactive crosslinker that can be used to modify amine-containing compounds through its NHS ester end and subsequently photoactivated to initiate coupling with nucleophiles (after ring expansion to an intermediate dehydroazepine derivative). The crosslinks may be selectively cleaved at the internal disulfide group using DTT.

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... 

In use, SAND is first reacted with an amine-containing protein or other molecule—being careful to protect the photoreactive group from inadvertent degradation by exposure to excessive room light or sun. The modified intermediate then is allowed to interact with a target molecule. Finally, the photolyzing process is done to effect a nonselective crosslink between the... 

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. 

SADP or sulfo-SADP also have been used to study the phenylalanine-methionine-arginine-phenylalanine-amide-activated sodium channel (Coscoy et al., 1998), various apolipoprotein E isoforms (Mann et al., 1995), the high-affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig (Moebius et al., 1994), the interaction of non-histone proteins with nucleosome core particles (Reeves and Nissen, 1993), and the interactions among cytochromes P-450 in the endoplasmic reticulum (Alston et al., 1991). See Chapter 28 for methods of using photoreactive heterobifunctional crosslinkers to study protein interactions. 

Sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-l,3 -dithiopropionate (SAED) is a photoreactive heterobifunctional crosslinking agent that also contains a fluorescent group (Thermo Fisher). The sulfo-NHS ester end of the reagent reacts with primary amines in proteins and other molecules to form stable amide linkages. The photoreactive end is an AMCA... 

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