Ligation Bioorthogonal Reagents

Some reports indicated that the Diels-Alder reaction could be done in aqueous environments with a potential for accelerated reaction rates under the right conditions (Rideout and Breslow, 1980 Blokzijl and Engberts, 1992 Pai and Smith, 1995 Otto et al., 1996 Wijnen and Engberts, 1997), and the addition of InCl3 was determined to act as a catalyst in aqueous environments (Loh et al., 1996). For a review of organic reactions that can be done in aqueous media, see Li (2005). 

Where D is an electron donating group and W is an electron withdrawing group. 

In addition, it has been discovered that there are naturally occurring enzymes that facilitate Diels-Alder type reactions within certain metabolic pathways and that enzymes are also instrumental in forming polyketides, isoprenoids, phenylpropanoids, and alkaloids (de Araujo et al., 2006). Agresti et al. (2005) identified ribozymes from RNA oligo libraries that catalyzed multiple-turnover Diels-Alder cycloaddition reactions. 

The reaction kinetics between a maleimide derivative and a 3,5-hexadiene derivative varies depending on the maleimide compound being reacted. Cycloaddition yields of greater than 80 percent and often as much as 90-95 percent can be expected within 1-18 hours at room temperature or slightly elevated reaction conditions (e.g., 30°C). 

The conjugation of oligonucleotides with peptides also can be done using Diels-Alder cycloadditions in water (Tona and Haner, 2005). Marchan et al. (2006) used the same 3,5-hexadiene phosphoramidite derivative as Hill et al. (2001), but in this case used a maleimide-modified 

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The use of the alkyne-containing E64-based ABP (Fig. 1), followed by bioorthogonal ligation to a novel doubly labelled reagent containing a TAMRA... 

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