Iodoacetyl group

SIAX, succinimidyl-6-((iodoacetyl)amino)hexanoate, is a heterobifunctional reagent containing an NHS ester on one end and an iodoacetyl group on the other end (Brinkley, 1992)... 

Figure 5.11 NPIA is one of the shortest heterobifunctional reagents. It reacts with amine-containing molecules through its p-nitrophenyl ester end to produce amide bonds. The iodoacetyl group then can be used to couple with thiol compounds to give stable thioether linkages.

The iodoacetamido derivatives of fluorescein possess a sulfhydryl-reactive iodoacetyl group (Chapter 1, Section 4.2 and Chapter 2, Section 2.1) at either the 5- or 6-carbon position on their lower ring. The isomers are commercially available in purified form, since some reactivity and specificity differences between the 5- and 6-derivatives toward various sulfhydryl sites in proteins may be observed. Both iodoacetamido derivatives are among the most intense fluoro-phores available for labeling biomolecules due to high QY. 

The iodoacetyl group of both isomers reacts with sulfhydryls under slightly alkaline conditions to yield stable thioether linkages (Figure 9.7). They do not react with unreduced disulfides in cystine residues or with oxidized glutathione (Gorman et al., 1987). The thioether bonds will be hydrolyzed under conditions necessary for complete protein hydrolysis prior to amino acid analysis. 

DCIA is 7-diethylamino-3-[(4 -(iodoacetyl)amino)phenyl]-4-methylcoumarin, a derivative of the basic aminomethylcoumarin structure that contains a sulfhydryl-reactive iodoacetyl group and a diethyl substitution on its amine. This particular coumarin derivative is among the most fluorescent UV-excitable iodoacetamide probes available (Sippel, 1981) (Invitrogen). 

One Lucifer Yellow derivative is available for labeling sulfhydryl-containing molecules. Lucifer Yellow iodoacetamide is a 4-ethyliodoacetamide derivative of the basic disulfonate aminonaph-thalimide fluorophore structure (Invitrogen). The iodoacetyl groups react with —SH groups in proteins and other molecules to form stable thioether linkages (Figure 9.42). 

Figure 16.2 The original design of the ICAT compound. The iodoacetyl group provides reactivity with thiol groups. The isotopically labeled spacer arm typically is substituted with eight deuterium atoms.

Reduce disulfides in the two protein samples by the addition of 2 pi of 50 mM TCEP (Thermo Fisher) to each 100 pi aliquot of protein solution. Cover and boil the samples for 10 minutes in a water bath to completely denature and reduce the proteins. Avoid the use of thiol-containing reductants, such as DTT, as these will react with the iodoacetyl group on the ICAT compound. 

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

The use of a benzophenone photoactivatable group in the design of bioconjugate reagents is rare. Two sulfhydryl-reactive ones incorporating a maleimide group and a iodoacetyl group opposite the benzophenone are described in Chapter 5, Sections 4.3 and 4.4. 

---