Iodoacetyl

A variant of Method B is the cyclization of the phenolic 2-(chloroacetamido)benzophenones 9, produced by photo-Fries rearrangement (see Houben-Weyl, Vol. 7/2a, p 1148 ff) of the esters 8, with ammonia via the corresponding iodoacetyl compounds.195... 

Abbreviations EPR, electron paramagnetic resonance FITC, fluorescein-5 -isothiocyanate lAEDANS, iV-iodoacetyl-N -(5-sulfo-l-naphthyl)ethylenediamine NCD, fluorescent yV-cyclohexyl-N -(4-dimethyl-amino-a-naphthyl)carbodiimide RITC, rhodamine-5 -isothiocyanate DPPE, dipalmitoylphosphatidyl-ethanolamine PE, egg phosphatidyl-ethanolamine ANS, 8-anilino-l-naphthalene sulfonate DPH, diphenylhexatriene e-ADP, l,iV -ethanoadenosine-5 -diphosphate TNP-ADP, 2 [3 ]-0-(2,4,6-trinitrophe-nyl)adenosine-5 -diphosphate. 

An Ellman s assay comparing the unmodified protein to the iodoacetylated protein may be done to assess the degree of modification. 

Although the primary utility of active halogen compounds is to modify sulfhydryl groups in proteins or other molecules, the reaction is not totally specific. Iodoacetyl (and bromoacetyl) derivatives can react with a number of functional groups within proteins the sulfhydryl group of cysteine, both imidazolyl side chain nitrogens of histidine, the thioether of methionine, and... 

Figure 4.34 Several varieties of iodoacetylated diamine compounds have been investigated for crosslinking proteins through sulfhydryl groups.

To block any remaining iodoacetyl sites, add cysteine to a final concentration of 5 mM and react for an additional 15 minutes at room temperature. 

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)... 

SIACX, or succinimidyl-6-((((4-(iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino) hexanoate, is an analog of SIAC that contains an additional aminohexanoate spacer group next to its NHS ester end (Invitrogen). The result is the creation of an approximately 16-atom spacer arm between conjugated molecules. All other properties of SIACX are similar to SIAC. 

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 first two compounds discussed in this section are truly sulfhydryl-reactive, using the common iodoacetyl and maleimide functionalities, respectively. The third derivative, however, is not reactive directly with sulfhydryl groups, but contains a protected sulfhydryl which, after deprotection, can be used to react with other sulfhydryl-reactive crosslinkers. 

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). 

BODIPY FL IA is insoluble in aqueous solution, but may be dissolved in DMF or DMSO as a concentrated stock solution prior to addition of a small aliquot to a reaction mixture. Coupling to sulfhydryl-containing molecules occurs rapidly with the formation of a thioether linkage. The reaction may be done in 50mM sodium borate, 5mM EDTA, pH 8.3. The main consideration is to protect the iodoacetyl derivative from light which may generate iodine and... 

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). 

---