Handbook, Eighth Edition
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Section 3.3 — Amidation Reagents for Carboxylic Acids and Glutamine

Carboxylic acids can be converted either to esters, which are discussed in Section 3.4, or to amide derivatives or acylhydrazides, which are discussed in this section. Alternatively, the half-protected tert-butyloxycarbonyl (t-BOC) propylenediamine derivative (M-6248) is useful for converting organic solvent–soluble carboxylic acids into aliphatic amines (Figure 3.3). Following coupling of the half-protected aliphatic diamine to an activated carboxylic acid, the t-BOC group can be quantitatively removed with trifluoroacetic acid (Figure 3.3). The resultant aliphatic amine can then be modified with any of the amine-reactive reagents described in Chapter 1 or coupled to solid-phase matrices for affinity chromatography.

Coupling Hydrazines and Amines to Carboxylic Acids

Modification In Aqueous Solutions

The carboxylic acids of water-soluble biopolymers such as proteins have been coupled to hydrazines (Section 3.2) and amines in aqueous solution using water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, E-2247). Including N-hydroxysulfosuccinimide (H-2249) in the reaction mixture has been shown to improve the coupling efficiency of EDAC-mediated protein–carboxylic acid conjugations.ref To reduce intra- and interprotein coupling to lysine residues,ref which is a common side reaction, carbodiimide-mediated coupling should be performed in a concentrated protein solution at a low pH, using a large excess of the nucleophile.ref EDAC has been shown to be impermeable to membranes of live cells, which permits its use to distinguish between cytoplasmic and lumenal sites of reaction.ref EDAC may be useful for conjugating fluorescent aliphatic amines to cell-surface proteins.

Fluoresceinyl glycine amide (5-(aminoacetamido)fluorescein, A-1363) and various hydrazines may be the best probes for this application because they are more likely to remain reactive at a lower pH than are other aliphatic amines.ref Fluoresceinyl glycine amide has been coupled to the carboxylic acid of a cyclosporin derivative by EDAC.ref Quantitative analysis of carboxylic acids, including sugar carboxylates, in aqueous solution using 1-naphthylethylenediamine and o-phthaldialdehyde (P-2331, Section 1.7) has also been reported.ref

ANTS (A-350, Section 3.2) and FluoroPure grade 7-aminonaphthalene-1,3-disulfonic acid (ANDS, A-22840; Section 3.2) have high ionic charges, which permits electrophoretic separation of their products with complex oligosaccharides.ref Carboxylated polysaccharides have been coupled to the aromatic amine of ANDS preceding electrophoretic analysis.ref Several of the fluorescent hydrazine derivatives described in Section 3.2 should have similar utility for carbodiimide-mediated derivatization of carboxylic acids in polysaccharides.

Modification in Organic Solvents

Peptide synthesis research has led to the development of numerous methods for coupling carboxylic acids to amines in organic solution. One such method involves the conversion of carboxylic acids to succinimidyl esters or mixed anhydrides. Dicyclohexylcarbodiimide and diisopropylcarbodiimide are widely used to promote amide formation in organic solution. Another recommended derivatization method for coupling organic solvent–soluble carboxylic acids, including peptides, to aliphatic amines without racemization is the combination of 2,2'-dipyridyldisulfide and triphenylphosphine.ref Unlike fluorescent aliphatic amines, fluorescent aromatic amines such as those derived from 7-amino-4-methylcoumarin (A-191) and 2-aminoacridone (A-6289, Section 3.2) exhibit a shift in their absorption and emission (if any) to much shorter wavelengths upon forming carboxamides. This property makes these aromatic amines preferred reagents for preparing peptidase substrates (Section 10.4). Aromatic amines can generally be coupled to acid halides and anhydrides, with organic solvents usually required for efficient reaction. 5-Aminoeosin (A-117) is the key precursor to a wide variety of eosin-based probes.

Hydrazine and Aliphatic Amine Derivatives

Molecular Probes provides a wide selection of carboxylic acid–reactive reagents, including several different Dapoxyl, Alexa Fluor, BODIPY, fluorescein, Oregon Green, rhodamine, Texas Red and QSY 7 Hydrazine Derivatives and Amine Derivatives, all of which are particularly useful for synthesis of drug analogs and as probes for fluorescence polarization immunoassays.ref Some of the more important probes and their potential applications include:

Enzyme-Catalyzed Transamidation

A special enzyme-catalyzed transamidation reaction of glutamine residues in some proteins and peptides — including actin,ref melittin,ref rhodopsin ref and factor XIII ref — enables their selective modification by amine-containing probes. The NH2 group of certain glutamine residues can be replaced with an aliphatic amine to form a labeled glutamine amide, a reaction that can be catalyzed by a transglutaminase enzyme ref (Figure 3.4). This unique method for selective protein modification requires formation of a complex consisting of the glutamine residue, the aliphatic amine probe and the enzyme. It has been found that a short aliphatic spacer in the amine probe enhances the reaction. The cadaverine (–NH(CH2)5NH–) spacer is usually optimal. Although dansyl cadaverine (D-113) is probably the most widely used reagent,ref fluorescein cadaverine ref (A-10466), Oregon Green 488 cadaverine (O-10465), tetramethylrhodamine cadaverine ref (A-1318), Texas Red cadaverine (T-2425) and BODIPY TR cadaverine (D-6251) are the most fluorescent transglutaminase substrates available. The intrinsic transglutaminase activity in sea urchin eggs has been used to covalently incorporate dansyl cadaverine during embryonic development.ref Two biotin cadaverines (A-1594, B-1596; Section 4.2) are also available for transglutaminase reactions.ref Amine-terminated peptides and fluorescent and biotin hydrazides, including Cascade Blue hydrazide, have been successfully incorporated into protein fragments by transamidation during enzyme-catalyzed proteolysis.ref

Transamidation of cell-surface glutamine residues by the combination of a transglutaminase enzyme and a fluorescent or biotinylated aliphatic amine can form stable amides.ref Impermeability of the enzyme restricts this reaction to a limited number of proteins on the cell's surface. This technique was used to selectively label erythrocyte band 3 protein with dansyl cadaverine (D-113) and proteins of the extracellular matrix with fluorescein cadaverine ref (A-10466). Following protease treatment, the dansylated peptides were isolated using an anti-dansyl affinity column.ref

Modification of Proteins with Fluorescent Carbodiimides

When carboxylic acids are reacted with carbodiimides in the absence of a nucleophile, they may rearrange to form a stable N-acylurea (Figure 3.5). If the carbodiimide contains a fluorophore such as in the naphthyl carbodiimide NCD-4 (C-428), then the fluorophore will be specifically incorporated into the protein. This reaction has been used to label:

A similar mechanism of labeling may occur in some dicyclohexylcarbodiimide (DCC)–inhibited proteins, in which DCC appears to react with a carboxyl residue within a very hydrophobic sequence of the protein.ref

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Molecular Probes