Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3

Modification in Aqueous Solutions

The carboxylic acids of water-soluble biopolymers such as proteins can be coupled to hydrazines, hydroxylamines and amines (Molecular Probes' hydrazine, hydroxylamine and amine derivatives - Table 3.1) in aqueous solution using water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, E2247). Including N-hydroxysulfosuccinimide (H2249) in the reaction mixture has been shown to improve the coupling efficiency of EDAC-mediated protein–carboxylic acid conjugations (Figure 3.24). To reduce intra- and interprotein coupling to lysine residues, 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. 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. EDAC may also be useful for conjugating fluorescent aliphatic amines to cell-surface proteins.

Fluoresceinyl glycine amide (5-(aminoacetamido)fluorescein, A1363) and various hydrazines and hydroxylamines may be the best probes for this application because they are more likely to remain reactive at a lower pH than are aliphatic amines such as the cadaverines. Fluoresceinyl glycine amide has been coupled to the carboxylic acid of a cyclosporin derivative by EDAC. Quantitative analysis of carboxylic acids, including sugar carboxylates, in aqueous solution using 1-naphthylethylenediamine and o-phthaldialdehyde (P2331MP, Reagents for Analysis of Low Molecular Weight Amines - Section 1.8) has also been reported.

ANTS (A350, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) and 7-aminonaphthalene-1,3-disulfonic acid (ANDS; FluoroPure Grade - Note 19.2, A22840; Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) have high ionic charges, which permit electrophoretic separation of their products with complex oligosaccharides. Carboxylated polysaccharides have been coupled to the aromatic amine of ANDS preceding electrophoretic analysis. Several of the fluorescent hydrazine and hydroxylamine derivatives described in Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2 should have similar utility for carbodiimide-mediated derivatization of carboxylic acids.

Figure 3.24 Stabilization of an unstable O-acylisourea intermediate by N-hydroxysulfosuccinimide (NHSS, H2249) in a carbodiimide-mediated (EDAC, E2247) modification of a carboxylic acid with a primary amine.

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. Unlike fluorescent aliphatic amines, fluorescent aromatic amines such as those derived from 7-amino-4-methylcoumarin (A191) and 2-aminoacridone (A6289, Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - 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 (Detecting Peptidases and Proteases - Section 10.4). Aromatic amines can generally be coupled to acid halides and anhydrides, with organic solvents usually required for efficient reaction. 5-Aminoeosin (A117) is the key precursor to a wide variety of eosin-based probes.

Hydrazine, Hydroxylamine and Aliphatic Amine Derivatives

Molecular Probes provides a wide selection of carboxylic acid–reactive reagents (Molecular Probes' hydrazine, hydroxylamine and amine derivatives - Table 3.1), including several different Dapoxyl, Alexa Fluor, BODIPY, fluorescein, Oregon Green, rhodamine, Texas Red and QSY Hydrazine Derivatives, Hydroxylamine Derivatives and Amine Derivatives, all of which are particularly useful for synthesizing drug analogs and as probes for fluorescence polarization immunoassays (Fluorescence Polarization (FP) - Note 1.5). These probes all require a coupling agent such as a carbodiimide to react with carboxylic acids; they do not spontaneously react with carboxylic acids in solution. They do, however, react spontaneously with the common amine-reactive functional groups described in Introduction to Amine Modification - Section 1.1, including succinimidyl esters and isothiocyanates. Some of the more important probes and their potential applications include:

• Alexa Fluor hydrazides (A10436, A10437, A10438, A10439, A30634, A20501MP, A20502; Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2), Alexa Fluor hydroxylamines (A30627, A30629, A30632; Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) and Alexa Fluor cadaverines (A30674, A30675, A30676, A30677, A30678, A30679, A30680), our brightest and most photostable carboxylic acid–reactive probes
• BODIPY aliphatic amines (D2390, D6251), for preparing pH-insensitive probes, such as BODIPY FL etoposide, from carboxylic acid derivatives
• Isomeric aminomethylfluoresceins (A1351, A1353), which are readily coupled to activated carboxylic acids
• Dapoxyl (2-aminoethyl)sulfonamide (D10460) for preparing conjugates with strong UV absorption and a Stokes shift of ~200 nm ()
• Dansyl ethylenediamine (D112), dansyl cadaverine (D113), Dapoxyl (2-aminoethyl)sulfonamide (D10460) and Lissamine rhodamine B ethylenediamine (L2424), for carboxylic acid derivatization and glutamine transamidation reactions (Figure 3.26)
• Bimane amine (B30633), a small blue-fluorescent dye for carboxylic acid derivatization
• EDANS (A91), for preparing radioactive IAEDANS, energy transfer–quenched substrates for endopeptidases (Detecting Peptidases and Proteases - Section 10.4), labeled sugar carboxylates and an ATP substrate analog for DNA-dependent RNA polymerase
• 1-Pyrenemethylamine (P2421), for synthesizing new probes that have excited-state lifetimes of ~100 nanoseconds and also for preparing derivatives of carboxylic acids for chromatographic analysis
• QSY 7 amine (Q10464, ) and QSY 35 methylamine (Q20540), which are essentially nonfluorescent dyes with strong visible absorption (Figure 1.70) for preparing highly efficient quenchers (Molecular Probes' amine-reactive dyes - Table 1.1) for bioassays based on fluorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 )
• Hydrazine (Hydrazines, Hydroxylamines and Aromatic Amines for Modifying Aldehydes and Ketones - Section 3.2) and amine derivatives of lucifer yellow (A1339), Alexa Fluor 405 (A30675) and Cascade Blue (C621) dyes, which are precursors of highly fluorescent, water-soluble probes
• Hydrazine and amine derivatives of biotin and desthiobiotin (Biotinylation and Haptenylation Reagents - Section 4.2), which are versatile intermediates for synthesizing biotin- and desthiobiotin-containing probes
• Fluo-4 cadaverine (F36201), an amine-containing Ca²⁺ indicator (Fluorescent Ca{2+} Indicators Excited with Visible Light - Section 19.3) that can react with aldehydes, ketones and activated esters to form unique Ca²⁺-sensitive fluorescent probes

Enzyme-Catalyzed Transamidation

A special enzyme-catalyzed transamidation reaction of glutamine residues in some proteins and peptides — including actin, melittin, rhodopsin and factor XIII — 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 (Figure 3.26). 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 (D113) has been probably the most widely used reagent, Alexa Fluor cadaverines (A30674, A30675, A30676, A30677, A30678, A30679, A30680), Oregon Green 488 cadaverine (O10465), fluorescein cadaverine (A10466), tetramethylrhodamine cadaverine (A1318), Texas Red cadaverine (T2425) and BODIPY TR cadaverine (D6251) 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. Two biotin cadaverines (A1594, B1596; Biotinylation and Haptenylation Reagents - Section 4.2) are also available for transglutaminase-mediated reactions. 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.

Transamidation of cell-surface glutamine residues by the combination of a transglutaminase enzyme and a fluorescent or biotinylated aliphatic amine can form stable amides. Impermeability of the enzyme restricts this reaction to a limited number of proteins on the cell surface. This technique was used to selectively label erythrocyte band 3 protein with dansyl cadaverine (D113) and proteins of the extracellular matrix with fluorescein cadaverine (A10466). Following protease treatment, the dansylated peptides were isolated using an anti-dansyl affinity column.

Figure 3.26 Transglutaminase-mediated labeling of a protein using dansyl cadaverine (D113).

When carboxylic acids are reacted with carbodiimides in the absence of a nucleophile, they may rearrange to form a stable N-acylurea (Figure 3.27). If the carbodiimide contains a fluorophore such as in the naphthyl carbodiimide NCD-4 (C428), then the fluorophore will be specifically incorporated into the protein. This reaction has been used to label:

• Chloroplast-coupling factor
• Cytochrome bc1 complex
• Mitochondrial proton-channel protein
• Plant tonoplast ATPase
• Proteins in the sarcoplasmic reticulum
• An inorganic pyrophosphatase

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.

Figure 3.27 Carbodiimide modification of a carboxylic acid group in a protein, followed by rearrangement to yield a stable N-acylurea.

Biologically important molecules, especially the nonchromophoric fatty acids, bile acids and prostaglandins, are typically esterified by carboxylic acid–reactive reagents in organic solvents. Esterification of carboxylic acids in aqueous solution is usually not possible, and esters tend to be unstable in water. Fluorescent derivatization reagents for biomedical chromatography have been extensively discussed in reviews.

Fluorescent Diazoalkanes

HPLC derivatization reagents for carboxylic acids include two fluorescent analogs of the common esterification reagent diazomethane. Diazoalkanes react without the addition of catalysts and may be useful for direct carboxylic acid modification of proteins and synthetic polymers. Fluorescent diazoalkanes also react with phosphates and potentially with lipid-associated carboxylic acids in membrane-bound proteins or with free fatty acids.

The fluorescent diazomethyl derivative 9-anthryldiazomethane (ADAM, A1400) has been commonly used to derivatize biomolecules. Unfortunately, ADAM is not very stable and may decompose during storage. 1-Pyrenyldiazomethane (PDAM, P1405) is recommended as a replacement for ADAM because it has much better chemical stability. Moreover, the detection limit for PDAM conjugates is reported to be about 20–30 femtomoles, which is five times better than reported for detection of ADAM conjugates. ADAM and PDAM have been used to detect several types of acids, including:

• Amino acids
• Arachidonic acid
• Bile acids
• Fatty acids
• Okadaic acid
• Prostaglandins
• Steroid acids

In addition, fatty acids derivatized with these reagents have been used to measure phospholipase A₂ activity (Probes for Lipid Metabolism and Signaling - Section 17.4). It has been reported that photolysis of pyrenemethyl esters liberates the free carboxylic acid, making PDAM a potential protecting group for carboxylic acids. To optimize solid-phase organic synthesis, PDAM has been used to quantitate the absolute amount of resin-bound carboxyl groups directly on solid support.

The low nucleophilicity of carboxylic acids requires that they be converted to anions (typically cesium or quaternary ammonium are used as counterions) before they can be esterified with alkyl halides in organic solvents. Panacyl bromide (A1122) has been used to derivatize prostaglandins, fatty acids and biotin, and it also reacts with phosphonic acids. Conjugates of 6-bromoacetyl-2-dimethylaminonaphthalene (badan, B6057) have a high Stokes shift, as well as spectral properties that are very sensitive to their environment. 5-(Bromomethyl)fluorescein (B1355), BODIPY 493/503 methyl bromide (B2103) and BODIPY 630/650 methyl bromide (B22802) have the strongest absorptivity and fluorescence of the currently available carboxylic acid–derivatization reagents. Molecular Probes' BODIPY 493/503 methyl bromide and BODIPY 630/650 methyl bromide may react with anions of carboxylic acids during heating in an organic solvent such as methanol or acetonitrile. The high absorptivity, electrical neutrality and intense fluorescence of their conjugates may make the BODIPY 493/503 and BODIPY 630/650 methyl bromides the preferred reagents for carboxylic acid determinations. Esters and thioethers of BODIPY 630/650 methyl bromide can be excited by the red He–Ne laser and 635 nm laser diodes and have near-infrared fluorescence emission.

All of the alkyl halides in this section also react with thiol groups, including those in proteins. Although more commonly used as thiol-reactive reagents, the monobromobimanes (M1378, M1380, M20381; Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3) have been reported to react with carboxylic acids in organic solvents. The coumarin iodoacetamide DCIA (D404, Thiol-Reactive Probes Excited with Ultraviolet Light - Section 2.3) has also been used to derivatize carboxylic acids; other iodoacetamides in Thiol-Reactive Probes - Chapter 2 will probably react similarly.

2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate (N2461, ) reacts rapidly with the anions of carboxylic acids in acetonitrile to give adducts that are reported to be detectable by absorption at 259 nm down to 100 femtomoles and by fluorescence at 394 nm down to 4 femtomoles. This naphthalimide sulfonate ester will likely react with other nucleophiles too, including thiols, amines, phenols (e.g., tyrosine) and probably histidine. 2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate has been used for the sensitive fluorometric detection of carnitine and acylcarnitines in tissue.

4-Sulfo-2,3,5,6-tetrafluorophenol (STP, S10490) and N-hydroxysulfosuccinimide (NHSS, H2249) can be used to prepare water-soluble activated esters from various carboxylic acids (Figure 3.29). Coupling typically involves a carbodiimide such as EDAC (E2247) and is performed in an organic solvent. Scientists at Molecular Probes have found that the resulting STP esters are much easier to purify and more stable than activated esters prepared from N-hydroxysulfosuccinimide. NHSS esters of biotin and other derivatives considerably increase the aqueous solubility of the reagents. Molecular Probes offers a variety of amine-reactive STP Esters, which are discussed in Fluorophores and Their Amine-Reactive Derivatives - Chapter 1.

Figure 3.29 4-Sulfo-2,3,5,6-tetrafluorophenol (STP, S10490) can be used to prepare water-soluble activated esters from various carboxylic acids.