Carboxylic acids can be converted to esters, amides, acyl hydrazides or hydroxamic acids, all of which are discussed in this section. Alternatively, the half-protected tert-butyloxycarbonyl (t-BOC) propylenediamine derivative (M6248) is useful for converting organic solvent–soluble carboxylic acids into aliphatic amines. 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 3.4.1). The resultant aliphatic amine can then be modified with any of the amine-reactive reagents described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 or coupled to solid-phase matrices for affinity chromatography.

Conversion of a carboxylic acid
Figure 3.4.1 Conversion of a carboxylic acid group into an aliphatic amine. The activated carboxylic acid is derivatized with a half-protected aliphatic diamine (mono-N-(t-BOC)-propylenediamine, M6248), usually in an organic solvent, followed by removal of the t-BOC–protecting group with trifluoroacetic acid.

Coupling Hydrazines, Hydroxylamines and Amines to Carboxylic Acids

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 3.2) 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 ref (Figure 3.4.2). To reduce intra- and inter-protein 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. EDAC-mediated coupling has been used to functionalize Qdot 605 ITK carboxyl quantum dots (Q21301MP, Qdot Nanocrystals—Section 6.6) with the GABA receptor agonist muscimol.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

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.ref Fluoresceinyl glycine amide has been coupled to the carboxylic acid of a cyclosporin derivative by EDAC.ref

ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, A350; Reagents for Modifying Aldehydes and Ketones—Section 3.3) has a high ionic charge, which permits electrophoretic separation of its products with complex oligosaccharides.ref Several of the fluorescent hydrazine and hydroxylamine derivatives described in Reagents for Modifying Aldehydes and Ketones—Section 3.3 should have similar utility for carbodiimide-mediated derivatization of carboxylic acids.

Stabilization of an unstable O-acylisourea
Figure 3.4.2 Stabilization of an unstable O-acylisourea intermediate by N-hydroxysuccinimide 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.ref Unlike fluorescent aliphatic amines, fluorescent aromatic amines such as those derived from 7-amino-4-methylcoumarin (A191) and 2-aminoacridone (A6289, Reagents for Modifying Aldehydes and Ketones—Section 3.3) 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.

Hydrazine, Hydroxylamine and Aliphatic Amine Derivatives

We provide a wide selection of carboxylic acid–reactive reagents (Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2), 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 ref (Fluorescence Polarization (FP)—Note 1.4). 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 applications include:

Enzyme-Catalyzed Transamidation

A transglutaminase-catalyzed transamidation reaction of glutamine residues in some proteins and peptides enables their selective modification by amine-containing probes ref (Figure 3.4.3). 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,ref Alexa Fluor cadaverines ref (A30674, A30675, A30676, A30677, A30678, A30679, A30680), Oregon Green 488 cadaverine (O10465), fluorescein cadaverine ref (A10466), tetramethylrhodamine cadaverine ref (A1318), Texas Red cadaverine (T2425) and BODIPY TR cadaverine (D6251) are among 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 (A1594, B1596; Biotinylation and Haptenylation Reagents—Section 4.2) are also available for transglutaminase-mediated 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 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 ref (A10466). Following protease treatment, the dansylated peptides were isolated using an anti-dansyl affinity column.ref

Transglutaminase-mediated labeling
Figure 3.4.3 Transglutaminase-mediated labeling of a protein using dansyl cadaverine (D113).

Esterification of Carboxylic Acids with Fluorescent Diazoalkanes

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.ref

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 ref 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 ref (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.ref In addition, fatty acids derivatized with these reagents have been used to measure phospholipase A2 activity ref (Probes for Lipid Metabolism and Signaling—Section 17.4). It has been reported that photolysis of pyrenemethyl esters liberates the free carboxylic acid,ref making PDAM a potential protecting group for carboxylic acids.

Fluorescent Alkyl Halides

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. 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, structure) and BODIPY 493/503 methyl bromide (B2103, structure) have the strongest absorptivity and fluorescence of the currently available carboxylic acid–derivatization reagents.ref An analytical method for estimating the degree of EDAC crosslinking of collagen has been developed based on derivatization of residual carboxyl groups by 5-bromomethylfluorescein.ref

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

Fluorescent Trifluoromethanesulfonate

2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate (N2461, structure) 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.ref 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 reverse-phase HPLC detection of eicosanoids in brain tissue.ref

4-Sulfo-2,3,5,6-Tetrafluorophenol (STP) and N-Hydroxysulfosuccinimide (NHSS)

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 3.4.4). Coupling typically involves a carbodiimide such as EDAC (E2247) and is performed in an organic solvent. We have found that the resulting STP esters are much easier to purify and more stable than activated esters prepared from N-hydroxysulfosuccinimide.ref NHSS esters of biotin and other derivatives considerably increase the aqueous solubility of the reagents.ref We offer a variety of amine-reactive STP esters, which are discussed in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1.

Figure 3.4.4 4-Sulfo-2,3,5,6-tetrafluorophenol (STP, S10490) can be used to prepare water-soluble activated esters from various carboxylic acids.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A91 icon 288.30 L pH >10, DMF 335 5900 493 pH 8  
A191 icon icon 175.19 L DMF, DMSO 351 18,000 430 MeOH 1
A1318 icon 514.62 F,D,L DMF, EtOH 544 78,000 571 MeOH  
A1339 icon icon 491.57 L H2O 425 12,000 532 H2O  
A1340 icon icon 533.65 L H2O 426 11,000 531 H2O  
A1351 icon 397.81 L pH >6, DMF 492 80,000 516 pH 9 2
A1353 icon 397.81 L pH >6, DMF 492 68,000 516 pH 9 2
A1363 icon 404.38 L pH >6, DMF 491 80,000 515 pH 9 2
A1400 icon 218.26 FF,D,L DMF, MeCN 364 6100 411 MeOH  
A10466 icon 653.38 D,L pH >6, DMF 493 82,000 517 pH 9 2
A30674 icon icon 397.45 F,D,L H2O 353 20,000 437 MeOH  
A30675 icon icon 666.58 F,D,L H2O 399 29,000 422 H2O 3
A30676 icon icon 640.61 F,D,L H2O 493 73,000 516 pH 7 4, 5
A30677 icon ~950 F,D,L H2O 555 155,000 572 MeOH  
A30678 icon icon 806.94 F,D,L H2O 588 105,000 612 pH 7  
A30679 icon ~1000 F,D,L H2O 651 245,000 672 MeOH  
A30680 icon icon 812.95 F,D,L H2O 578 93,000 602 pH 7  
B1355 icon 425.23 F,D,L pH >6, DMF 492 81,000 515 pH 9  
B2103 icon 341.00 F,D,L DMSO, MeCN 533 62,000 561 CHCl3  
B6057 icon 292.17 F,L DMF, MeCN 387 21,000 520 MeOH  
B30633 icon 207.23 F,D,L DMSO 375 6000 458 MeOH  
C621 icon icon 624.49 L H2O 399 30,000 423 H2O 3
D112 icon icon 293.38 L EtOH, DMF 335 4600 526 MeOH  
D113 icon icon 335.46 L EtOH, DMF 335 4600 518 MeOH  
D2390 icon icon 370.64 F,D,L DMSO, MeCN 503 76,000 510 MeOH 6
D6251 icon icon 544.85 F,D,L DMSO, MeCN 588 64,000 616 MeOH 6
D10460 icon icon 386.47 L DMF, DMSO 373 23,000 571 MeOH 7
E2247 icon 191.70 F,D H2O <300   none    
H2249 icon 217.13 D H2O <300   none    
L2424 icon 600.75 L DMF, DMSO 561 122,000 581 MeOH  
M6248 icon 174.24 D,A DMF, MeCN <300   none    
N2461 icon 373.30 FF,DD,L DMF, CHCl3 260 59,000 395 MeOH  
O10465 icon 496.47 F,D,L pH >6, DMF 494 75,000 521 pH 9 8
P1405 icon 242.28 FF,L DMF, MeCN 340 41,000 375 MeOH  
Q10464 icon icon 814.87 L DMSO 560 92,000 none MeOH  
S10490 icon 268.11 D H2O <300   none    
T2425 icon 690.87 L DMF 591 85,000 612 pH 9  
  1. A191 in aqueous solution (pH 7.0): Abs = 342 nm (EC = 16,000 cm-1M-1), Em = 441 nm.
  2. Absorption and fluorescence of fluorescein derivatives are pH dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.
  3. The Alexa Fluor 405 and Cascade Blue dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.
  4. The fluorescence lifetime (τ) of the Alexa Fluor 488 dye in pH 7.4 buffer at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  5. Abs and Em of the Alexa Fluor 488 dye are red-shifted by as much as 16 nm and 25 nm respectively on microarrays relative to aqueous solution values. The magnitude of the spectral shift depends on the array substrate material.ref
  6. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
  7. Fluorescence emission spectrum shifts to shorter wavelengths in nonpolar solvents.8. Absorption and fluorescence of Oregon Green 488 derivatives are pH dependent only in moderately acidic solutions (pH <5).