Handbook, Eighth Edition
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Section 3.4 — Esterification Reagents for Carboxylic Acids

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

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 ref and potentially with lipid-associated carboxylic acids in membrane-bound proteins or with free fatty acids.

The most common fluorescent diazomethyl derivative has been 9-anthryldiazomethane (ADAM, A-1400), a reagent that has been used to derivatize biomolecules. Unfortunately, ADAM is not very stable and may decompose during storage. 1-Pyrenyldiazomethane ref (PDAM, P-1405) 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 for ADAM conjugates.ref ADAM and PDAM have been used to detect several types of acids, including:

In addition, fatty acids derivatized with these reagents have been used to measure phospholipase A2 activity ref (Section 18.4). It has been reported that photolysis of pyrenemethyl esters liberates the free carboxylic acid,ref making PDAM a potential protecting group for carboxylic acids. To optimize solid-phase organic synthesis, PDAM has been used to quantitate the absolute amount of resin-bound carboxyl groups directly on solid support.ref

Fluorescent alcohols such as 1-pyrenebutanol (P-244) react with acid halides and isocyanates to yield aliphatic esters and urethanes. Like diazoalkanes, these reagents are primarily useful as synthetic precursors.

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. Panacyl bromide (A-1122) has been used to derivatize prostaglandins,ref fatty acids ref and biotin,ref and it also reacts with phosphonic acids.ref Conjugates of 6-bromoacetyl-2-dimethylaminonaphthalene (badan, B-6057) have a high Stokes shift, as well as spectral properties that are very environmentally sensitive. 5-(Bromomethyl)fluorescein (B-1355), BODIPY 493/503 methyl bromide (B-2103) and BODIPY 630/650 methyl bromide (B-22802) have the strongest absorptivity and fluorescence of the currently available carboxylic acid–derivatization reagents.ref 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 green fluorescence of its conjugates may make this BODIPY methyl bromide the preferred reagent 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-IR fluorescence emission.

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 (M-1378, M-1380, M-20381; Section 2.3) have been reported to react with carboxylic acids in organic solvents.ref The coumarin iodoacetamide DCIA (D-404, Section 2.3) has also been used to derivatize carboxylic acids;ref other iodoacetamides in Chapter 2 will probably react similarly.

A Fluorescent Trifluoromethanesulfonate

2-(2,3-Naphthalimino)ethyl trifluoromethanesulfonate structure (N-2461) 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 has also been used for the sensitive fluorometric detection of carnitine and acylcarnitines in tissue.ref This reagent will likely react with other nucleophiles too, including thiols, amines, phenols (e.g. tyrosine) and probably histidine.

4-Sulfo-2,3,5,6-Tetrafluorophenol (STP)

4-Sulfo-2,3,5,6-tetrafluorophenol (STP, S-10490) can be used to prepare water-soluble activated esters from various carboxylic acids. Coupling typically involves a carbodiimide such as EDAC (E-2247, Section 3.3) 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, H-2249; Section 3.3). Molecular Probes offers a variety of amine-reactive STP Esters (Figure 1.1), which are discussed in Chapter 1.

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Molecular Probes