Aldehydes and ketones are present in a number of low molecular weight molecules such as drugs, steroid hormones, reducing sugars and metabolic intermediates (e.g., pyruvate and α-ketoglutarate). Except for polysaccharides containing free reducing sugars, however, biopolymers generally lack aldehyde and ketone groups. Even those aldehydes and ketones that are found in the open-ring form of simple carbohydrates are usually in equilibrium with the closed-ring form of the sugar.
The infrequent occurrence of aldehydes and ketones in biomolecules has stimulated the development of techniques to selectively introduce these functional groups, thus providing unique sites for chemical modification and greatly extending the applications of the probes found in this section. Fluorescent modification of aldehyde or carboxylic acid groups in carbohydrates is also frequently utilized for their analysis by HPLC, capillary electrophoresis and other methods.
The most common method for introducing aldehydes and ketones into polysaccharides and glycoproteins (including antibodies) is by periodate-mediated oxidation of vicinal diols. These introduced aldehydes and ketones can then be modified with fluorescent or biotinylated hydrazine, hydroxylamine or amine derivatives to label the polysaccharide or glycoprotein. For example, some of the hydrazine derivatives described in this section have been used to detect periodate-oxidized glycoproteins in gels. The Pro-Q Emerald 300 and Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kits (P21855, P21857, M33307; Detecting Protein Modifications—Section 9.4) are based on periodate oxidation of glycoproteins and subsequent labeling with a Pro-Q Emerald dye.
Periodate oxidation of the 3'-terminal ribose provides one of the few methods of selectively modifying RNA; periodate-oxidized ribonucleotides can subsequently be converted to fluorescent nucleic acid probes by reaction with fluorescent hydrazines, hydroxylamines and amines. Alkenes from unsaturated fatty acids and ceramides can also be converted to glycols by osmium tetroxide and then oxidized by periodate to aldehydes, and periodate will oxidize certain β-aminoethanol derivatives such as the hydroxylysine residues in collagen, as well as methionine (to its sulfoxide) and certain thiols (usually to disulfides). These other reactions, however, usually occur at a slower rate than oxidation of vicinal diols.
In addition to vicinal diols, N-terminal serine and threonine residues of peptides and proteins can be selectively oxidized by periodate to aldehyde groups (Figure 3.3.1). Moreover, because antibodies are glycosylated at sites distant from the antigen-binding region, modification of periodate-oxidized antibodies by hydrazines and hydroxylamines usually does not inactivate the antibody, as sometimes occurs with amine-reactive labeling.
Figure 3.3.1 Sodium periodate oxidation of an N-terminal serine residue to an aldehyde, with the release of formaldehyde. The aldehyde thus formed from the protein can be subsequently modified with a variety of hydrazine, hydroxylamine or amine derivatives.
Galactose Oxidase–Mediated Oxidation and Other Methods
Galactose oxidase oxidizes terminal galactose residues to aldehydes, particularly in glycoproteins. The introduction of galactose residues can be especially advantageous for structural studies because it provides a means of selectively labeling specific sites on biomolecules. For example, 2-keto-galactose has been specifically inserted into the Fc glycans of therapeutic antibodies, including Herceptin and Avastin, enabling site-specific labeling with Alexa Fluor 488 hydroxylamine (A30629). Galactose oxidase–modified lipopolysaccharides (LPS) have been modified with Alexa Fluor 488 hydrazide (A10436) to probe for LPS-binding sites on cells. Because galactose oxidase–mediated oxidation liberates a molecule of hydrogen peroxide for each molecule of aldehyde that is formed (Figure 3.3.2), horseradish peroxidase–catalyzed oxidation of the Amplex Red reagent to red-fluorescent resorufin by hydrogen peroxide provides a ready means by which the number of aldehyde residues introduced into a biomolecule, including on a cell surface, can be quantitated. The Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179, Substrates for Oxidases, Including Amplex Red Kits—Section 10.5) provides the reagents and a general protocol for this assay of introduced aldehyde residues. Other methods for aldehyde and ketone introduction include selective N-terminal transamination in the presence of pyridoxal-5'-phosphate, ligation of a ketone analog of biotin to proteins with a biotin acceptor peptide (BAP) fusion tag by biotin ligase (BirA) and co-translational modification of recombinantly tagged proteins by formylglycine-generating enzyme (FGE).
Figure 3.3.2 Oxidation of the terminal galactose residue of a glycoprotein, glycolipid or polysaccharide results in the generation of an aldehyde, which can react with hydrazines, hydroxylamines or primary amine–containing compounds.
Coupling Hydrazines and Amines to Amine-Containing Biomolecules without Introducing Aldehydes and Ketones
Common tissue fixatives such as formaldehyde and glutaraldehyde can be used to couple hydrazine and amine derivatives to proteins and other amine-containing polymers. For example, lucifer yellow CH (L453) can be conjugated to surrounding biomolecules by common aldehyde-based fixatives in order to preserve the dye's staining pattern during subsequent tissue manipulations.
The tetrafluorophenyl (TFP) ester of N-(t-BOC)-aminooxyacetic acid (B30300, ) is an amine-reactive protected hydroxylamine that is useful for synthesizing new aldehyde- and ketone-reactive probes in an organic solvent. Following coupling to aliphatic amines, the t-BOC group can be quantitatively removed with trifluoroacetic acid. The resultant hydroxylamine probe can then spontaneously react with aldehydes, with the reducing ends of saccharides and oligosaccharides, and with abasic sites in oligonucleotides to form stable adducts.
Reactivity of Hydrazine and Hydroxylamine Derivatives
Although certain aromatic amines such as 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS, A350), 2-aminoacridone (A6289) and 8-aminopyrene-1,3,6-trisulfonic acid (APTS, A6257; ) have been extensively utilized to modify reducing sugars for analysis and sequencing, the most reactive reagents for forming stable conjugates of aldehydes and ketones are usually hydrazine derivatives, including hydrazides, semicarbazides and carbohydrazides (Figure 3.3.3), as well as hydroxylamine derivatives. Hydrazine derivatives react with ketones to yield relatively stable hydrazones (Figure 3.3.4), and with aldehydes to yield hydrazones that are somewhat less stable, though they may be formed faster. Hydroxylamine derivatives (aminooxy compounds) react with aldehydes and ketones to yield oximes. Oximes are superior to hydrazones with respect to hydrolytic stability. Both hydrazones and oximes can be reduced with sodium borohydride (NaBH4) to further increase the stability of the linkage. Rates and yields of aldehyde reactions with hydrazine and hydroxylamine derivatives are substantially enhanced by aniline catalysis. This chemistry is sufficiently mild and efficient to be applicable for labeling periodate-oxidized sialylated glycoproteins on the surface of live cells.
Figure 3.3.3 Structures of A) a hydrazide, B) a semicarbazide and C) a carbohydrazide.
Figure 3.3.4 Modifying aldehydes and ketones with hydrazine derivatives.
Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with Visible Light
We offer a large number of fluorescent hydrazine and hydroxylamine derivatives for reaction with aldehydes or ketones (Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2). Because they are more photostable than the fluorescein derivatives, the Alexa Fluor, BODIPY and Texas Red hydrazides should be among the most sensitive reagents for detecting aldehydes and ketones in laser-excited chromatographic methods. However, with the exception of the Alexa Fluor 555 and Alexa Fluor 647 hydrazides and the Alexa Fluor 647 hydroxylamine, the Alexa Fluor reagents are mixed isomers and may resolve into multiple peaks when analyzed with high-resolution separation techniques. Fluorescent hydrazides and hydroxylamines are extensively used for labeling glycans via derivatization of aldehydes generated after periodate oxidation or via coupling to the reducing terminus. Alexa Fluor 488 hydroxylamine (A30629, ) is particularly useful for detecting aldehyde groups at abasic DNA lesions, similar to the biotinylated hydroxylamine ARP described later in this section.
Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with UV Light
Dansyl hydrazine (D100) has been by far the most widely used UV light–excitable hydrazine probe for derivatizing aldehydes and ketones for chromatographic analysis and mass spectrometry. A unique application that has been reported for dansyl hydrazine, but that is likely a general reaction of hydrazine derivatives, is the detection of N-acetylated or N-formylated proteins through transfer of the acyl group to the fluorescent hydrazide. Although dansyl hydrazine has been widely used as a UV light–excitable derivatization reagent, our 7-diethylaminocoumarin and pyrene hydrazides (D355, P101) have much higher absorptivity and fluorescence, which should make their conjugates more detectable than those of dansyl hydrazine.
Polar Fluorescent Hydrazides and Hydroxylamines
Lucifer yellow CH (L453) is most commonly used as an aldehyde-fixable neuronal tracer with visible absorption and emission (). This membrane-impermeant hydrazide also reacts with periodate-oxidized cell-surface glycoproteins, oxidized ribonucleotides and gangliosides. Cascade Blue hydrazide (C687) exhibits high absorptivity (EC >28,000 cm-1M-1), fluorescence quantum yield (0.54) and water solubility (~1%). Like Cascade Blue hydrazide, Alexa Fluor 350 hydrazide (A10439) and Alexa Fluor 350 hydroxylamine (A30627) also have high water solubility and bright blue fluorescence. These sulfonated pyrene and coumarin derivatives have applications similar to those of lucifer yellow CH, including as aldehyde-fixable polar tracers; see Polar Tracers—Section 14.3 for a more complete discussion of this application.
Cell membrane–impermeant aldehyde- and ketone-reactive reagents are also important probes for assessing the topology of peptide and protein exposure on the surface of live cells. Periodate- or galactose oxidase–mediated oxidation of cell-surface glycoproteins and polysaccharides can be used to selectively introduce aldehyde residues on the cell surface, and these aldehydes can then be reacted with a membrane-impermeant hydrazide. The high polarity of our Alexa Fluor hydrazides (A10436, A10437, A10438, A10439, A20501MP, A20502, A30634), Alexa Fluor hydroxylamines (A30627, A30629, A30632), lucifer yellow CH (L453) and Cascade Blue hydrazide (C687) make them the preferred labeling reagents.
NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M20490) has been used to monitor aldehydes and ketones in tobacco smoke and automobile exhaust and also to measure nitrite in water (Detecting Chloride, Phosphate, Nitrite and Other Anions—Section 21.2). NBD methylhydrazine reacts with carbonyl compounds in acidic media, forming the corresponding hydrazones (Figure 3.3.5). Following separation by HPLC, the hydrazones can be detected either by spectrophotometry (using wavelengths corresponding to the absorption maxima of the relevant hydrazone) or by fluorescence spectroscopy using excitation/emission at ~470/560 nm.
Figure 3.3.5 Reaction scheme illustrating the principle of ketone and aldehyde detection by NBD methylhydrazine (M20490).
Biotin Hydrazides and Biotin Hydroxylamine
In addition to the fluorescent hydrazine and hydroxylamine derivatives, we offer several nonfluorescent biotin and DSB-X biotin hydrazides (B1603, B2600, D20653; Biotinylation and Haptenylation Reagents—Section 4.2) and the biotin hydroxylamine derivative ARP (A10550, Biotinylation and Haptenylation Reagents—Section 4.2), each of which can be detected using fluorescent dye– or enzyme-labeled avidin or streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). DSB-X biotin hydrazide, which has moderate affinity for avidin and streptavidin that is rapidly reversed by low concentrations of free biotin, can be used to produce a DSB-X biotin–labeled molecule that reversibly binds avidin or streptavidin affinity matrices (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).
We recommend the biotin hydroxylamine derivative ARP (aldehyde-reactive probe, A10550; ) as our most efficient reagent for incorporating biotins into aldehyde- or ketone-containing cell surfaces. ARP has been used extensively to modify the exposed aldehyde group at abasic lesions in DNA (Figure 3.3.6). A quick and sensitive microplate assay for abasic sites can be performed using ARP. In addition, ARP is membrane permeant, permitting detection of abasic sites in live cells. Once the aldehyde groups in abasic sites are modified by ARP and the cells are fixed and permeabilized, the resulting biotinylated DNA can be detected with fluorescent dye–, Qdot nanocrystal– or enzyme-conjugated streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). Likewise, ARP can be used to detect and capture 4-hydroxynonenal (HNE)–modified proteins. ARP has also been used to immobilize IgG antibodies on streptavidin-coated monolayer surfaces with their binding sites oriented toward the solution phase. An alternative to ARP for detection of protein carbonyls is dinitrophenylhydrazine derivatization followed by immunolabeling with our Alexa Fluor 488 dye–labeled anti-dinitrophenyl antibody (A11097, Anti-Dye and Anti-Hapten Antibodies—Section 7.4).
Figure 3.3.6 Aldehyde-reactive probe (ARP) used to detect DNA damage. The biotin hydroxylamine ARP (A10550) reacts with aldehyde groups formed when reactive oxygen species depurinate DNA. This reaction forms a covalent bond linking the DNA to biotin. The biotin can then be detected using fluorophore- or enzyme-linked streptavidin.
Primary aliphatic and aromatic amines (Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2) can be coupled reversibly to aldehydes and ketones to form hydrolytically unstable Schiff bases (Figure 3.3.7). The reversibility of this modification makes reagents that contain amines less desirable unless the Schiff base is reduced by sodium borohydride or sodium cyanoborohydride. Chemical reduction also retains the amine's original charge. Sequencing of carbohydrate polymers using fluorescent derivatives has usually relied on derivatization of the reducing end of the polymer with a fluorescent amine. Certain aromatic amines have been extensively utilized for coupling to aldehydes, ketones, monosaccharides and the reducing end of carbohydrate polymers:
- 2-Aminoacridone (A6289) forms conjugates that can be separated by HPLC or, as their borate complexes, by polyacrylamide gel electrophoresis, capillary electrophoresis and micellar electrokinetic capillary chromatography (MECC). Starting with as little as 25 µg of a glycoprotein, researchers have efficiently released and purified the carbohydrates, and then derivatized them with 2-aminoacridone for subsequent structural analysis. 2-Aminoacridone derivatives of oligosaccharides have been directly analyzed by MALDI-TOF mass spectrometry. 2-Aminoacridone is also used to prepare fluorogenic substrates for proteases.
- 7-Amino-4-methylcoumarin (A191), which is a common base of protease substrates (Detecting Peptidases and Proteases—Section 10.4), can be used for the reductive derivatization of oligosaccharides.
- 8-Aminopyrene-1,3,6-trisulfonic acid (APTS, A6257) has been extensively used to derivatize carbohydrates prior to separation by gel or capillary electrophoresis. Among the amines we offer, APTS is the aromatic amine that has the most favorable combination of strong absorbance, high quantum yield and ionic charge.
- ANTS (A350) has a high ionic charge, permitting electrophoretic separation of its products with complex oligosaccharides.
The aromatic diamine 1,2-diamino-4,5-dimethoxybenzene (DDB, D1463), which forms heterocyclic compounds with certain aldehydes and ketones, has been used to selectively detect aromatic aldehydes in the presence of aliphatic aldehydes, including carbohydrates. DBB has proven to be a useful reagent for HPLC analysis of the cytotoxic metabolic by-product methylglyoxal in blood samples from diabetic patients.
Alternatively, aldehydes and ketones can be transformed into primary aliphatic amines by reductive amination with ammonia, ethylenediamine or other nonfluorescent diamines. This chemistry is particularly useful because the products can then be coupled with any of the amine-reactive reagents described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 such as the succinimidyl esters of TAMRA dye (C1171, C6121, C6122; Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6). Derivatization by succinimidyl esters has been extensively utilized for tagging oligosaccharides that are to be separated by capillary zone electrophoresis with laser-induced fluorescence detection.
Figure 3.3.7 Modifying aldehydes and ketones with amine derivatives.
|A10439||349.29||L||H2O, DMSO||345||13,000||445||pH 7|
|A30629||895.07||F,D,L||H2O, DMSO||494||77,000||518||pH 7||3, 4, 5|
|A30634||~950||D,L||H2O, DMSO||624||110,000||643||pH 7|
|C356||493.49||L||pH >7, DMF||492||78,000||516||pH 8||6|
|F121||421.43||D,L||pH >7, DMF||492||85,000||516||pH 9||6|