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Section 3.2 — Hydrazines and Aromatic Amines for Modifying Aldehydes and Ketones

Aldehydes and ketones are present in a number of low molecular weight molecules such as drugs, steroid hormones, reducing sugars and some metabolic intermediates such as pyruvate and alpha-ketoglutarate. Except for polysaccharides containing free reducing sugars, biopolymers generally lack aldehyde and ketone groups, although there are recent efforts using genetic engineering and metabolic methods to directly express ketone-containing carbohydrates on cell surfaces that have resulted in the reagents ManLev and ManLev tetraacetate (see below) for metabolic labeling of cell surface glycoproteins.ref 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 has stimulated the development of techniques to selectively introduce these functional groups into certain biomolecules, 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 frequently utilized for their analysis by capillary electrophoresis ref and other methods.

Introducing Aldehydes and Ketones into Biomolecules

Periodate Oxidation

The most common method for introducing aldehydes and ketones into polysaccharides and glycoproteins (including antibodies) is by periodate-mediated oxidation of vicinal diols. Alkenes from unsaturated fatty acids and ceramides can be converted to glycols by osmium tetroxide and these oxidized by periodate to aldehydes. Periodate will also oxidize certain beta-aminoethanol derivatives such as the hydroxylysine residues in collagen, as well as methionine (to its sulfoxide) and certain thiols (usually to disulfides). These reactions, however, usually occur at a slower rate than oxidation of vicinal diols. Periodate oxidation of the 3'-terminal ribose provides one of the few methods of selectively modifying RNA.ref Periodate-oxidized ribonucleotides can subsequently be converted to fluorescent nucleotide probes by reaction with fluorescent hydrazines and amines.ref In addition, N-terminal serine and threonine residues of peptides and proteins can be selectively oxidized to aldehyde groups,ref thus allowing highly selective modification of certain proteins such as corticotrophin ref and beta-lactamase.ref Moreover, because antibodies are glycosylated at sites distant from the antigen-binding region, modification of periodate-oxidized antibodies by hydrazines usually does not inactivate the antibody, as sometimes occurs with FITC, TRITC and Texas Red sulfonyl chloride labeling. Researchers have also used the hydrazine derivatives described in this section to detect periodate-oxidized glycoproteins in gels.ref Our Pro-Q Emerald 300 and Pro-Q Fuchsia Glycoprotein Stain Kits for Gels and for Blots (Section 9.2) are based on periodate oxidation of glycoproteins. The Pro-Q Emerald 300 reagent provides sensitivity and linearity of response for glycoprotein detection that is superior to any previous technology.

Enzyme-Mediated Oxidation with Galactose Oxidase

A second specific method for introducing aldehydes into biomolecules is through the use of galactose oxidase, an enzyme that oxidizes galactose residues to aldehydes, particularly in glycoproteins.ref This method was used to label live corn and rose protoplasts with hydrazine derivatives and aromatic amines.ref 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, galactose has been specifically inserted into the carbohydrate moiety of rhodopsin using a galactosyl transferase.ref Because galactose oxidase–mediated oxidation liberates a molecule of hydrogen peroxide for each molecule of aldehyde that is formed, horseradish peroxidase–catalyzed oxidation of Amplex Red 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. Our Amplex Red Galactose/Galactose Oxidase Assay Kit (A-22179, Section 10.2) can be used for this assay.

Metabolic Incorporation of Ketone-Containing Carbohydrates

The oligosaccharide components of cell-surface glycoproteins play a role in the interactions that regulate many important biological processes, from cell–cell adhesion to signal transduction. Because modification of these surface groups affects the behavior of the cell,ref strategies that introduce alternative surface groups to the cell provide researchers with novel methods for tagging specific cell populations.

Sialic acids are the most abundant terminal components of oligosaccharides on mammalian cell-surface glycoproteins and are synthesized from the six-carbon precursor N-acetylmannosamine.ref When cells in culture are incubated with 25 mM N-levulinoyl-D-mannosamine structurenote (ManLev; L-20492) or — much more efficiently — with 25 µM ManLev tetraacetate (L-20493), this ketone-containing monosaccharide serves as a substrate in the oligosaccharide synthesis pathway, resulting in ketone-tagged cell-surface oligosaccharides.ref And because ketones are rare in cells, reaction with 2 µM biotinylated aldehyde-reactive probe structure (ARP, A-10550) followed by a fluorescent avidin or streptavidin conjugate (Section 7.5, Table 7.2) provides a means of identifying and tracing tagged cells by either imaging photo or flow cytometry. We find ARP to be much more reactive than other biotin hydrazides and that fluorescent hydrazides usually are not suitable for this detection because the concentrations that are required can result in their internalization in live cells by pinocytosis.

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 (L-453) can be conjugated to surrounding biomolecules by common aldehyde fixatives in order to preserve the dye's staining pattern during subsequent tissue manipulations.ref Glutaraldehyde has also been used to couple biotin hydrazides (Section 4.2) directly to nucleic acids,ref a reaction that is potentially useful for conjugating fluorescent hydrazine derivatives to DNA.

Hydrazines

Reactivity of Hydrazine Derivatives

Although certain aromatic amines such as 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS, A-350), 2-aminoacridone (A-6289) and 8-aminopyrene-1,3,6-trisulfonic acid structure (APTS, A-6257) 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. Hydrazine derivatives react with ketones to yield relatively stable hydrazones (Figure 3.2), and with aldehydes to yield hydrazones that are somewhat less stable though they may be formed faster. These hydrazones can be reduced with sodium borohydride to further increase the stability of the linkage. Hydrazine derivatives also have amine-like reactivity and, in some cases, can be coupled to water-soluble carbodiimide-activated carboxylic acid groups in drugs, peptides and proteins;ref see Section 3.3 for more details. Because the fluorescence of the hydrazide-containing reagents usually does not change significantly upon reaction with aldehydes or ketones, especially after chemical reduction, the excess reagent required to drive the equilibrium reaction must be separated from the conjugate by washing, gel filtration, dialysis or other separation techniques.

Fluorescent Hydrazine Derivatives Excited with Visible Light

Molecular Probes offers a large number of fluorescent hydrazine derivatives for reaction with aldehydes or ketones (Table 3.1). The most extensively used visible light–excitable fluorescent hydrazine derivative, fluorescein-5-thiosemicarbazide structure (F-121), has been coupled to a wide variety of biomolecules, including:

Probably the brightest hydrazides, however, are the Alexa Fluor 488,photo Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 633 hydrazides (A-10436, A-10437, A-10438, A-20500), the fluorescein hydrazide with the extra spacer between the fluorophore and the reactive group ref (C-356), BODIPY FL hydrazide (D-2371) and Texas Red hydrazide (T-6256), which is >90% single isomer by HPLC. HPLC detection of progesterone and 17-hydroxyprogesterone after derivatization with BODIPY FL hydrazide proved to be over 50 times more sensitive than reported HPLC methods with dansyl hydrazine.ref Texas Red hydrazide was used to assay the extent of periodate oxidation of saccharide units in glycoproteins ref and to detect proteins on blots.ref Because they are more photostable than the fluorescein derivatives, the Alexa Fluor, BODIPY and Texas Red hydrazides should be the best reagents for detecting aldehydes and ketones in laser-excited chromatographic methods. However, the Alexa Fluor hydrazides are mixed isomers and may resolve into multiple peaks when analyzed with high-resolution techniques.

Fluorescent Hydrazine Derivatives Excited with UV Light

Dansyl hydrazine (D-100) has been by far the most widely used UV light–excitable hydrazine probe for derivatizing aldehydes and ketones for chromatographic analysis. It has been used to modify:

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.ref Although dansyl hydrazine has been widely used as a derivatization reagent, our Marina Blue probes, 7-diethylaminocoumarin, pyrene and Dapoxyl hydrazides (M-10432, D-355, P-101, D-10430) have much higher absorptivity and fluorescence, which should make their conjugates about 10 times more detectable than those of dansyl hydrazine. Dapoxyl sulfonyl hydrazine (D-10430) combines good UV absorption (epsilon~27,000 cm-1M-1 at 374 nm) with a huge Stokes shift (up to ~200 nm), permitting detection of its conjugates at wavelengths where autofluorescence is minimal. The fluorescence of Dapoxyl derivatives is also exceptionally sensitive to the environment ref (Figure 1.15).

We also offer the shorter-wavelength derivatization reagent, FMOC hydrazine (F-6290), which was employed in the HPLC determination of neutral and amino monosaccharides in glycoproteins.ref The FMOC-derivatized sugar hydrazones were detected by fluorescence (excitation/emission wavelengths 270/320 nm) with a detection limit of 0.05–0.4 picomoles and by UV absorption (at 263 nm) with a detection limit of 1–3 picomoles.ref FMOC hydrazine has been used to analyze blood sugars ref and reducing sugars in aqueous samples such as urine.ref

Lucifer Yellow, Cascade Blue and Alexa Fluor 350 Hydrazides

Lucifer yellow CH (L-453) is most commonly used as an aldehyde-fixable neuronal tracer. This membrane-impermeant hydrazide also reacts with periodate-oxidized cell-surface glycoproteins,ref oxidized ribonucleotides ref and gangliosides.ref Cascade Blue hydrazide (C-687, Patents) exhibits high absorptivity (epsilon >28,000 cm-1M-1), fluorescence quantum yield (0.54) and water solubility (~1%).ref Like Cascade Blue hydrazide, our Alexa Fluor 350 hydrazide (A-10439) also has high water solubility and bright blue fluorescence. These sulfonated pyrene and coumarin derivatives have applications similar to those of lucifer yellow CH.ref See Section 14.3 for a discussion of the use of these probes as aldehyde-fixable polar tracers.

NBD Methylhydrazine

NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M-20490) has been used to monitor aldehydes and ketones in automobile exhaust ref and also to measure nitrite in water ref (Section 22.2). NBD methylhydrazine reacts with carbonyl compounds in acidic media, forming the corresponding hydrazones (Figure 3.6). Following separation by HPLC, the hydrazones can be detected either by UV/VIS spectroscopy (using wavelengths corresponding to the absorption maxima of the relevant hydrazone), or by fluorescence spectroscopy using excitation/emission maxima of ~470/560 nm.

Biotin Hydrazine and Hydroxylamine Derivatives

In addition to the fluorescent hydrazine derivatives, we offer several nonfluorescent biotin hydrazides and a biotin hydroxylamine derivative (Section 4.2) that can be detected using fluorescent or enzyme-labeled avidin or streptavidin (Section 7.5). We recommend the biotinylated aldehyde-reactive probe ARP structure (A-10550) as the most efficient reagent for incorporation of biotins into aldehyde- or ketone-containing cell surfaces.

Labeling Cell-Surface Carbohydrates

Cell membrane–impermeant reagents are important probes for assessing the topology of peptide and protein exposure on the surface of live cells. Some of the reagents in this chapter are useful for this application. Periodate- or galactose oxidase–mediated oxidation of cell-surface glycoproteins and polysaccharides can be used to selectively introduce aldehyde residues on the cell's surface; these can then be reacted with a membrane-impermeant hydrazide. The high polarity of our Alexa Fluor hydrazides (A-10436, A-10437, A-10438, A-10439, A-20500), lucifer yellow CH (L-453) and Cascade Blue hydrazide (C-687) make them the preferred labeling reagents. These methods have been used to label plant protoplasts with lucifer yellow CH and Texas Red hydrazide ref (T-6256) and to label erythrocyte ghosts with fluorescein-5-thiosemicarbazide (F-121), lucifer yellow CH, 1-pyrenebutanoic acid hydrazide (P-101) and several other dyes.ref Our anti-dye antibodies and avidin derivatives (Section 7.3, Section 7.5) may facilitate isolation and detection of the modified residues.

Staining Lipopolysaccharides in Gels

Molecular Probes' Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P-20495) provides a simple, rapid and highly sensitive method for staining lipopolysaccharides (LPS) in gels. LPS, also known as endotoxins, are a family of complex glycolipid molecules located on the surface of gram-negative bacteria. LPS play a large role in protecting the bacterium from host defense mechanisms and antibiotics. The structure of this important class of molecules can be analyzed by SDS polyacrylamide gel electrophoresis, during which the heterogeneous mixture of polymers separates into a characteristic ladder pattern. This ladder has conventionally been detected using silver staining.ref However, despite the long and complex procedures required, silver staining provides poor sensitivity and cannot differentiate LPS from proteins in the sample. An alternative staining method that makes use of the reaction of the carbohydrates with detectable hydrazides obtains higher sensitivity, but requires blotting to a membrane and time and labor-intensive procedures.

By comparison, the staining technology used in the Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit vastly simplifies detection of LPS in SDS-polyacrylamide gels. The key to this novel methodology is our bright green–fluorescent Pro-Q Emerald 300 dye, which covalently binds to periodate-oxidized carbohydrates of LPS. Using this dye, it is possible to detect as little as 200 pg of LPS in just a few hours. The sensitivity is at least 50–100 times that of silver staining and requires much less hands-on time. This dye is also used in our Pro-Q Emerald 300 Glycoprotein Stain Kits (P-21855, P-21856, P-21857; Section 9.2) and may be useful for detection of other molecules containing carbohydrates or aldehydes. The bright green fluorescence is easy to visualize using a simple UV transilluminator. To detect contaminating proteins in the sample, the same gel can also be stained with SYPRO Ruby protein gel stain (S-12000, S-12001; Section 9.2). The Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit contains:

Sufficient materials are supplied to stain ten 8 cm times 10 cm gels, 0.5–0.75 mm thick.

Aromatic Amines

Primary aliphatic and aromatic amines (Table 3.1) can couple reversibly with aldehydes and ketones to form hydrolytically unstable Schiff bases ref (Figure 3.2). The reversibility of this modification makes reagents that contain amines less desirable unless the Schiff base is reduced to a stable amine derivative by sodium borohydride ref or sodium cyanoborohydride.ref 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.ref Certain aromatic amines have been extensively utilized for coupling to aldehydes, ketones, monosaccharides and the reducing end of carbohydrate polymers:

The aromatic diamine 1,2-diamino-4,5-dimethoxybenzene (DDB, D-1463), 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.ref It has also been employed for fluorometric determination of ascorbic acid,ref lactic acid,ref alpha-keto acids ref and N-acetylneuraminic acids ref by HPLC. Endogenous leucine-enkephalin and other tyrosine-containing peptides can be detected by HPLC at concentrations as low as 5.6 picomoles per gram of rat brain tissue using a Reimer–Tiemann reaction followed by derivatization with DDB.ref The dimly fluorescent aromatic diamine, 2,3-diaminonaphthalene (D-7918), may be similarly useful for detection of ketones and alpha-keto acids through formation of fluorescent heterocycles.

Alternatively, aldehydes and ketones can be transformed into primary aliphatic amines by reductive amination with ammonia, ethylenediamine or other nonfluorescent diamines.ref This chemistry is particularly useful because the products can then be coupled with any of the amine-reactive reagents described in Chapter 1 such as the succinimidyl esters of TAMRA (C-1171, C-6121, C-6122; Section 1.6). Derivatization by succinimidyl esters has been extensively utilized for tagging of oligosaccharides that are to be separated by capillary zone electrophoresis with laser-induced fluorescence detection.ref

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