Related Tables

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 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. 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.ref The Pro-Q Emerald 300 and Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain Kits (P21855, P21857; Detecting Protein Modifications—Section 9.4) are based on periodate oxidation of glycoproteins and subsequent labeling with a Pro-Q Emerald dye.ref

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.ref 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 ref (Figure 3.3.1). Moreover, because antibodies are glycosylated at sites distant from the antigen-binding region, modification of periodate-oxidized antibodies by hydrazines ref and hydroxylamines usually does not inactivate the antibody, as sometimes occurs with amine-reactive labeling.

Sodium periodate oxidation of an N-terminal serine
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.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, 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 ref (A30629). Galactose oxidase–modified lipopolysaccharides (LPS) have been modified with Alexa Fluor 488 hydrazide (A10436) to probe for LPS-binding sites on cells.ref 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,ref ligation of a ketone analog of biotin to proteins with a biotin acceptor peptide (BAP) fusion tag by biotin ligase (BirA) ref and co-translational modification of recombinantly tagged proteins by formylglycine-generating enzyme ref (FGE).

Oxidation of the terminal galactose 
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.ref

Introducing a Hydroxylamine into a Biomolecule

The tetrafluorophenyl (TFP) ester of N-(t-BOC)-aminooxyacetic acid 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.

Hydrazines and Hydroxylamines

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.ref 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.ref This chemistry is sufficiently mild and efficient to be applicable for labeling periodate-oxidized sialylated glycoproteins on the surface of live cells.ref

Structures of A) a hydrazide, B) a semicarbazide and C) a carbohydrazide 
Figure 3.3.3 Structures of A) a hydrazide, B) a semicarbazide and C) a carbohydrazide.
Modifying aldehydes and ketones with hydrazine derivatives
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.ref 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.ref Alexa Fluor 488 hydroxylamine (A30629) is particularly useful for detecting aldehyde groups at abasic DNA lesions,ref similar to the biotinylated hydroxylamine ARP described later in this section.

Fluorescent Hydrazine and Hydroxylamine Derivatives Excited with UV Light

Dansyl hydrazine has been by far the most widely used UV light–excitable hydrazine probe for derivatizing aldehydes and ketones for chromatographic analysis and mass spectrometry.ref 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 UV light–excitable derivatization reagent, our 7-diethylaminocoumarin and pyrene hydrazides 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,ref oxidized ribonucleotides ref and gangliosides.ref Cascade Blue hydrazide (C687) exhibits high absorptivity (EC >28,000 cm-1M-1), fluorescence quantum yield (0.54) and water solubility ref (~1%). Like Cascade Blue hydrazide, Alexa Fluor 350 hydrazide (A10439) and Alexa Fluor 350 hydroxylamine 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;ref 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 (A30629, A30632), lucifer yellow CH (L453) and Cascade Blue hydrazide (C687) make them the preferred labeling reagents.

NBD Methylhydrazine

NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan) has been used to monitor aldehydes and ketones in tobacco smoke ref and automobile exhaust ref and also to measure nitrite in water ref (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.

Reaction scheme illustrating the principle of ketone and aldehyde detection
Figure 3.3.5 Reaction scheme illustrating the principle of ketone and aldehyde detection by NBD methylhydrazine.

Biotin Hydrazides and Biotin Hydroxylamine

In addition to the fluorescent hydrazine and hydroxylamine derivatives, we offer biocytin hydrazides (B1603, 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).

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 ref (Figure 3.3.6). A quick and sensitive microplate assay for abasic sites can be performed using ARP.ref In addition, ARP is membrane permeant, permitting detection of abasic sites in live cells.ref 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.ref ARP has also been used to immobilize IgG antibodies on streptavidin-coated monolayer surfaces with their binding sites oriented toward the solution phase.ref 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 ref (A11097, Anti-Dye and Anti-Hapten Antibodies—Section 7.4).

Aldehyde-reactive probe (ARP) used to detect DNA damage 
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.

Aliphatic and Aromatic Amines

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 ref (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 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:

  • 2-Aminoacridone (A6289) forms conjugates that can be separated by HPLC ref or, as their borate complexes, by polyacrylamide gel electrophoresis,ref capillary electrophoresis ref and micellar electrokinetic capillary chromatography ref (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.ref 2-Aminoacridone derivatives of oligosaccharides have been directly analyzed by MALDI-TOF mass spectrometry.ref 2-Aminoacridone is also used to prepare fluorogenic substrates for proteases.ref
  • 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.ref
  • 8-Aminopyrene-1,3,6-trisulfonic acid (APTS, A6257) has been extensively used to derivatize carbohydrates prior to separation by gel or capillary electrophoresis.ref 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.ref

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.ref DBB has proven to be a useful reagent for HPLC analysis of the cytotoxic metabolic by-product methylglyoxal in blood samples from diabetic patients.ref

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 Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 such as the succinimidyl esters of TAMRA dye ref (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.ref

Modifying aldehydes and ketones with amine derivatives
Figure 3.3.7
Modifying aldehydes and ketones with amine derivatives.

Data Table

For a detailed explanation of column headings, see Definitions of Data Table Contents

Cat #MWStorageSolubleAbsECEmSolventNotes
175.19LDMF, DMSO35118,000430MeOH 
523.39D,LH2O42419,000505pH 7 
246.70D,LDMF, DMSO4255200531MeOH1
Alexa Fluor 488 hydrazide
570.48D,LH2O49371,000517pH 7 
Alexa Fluor 568 hydrazide
730.74D,LH2O57686,000599pH 72
Alexa Fluor 594 hydrazide
758.79D,LH2O58897,000613pH 72
Alexa Fluor 350 hydrazide
349.29LH2O, DMSO34513,000445pH 7 
Alexa Fluor 555 hydrazide
~1150D,LH2O554150,000567pH 7 
Alexa Fluor 647 hydrazide
~1200D,LH2O649250,000666pH 7 
Alexa Fluor 350 hydroxylamine584.52F,D,LH2O, DMSO35320,000437MeOH3
Alexa Fluor 488 hydroxylamine
895.07F,D,LH2O, DMSO49477,000518pH 73, 4, 5
Alexa Fluor 647 hydroxylamine
~1220F,D,LH2O, DMSO651250,000672MeOH3
Alexa Fluor 633 hydrazine
~950D,LH2O, DMSO624110,000643pH 7 
N-(t-BOC)-aminooxyacetic acid, TFP339.24F,DDMSO<300 none  
5-(((2-(carbohydrazino)methyl)thio)acetyl)aminofluorescein493.49LpH >7, DMF49278,000516pH 86
Cascade Blue hydrazide
596.44LH2O39930,000421H2O7, 8
dansyl hydrazide265.33LEtOH3364400534MeOH 
DCCH275.31D,LMeCN, DMF42046,000468MeOH 
BODIPY FL hydrazide
306.12F,D,LMeOH, MeCN50371,000510MeOH9
2,3-diaminonaphthalene158.20LDMSO, MeOH3405100377MeOH10
421.43D,LpH >7, DMF49285,000516pH 96
lucifer yellow CH
457.24LH2O42812,000536H2O11, 12
NBD methylhydrazine209.16F,LMeCN48724,000noneMeOH13
1-pyrenebutanoic acid, hydrazide302.38D,LMeCN, DMF34143,000376MeOH14
Texas Red hydrazide
  1. Spectra of this compound are in methanol containing a trace of KOH.
  2. Maximum solubility in water is ~8% for A10437 and A10438.
  3. Aqueous stock solutions should be used within 24 hours; long-term storage is NOT recommended.
  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. Absorption and fluorescence of fluorescein derivatives are pH dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.
  7. 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.
  8. Maximum solubility in water is ~1% for Cascade Blue hydrazide, trisodium salt (C687), ~1% for Cascade Blue hydrazide, tripotassium salt and ~8% for Cascade Blue hydrazide, trilithium salt (C3239).
  9. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
  10. Fluorescence of 2,3-diaminonaphthalene is weak. Reaction with α-ketoaldehydes yields fluorescent benzoquinoxaline derivatives (Abs = 365 nm, Em = 540 nm in H2O at pH 8).ref
  11. The fluorescence quantum yield of lucifer yellow CH in H2O is 0.21.ref
  12. Maximum solubility in water is ~8% for lucifer yellow CH, lithium salt (L453), ~6% for lucifer yellow CH, ammonium salt and ~1% for lucifer yellow CH potassium salt (L1177).
  13. NBD methylhydrazine reacts with aldehydes and ketones in the presence of strong acid, yielding weakly fluorescent hydrazone products.ref Abs = 493 nm, Em = 552 nm in MeOH for reaction product with acetone.
  14. Pyrene derivatives exhibit structured spectra. The absorption maximum is usually about 340 nm with a subsidiary peak at about 325 nm. There are also strong absorption peaks below 300 nm. The emission maximum is usually about 376 nm with a subsidiary peak at 396 nm. Excimer emission at about 470 nm may be observed at high concentrations.

For Research Use Only. Not for use in diagnostic procedures.