104 Chapter 3 — Reagents for Modifying Groups Other Than Thiols or Amines www.probes.com

3.2 Hydrazines and Aromatic Amines forModifying Aldehydes and Ketones

Aldehydes and ketones are present in a number of low molecular weight moleculessuch as drugs, steroid hormones, reducing sugars and some metabolic intermediates suchas pyruvate and α-ketoglutarate. Except for polysaccharides containing free reducingsugars, biopolymers generally lack aldehyde and ketone groups. Even those aldehydesand ketones that are found in the open-ring form of simple carbohydrates are usually inequilibrium with the closed-ring form of the sugar. The infrequent occurrence of alde-hydes and ketones has stimulated the development of techniques to selectively introducethese functional groups into certain biomolecules, thus providing unique sites for chemi-cal modification and greatly extending the applications of the probes found in this sec-tion. Recent efforts to directly express ketone-containing carbohydrates on cell surfaceshave led to the introduction of the reagents ManLev and ManLev tetraacetate (see below)for metabolic labeling of cell-surface glycoproteins.1–6 Fluorescent modification of alde-hyde or carboxylic acid groups in carbohydrates is frequently utilized for their analysisby capillary electrophoresis 7 and other methods.

Introducing Aldehydes and Ketones into Biomolecules

Periodate OxidationThe most common method for introducing aldehydes and ketones into polysaccha-

rides and glycoproteins (including antibodies) is by periodate-mediated oxidation ofvicinal diols. In addition, alkenes from unsaturated fatty acids and ceramides can beconverted to glycols by osmium tetroxide and then oxidized by periodate to aldehydes.Periodate will also oxidize certain β-aminoethanol derivatives such as the hydroxylysineresidues in collagen, as well as methionine (to its sulfoxide) and certain thiols (usually todisulfides). These reactions, however, usually occur at a slower rate than oxidation ofvicinal diols. Periodate oxidation of the 3′-terminal ribose provides one of the few meth-ods of selectively modifying RNA.8,9 Periodate-oxidized ribonucleotides can subsequent-ly be converted to fluorescent nucleotide probes by reaction with fluorescent hydrazinesand amines.10 In addition, N-terminal serine and threonine residues of peptides and pro-teins can be selectively oxidized by periodate to aldehyde groups 11–14 (Figure 3.1), thusallowing highly selective modification of certain proteins such as corticotrophin 15,16 andβ-lactamase.17 Moreover, because antibodies are glycosylated at sites distant from theantigen-binding region, modification of periodate-oxidized antibodies by hydrazinesusually does not inactivate the antibody, as sometimes occurs with FITC, TRITC andTexas Red sulfonyl chloride labeling. Researchers have also used the hydrazine deriva-tives described in this section to detect periodate-oxidized glycoproteins in gels.18 OurPro-Q Emerald 300, Pro-Q Emerald 488 and Pro-Q Fuchsia Glycoprotein Stain Kits forGels and for Blots (Section 9.4, Table 9.6) are based on periodate oxidation of glycopro-teins. The Pro-Q Emerald 300 and Pro-Q Emerald 488 reagents provide sensitivity andlinearity of response for glycoprotein detection that is superior to any previous techno-logy (Figure 9.56).

Figure 3.8 Oxidation of the terminal galactose resi-due of a glycoprotein, glycolipid or polysaccharideresults in the generation of an aldehyde, which canreact with hydrazines or primary amine–containingcompounds.

Figure 3.9 L-20492 N-levulinoyl-D-mannosamine(ManLev).

Figure 3.10 A-10550 N-(aminooxyacetyl)-N ′-(D-biotinoyl) hydrazine, trifluoroacetic acid salt(ARP).

Product List — 3.1 Reagents for Modifying Alcohols

Used in combination with ARP andstreptavidin reagents, ManLev andManLev tetraacetate provide a totallyunique metabolic method for labelingcell surface glycoproteins in live cells.

Cat # Product Name Unit SizeA-1440 9-anthroylnitrile .................................................................................................................................................................................................... 25 mgD-2281 m-dansylaminophenylboronic acid ....................................................................................................................................................................... 100 mgD-16 5-(4,6-dichlorotriazinyl)aminofluorescein (5-DTAF) *single isomer* ................................................................................................................... 100 mgD-1446 7-diethylaminocoumarin-3-carbonyl azide ........................................................................................................................................................... 25 mgF-6218 fluorescein-5-carbonyl azide, diacetate ................................................................................................................................................................. 10 mgM-1445 7-methoxycoumarin-3-carbonyl azide .................................................................................................................................................................. 25 mgM-25 N-methylisatoic anhydride *high purity* .............................................................................................................................................................. 1 gT-6219 tetramethylrhodamine-5-carbonyl azide ............................................................................................................................................................... 5 mg

105

Enzyme-Mediated Oxidation with Galactose OxidaseA second specific method for introducing aldehydes into biomolecules is through the

use of galactose oxidase, an enzyme that oxidizes terminal galactose residues to alde-hydes, particularly in glycoproteins.19–23 This method was used to label live corn and roseprotoplasts with hydrazine derivatives and aromatic amines.24 The introduction of galac-tose residues can be especially advantageous for structural studies because it provides ameans of selectively labeling specific sites on biomolecules. For example, galactose hasbeen specifically inserted into the carbohydrate moiety of rhodopsin using a galactosyltransferase.23 Galactose oxidase–modified lipopolysaccharides (LPS) have been modifiedwith Alexa Fluor 488 hydrazide (A-10436) to probe for LPS-binding sites on cells.25

Because galactose oxidase–mediated oxidation liberates a molecule of hydrogen peroxidefor each molecule of aldehyde that is formed (Figure 3.8), horseradish peroxidase–catalyzed oxidation of the Amplex Red reagent to red-fluorescent resorufin by hydrogenperoxide provides a ready means by which the number of aldehyde residues introducedinto a biomolecule, including on a cell surface, can be quantitated. Our Amplex RedGalactose/Galactose Oxidase Assay Kit (A-22179, Section 10.5) can be used for this assay.

Metabolic Incorporation of Ketone-Containing CarbohydratesThe oligosaccharide components of cell-surface glycoproteins play a role in the inter-

actions that regulate many important biological processes, from cell–cell adhesion tosignal transduction. Because modification of these surface groups affects the behavior ofthe cell,26 strategies that introduce alternative surface groups to the cell provide research-ers with novel methods for tagging specific cell populations.

Sialic acids are the most abundant terminal components of oligosaccharides onmammalian cell-surface glycoproteins and are synthesized from the six-carbon precursorN-acetylmannosamine.27 When cells in culture are incubated with 25 mM N-levulinoyl-D-mannosamine 28 (ManLev, L-20492; Figure 3.9) or — much more efficiently — with25 µM ManLev tetraacetate (L-20493), this ketone-containing monosaccharide serves asa substrate in the oligosaccharide synthesis pathway, resulting in ketone-tagged cell-surface oligosaccharides.1,3,5,6,29–31 And because ketones are rare in cells, reaction with2 mM biotinylated aldehyde-reactive probe (ARP, A-10550; Figure 3.10) followed by afluorescent avidin or streptavidin conjugate (Section 7.6, Table 7.17) provides a means ofidentifying and tracing tagged cells either by imaging (Figure 3.11) or by flow cytometry.We find that ARP is much more reactive than the biotin hydrazides and that fluorescenthydrazides usually are not suitable for this detection because the required concentrationscan result in their internalization in live cells by pinocytosis.

Coupling Hydrazines and Amines to Amine-Containing Biomolecules WithoutIntroducing Aldehydes and Ketones

Common tissue fixatives such as formaldehyde and glutaraldehyde can be used tocouple hydrazine and amine derivatives to proteins and other amine-containing polymers.For example, lucifer yellow CH (L-453) can be conjugated to surrounding biomoleculesby common aldehyde-based fixatives in order to preserve the dye’s staining pattern dur-ing subsequent tissue manipulations.32 Glutaraldehyde has also been used to couplebiotin hydrazides (Section 4.2) directly to nucleic acids,33 a reaction that is potentiallyuseful for conjugating fluorescent hydrazine derivatives to DNA.

Hydrazines

Reactivity of Hydrazine DerivativesAlthough 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(APTS, A-6257; Figure 3.12) have been extensively utilized to modify reducing sugarsfor analysis and sequencing, the most reactive reagents for forming stable conjugates ofaldehydes and ketones are usually hydrazine derivatives, including hydrazides, semicar-bazides and carbohydrazides (Figure 3.13). Hydrazine derivatives react with ketones toyield relatively stable hydrazones (Figure 3.14), and with aldehydes to yield hydrazonesthat are somewhat less stable, though they may be formed faster. These hydrazones canbe reduced with sodium borohydride (NaBH4) to further increase the stability of the

Figure 3.11 Metabolic labeling of cell-surface gly-coproteins. Jurkat cells were cultured for 24 hoursin the presence of 25 µM ManLev tetraacetate(L-20493). Following treatment, the cell surface–expressed, ketone-containing glycoproteins werereacted with a biotin-containing ketone-reactive re-agent, ARP (A-10550), and then visualized by incu-bation with Alexa Fluor 488 streptavidin (S-11223).The metabolically labeled glycoproteins were de-tected using a fluorescence microscope equippedwith a filter set appropriate for FITC. No fluores-cence was observed in control cells that were nottreated with ManLev tetraacetate (data not shown).

Figure 3.12 A-6257 8-aminopyrene-1,3,6-trisul-fonic acid, trisodium salt (APTS).

Figure 3.13 Structures of A) a hydrazide, B) asemicarbazide and C) a carbohydrazide.

A

B

C

Section 3.2

106 Chapter 3 — Reagents for Modifying Groups Other Than Thiols or Amines www.probes.com

linkage. Hydrazine derivatives also have amine-like reactivity and, in some cases, can becoupled to water-soluble carbodiimide–activated carboxylic acid groups in drugs, pep-tides and proteins;34–36 see Section 3.3 for more details. Because the fluorescence of thehydrazide-containing reagents does not usually change significantly upon reaction withaldehydes or ketones, especially after chemical reduction, the excess reagent required todrive the equilibrium reaction must be separated from the conjugate by washing, gelfiltration, dialysis or other separation techniques.

Fluorescent Hydrazine Derivatives Excited with Visible LightMolecular 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 (F-121,Figure 3.15), has been coupled to a wide variety of biomolecules, including:

• L-Aspartase-α-decarboxylase 37

• Enzyme-oxidized live plant protoplasts 24

• Immunoglobulins 38

• Na+/K+-ATPase glycoprotein 39

• Periodate-oxidized glycoproteins in gels 18

• Periodate-oxidized RNA 8,9,40–43

• Thrombin and antithrombin 44

Probably the brightest hydrazides, however, are the Alexa Fluor 488 (Figure 3.16),Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594 (Figure 14.21), Alexa Fluor 633and Alexa Fluor 647 hydrazides (A-10436, A-20501, A-10437, A-10438, A-20500,A-20502), the fluorescein hydrazide with the extra spacer between the fluorophore andthe reactive group 45 (C-356), BODIPY FL hydrazide (D-2371) and Texas Red hydrazide(T-6256), which is >90% single isomer by HPLC. HPLC detection of progesterone and17-hydroxyprogesterone after derivatization with BODIPY FL hydrazide proved to beover 50 times more sensitive than reported HPLC methods with dansyl hydrazine.46

Texas Red hydrazide was used to assay the extent of periodate oxidation of saccharideunits in glycoproteins 47 and to detect proteins on blots.48 Because they are more photo-stable than the fluorescein derivatives, the Alexa Fluor, BODIPY and Texas Red hy-drazides should be the best reagents for detecting aldehydes and ketones in laser-excitedchromatographic methods. However, with the exception of Alexa Fluor 555 and AlexaFluor 647 hydrazides, the Alexa Fluor hydrazides are mixed isomers and may resolveinto multiple peaks when analyzed with high-resolution techniques.

Fluorescent Hydrazine Derivatives Excited with UV LightDansyl hydrazine (D-100) has been by far the most widely used UV light–excitable

hydrazine probe for derivatizing aldehydes and ketones for chromatographic analysis. Ithas been used to modify:

• Bile acids 49–51

• Glycoproteins 52–54

• Oligosaccharides 55

Figure 3.16 The APR motor neuron of a larvalmoth, Manduca sexta, labeled by the intracellularinjection of Alexa Fluor 488 hydrazide, sodiumsalt (A-10436, A-10440). This pseudocolored im-age was created by combining 21 optical sectionsobtained using a scanning confocal microscopeequipped with a bandpass filter appropriate forfluorescein. Image contributed by Jack Gray andWalter K. Metcalfe, Institute of Neuroscience,University of Oregon.

Figure 3.14 Modifying aldehydes and ketones withhydrazine derivatives.

Figure 3.15 F-121 fluorescein-5-thiosemicarbazide.

• Reducing sugars 56–60

• RNA 61–63

• Steroids 51,64–67

A unique application that has been reported for dansyl hydrazine, but that is likely ageneral reaction of hydrazine derivatives, is the detection of N-acetylated or N-formylatedproteins through transfer of the acyl group to the fluorescent hydrazide.68,69 Althoughdansyl hydrazine has been widely used as a derivatization reagent, our 7-diethylamino-coumarin and pyrene hydrazides (D-355, P-101) have much higher absorptivity andfluorescence, which should make their conjugates about 10 times more detectable thanthose of dansyl hydrazine.

We also offer the shorter-wavelength derivatization reagent, FMOC hydrazine(F-6290), which was employed in the HPLC determination of neutral and aminomonosaccharides in glycoproteins.70,71 The FMOC-derivatized sugar hydrazones weredetected by fluorescence (excitation/emission wavelengths 270/320 nm) with a detectionlimit of 0.05–0.4 picomoles and by UV absorption (at 263 nm) with a detection limit of

Water-soluble hydrazine derivatives areextensively used as polar tracers, par-ticularly in neurons (see Section 14.3).Our Alexa Fluor hydrazides are un-doubtedly the best probes available forsuch applications.

107

1–3 picomoles.70 FMOC hydrazine has been used to analyze blood sugars 72 and reducingsugars in aqueous samples such as urine.73

Lucifer Yellow, Cascade Blue and Alexa Fluor 350 HydrazidesLucifer yellow CH (L-453) is most commonly used as an aldehyde-fixable neuronal

tracer with visible absorption and emission. This membrane-impermeant hydrazide alsoreacts with periodate-oxidized cell-surface glycoproteins,39,74 oxidized ribonucleotides 75

and gangliosides.76 Our patented Cascade Blue hydrazide (C-687) exhibits high absorp-

Derivative Hydrazines * Amines *

Acridone A-6289

Alexa Fluor 350 A-10439

Alexa Fluor 488 A-10436

Alexa Fluor 555 A-20501

Alexa Fluor 568 A-10437

Alexa Fluor 594 A-10438

Alexa Fluor 633 A-20500

Alexa Fluor 647 A-20502

ARP † ‡ A-10550

4-Azido-2,3,5,6-tetrafluorobenzyl

A-10662

Biotin ‡ B-1603, B-2600, D-20653 A-1593, A-1594, B-1592,B-1596, N-6356

t-BOC M-6248

BODIPY FL D-2371 D-2390

BODIPY TR D-6251

Cascade Blue C-687 C-621

Coumarin D-355 A-191

Dansyl D-100 D-112, D-113

Dapoxyl D-10460

2,3-Diaminonaphthalene D-7918

Dimethoxybenzene D-1463

Dinitrophenyl D-1552

Eosin A-117

Fluorescein C-356, F-121 A-10466, A-1351, A-1353,A-1363

FMOC F-6290

Lucifer yellow L-453 A-1339

Naphthalene A-91, A-350, A-22840

NBD M-20490

Oregon Green 488 O-10465

Pyrene P-101 A-6257, P-2421

QSY 7 Q-10464

QSY 35 Q-20540

Rhodamine A-1318, L-2424

Texas Red T-6256 T-2425

* Hydrazine derivatives are listed in Section 3.2 and amine derivatives are listed in Section 3.3, except forD-1552, which appears in Section 12.3, and A-10662, which appears in Section 5.3. † ARP is the abbreviationfor N-(aminooxyacetyl)-N′-(D-biotinoyl)hydrazine. ‡ Biotin and desthiobiotin DSB-X derivatives are listed inSection 4.2.

Table 3.1 Molecular Probes’ hydrazine and amine derivatives.

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Section 3.2

108 Chapter 3 — Reagents for Modifying Groups Other Than Thiols or Amines www.probes.com

tivity (ε >28,000 cm-1M-1), fluorescence quantum yield (0.54) and water solubility(~1%).77 Like Cascade Blue hydrazide, our Alexa Fluor 350 hydrazide (A-10439) alsohas high water solubility and bright-blue fluorescence. These sulfonated pyrene andcoumarin derivatives have applications similar to those of lucifer yellow CH.78 See Sec-tion 14.3 for a discussion of the use of these probes as aldehyde-fixable polar tracers.

NBD MethylhydrazineNBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M-20490)

has been used to monitor aldehydes and ketones in tobacco smoke 79 and automobileexhaust 80 and also to measure nitrite in water 81 (Section 22.2). NBD methylhydrazinereacts with carbonyl compounds in acidic media, forming the corresponding hydrazones(Figure 3.17). Following separation by HPLC, the hydrazones can be detected either byUV/Vis spectroscopy (using wavelengths corresponding to the absorption maxima of therelevant hydrazone) or by fluorescence spectroscopy using excitation/emission maximaof ~470/560 nm.

Biotin Hydroxylamine and Related DerivativesIn addition to the fluorescent hydrazine derivatives, we offer several nonfluorescent

biotin hydrazides and a biotin hydroxylamine derivative (Section 4.2) that can be detect-ed using fluorescent dye– or enzyme-labeled avidin or streptavidin (Section 7.6). Werecommend the biotinylated aldehyde-reactive probe ARP (A-10550, Figure 3.10) as themost efficient reagent for incorporation of biotins into aldehyde- or ketone-containingcell surfaces. DSB-X biotin hydrazide (D-20653, Section 4.2) has relatively low affinityfor avidin derivatives and can therefore be used to to produce a DSB-X biotin–labeledmolecule that reversibly binds avidin or streptavidin affinity matrices (Section 7.6) or thesuperparamagnetic Captivate ferrofluid conjugate of streptavidin (C-21476, Section 7.6).

Labeling Cell-Surface CarbohydratesCell 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 thischapter are useful for this application. Periodate- or galactose oxidase–mediated oxida-tion of cell-surface glycoproteins and polysaccharides can be used to selectively intro-duce aldehyde residues on the cell 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-20501, A-20502, A-20500), lucifer yellow CH (L-453)and Cascade Blue hydrazide (C-687) make them the preferred labeling reagents. Thesemethods have been used to label plant protoplasts with lucifer yellow CH and Texas Redhydrazide 24 (T-6256) and to label erythrocyte ghosts with fluorescein-5-thiosemicarba-zide (F-121), lucifer yellow CH, 1-pyrenebutanoic acid hydrazide (P-101) and severalother dyes.39 Our anti-dye antibodies and avidin derivatives (Section 7.4, Section 7.6)may facilitate isolation and detection of the modified residues.

Staining Lipopolysaccharides in GelsMolecular 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 (Figure 3.18, Figure 3.19, Figure 3.20). LPS, also known as endotoxins, area family of complex glycolipid molecules located on the surface of gram-negative bacte-ria. LPS play a large role in protecting the bacterium from host defense mechanisms andantibiotics. The structure of this important class of molecules can be analyzed by SDS-polyacrylamide gel electrophoresis, during which the heterogeneous mixture of polymersseparates into a characteristic ladder pattern. This ladder has conventionally been detect-ed using silver staining.82–84 However, despite the long and complex procedures required,silver staining provides poor sensitivity and cannot differentiate LPS from proteins in thesample. An alternative staining method that makes use of the reaction of the carbohy-drates with detectable hydrazides obtains higher sensitivity, but requires blotting to amembrane and time- and labor-intensive procedures.85–89

By comparison, the staining technology used in the Pro-Q Emerald 300 Lipopolysac-charide Gel Stain Kit vastly simplifies detection of LPS in SDS-polyacrylamide gels. Thekey to this novel methodology is our bright green–fluorescent Pro-Q Emerald 300 dye,

Figure 3.17 Reaction scheme illustrating the prin-ciple of ketone and aldehyde detection by NBDmethylhydrazine (M-20490).

Figure 3.18 Lipopolysaccharide staining with thePro-Q emerald 300 Lipopolysaccharide Gel StainKit. Lipopolysaccharides (LPS) were electrophore-sed through a 13% acrylamide gel and stained us-ing the Pro-Q Emerald 300 Lipopolysaccharide GelStain Kit (P-20495). From left to right, the lanescontain: CandyCane glycoprotein molecular weightstandards (~250 ng/band), blank, 4, 1 and 0.25 µgof LPS from Eschericia coli smooth serotype055:B5 and 4, 1 and 0.25 µg of LPS from E. colirough mutant EH100 (Ra mutant).

109

which covalently binds to periodate-oxidized carbohydrates of LPS. This dye allowsthe detection of as little as 200 pg of LPS in just a few hours. The sensitivity is at least50–100 times that of silver staining and requires much less hands-on time. This dyeis also used in our Pro-Q Emerald 300 Glycoprotein Stain Kits (P-21855, P-21856,P-21857; Section 9.4) and may be useful for detection of other molecules containingcarbohydrates or aldehydes. The bright-green fluorescence is easy to visualize using asimple UV transilluminator. To detect contaminating proteins in the sample, the samegel can also be stained with SYPRO Ruby protein gel stain (S-12000, S-12001, S-21900;Section 9.3). The Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit contains:

• Pro-Q Emerald 300 reagent• Pro-Q Emerald 300 staining buffer• Oxidizing reagent• Smooth LPS standard from Escherichia coli serotype 055-B5• A detailed protocol

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

Aromatic Amines

Primary aliphatic and aromatic amines (Table 3.1) can couple reversibly with alde-hydes and ketones to form hydrolytically unstable Schiff bases 19 (Figure 3.21). Thereversibility of this modification makes reagents that contain amines less desirable unlessthe Schiff base is reduced to a stable amine derivative by sodium borohydride 90,91 orsodium cyanoborohydride.92 Chemical reduction also retains the amine’s original charge.Sequencing of carbohydrate polymers using fluorescent derivatives has usually relied onderivatization of the reducing end of the polymer with a fluorescent amine.93 Certainaromatic amines have been extensively utilized for coupling to aldehydes, ketones,monosaccharides and the reducing end of carbohydrate polymers:

• 2-Aminoacridone (A-6289) forms conjugates that can be separated by HPLC 94,95 or,as their borate complexes, by polyacrylamide gel electrophoresis,93,96,97 capillaryelectrophoresis 98–100 and micellar electrokinetic capillary chromatography(MECC).101,102 Starting with as little as 25 µg of a glycoprotein, researchers haveefficiently released and purified the carbohydrates, and then derivatized them with2-aminoacridone for subsequent structural analysis.103 2-Aminoacridone derivatives ofoligosaccharides have been directly analyzed by MALDI-TOF mass spectrometry.104

2-Aminoacridone is also used to prepare fluorogenic substrates for proteases.105

• 7-Amino-4-methylcoumarin (A-191), which is a common base of protease substrates(Section 10.4), can be used for the reductive derivatization of oligosaccharides.106,107

• 8-Aminopyrene-1,3,6-trisulfonic acid (APTS, A-6257) has been extensively used toderivatize carbohydrates prior to separation by gel or capillary electrophoresis.108–115

Among the amines we offer, APTS is the aromatic amine that has the most favorablecombination of strong absorbance, high quantum yield and ionic charge.

• ANTS (A-350) and the FluoroPure Grade 7-aminonaphthalene-1,3-disulfonic acid(ANDS, A-22840) have high ionic charges, permitting electrophoretic separation ofthe degradation products of complex oligosaccharides.116 Carboxylated polysaccha-rides have been coupled to the aromatic amine of ANDS preceding electrophoreticanalysis.7,93,116–120

Figure 3.19 Characterization of lipopolysaccha-rides. Lipopolysaccharide (LPS) from Escherichiacoli smooth serotype 055:B5 was loaded onto a13% polyacrylamide gel. After electrophoresis, thegel was stained using the Pro-Q Emerald 300 Li-popolysaccharide Gel Stain Kit (P-20495) and thefluorescence signal measured for the lane. A plotof fluorescence signal versus the relative distancefrom the dye front shows a characteristic ladderingprofile for smooth-type LPS.

Figure 3.20 Linearity of the Pro-Q Emerald 300stain for lipopolysaccharide (LPS) detection. A di-lution series of lipopolysaccharide from Escheri-chia coli smooth serotype 055:B5 was loaded ontoa 13% polyacrylamide gel. After electrophoresis,the gel was stained using the Pro-Q Emerald 300Lipopolysaccharide Gel Stain Kit (P-20495) and thesame band from each lane was quantitated using aCCD camera. A plot of the fluorescence intensityversus the mass of LPS loaded shows a linearrange over two orders of magnitude.Figure 3.21 Modifying aldehydes and ketones with amine derivatives.

Section 3.2

Our PRO Q Emerald 300 lipopolysac-charide (LPS) detection technology isthe best available method for quantita-tive analysis of LPS in gels. Its ease ofuse and sensitivity for detection ofLPS is far superior to that of silverstaining.

110 Chapter 3 — Reagents for Modifying Groups Other Than Thiols or Amines www.probes.com

Data Table — 3.2 Hydrazines and Aromatic Amines for Modifying Aldehydes and Ketones

Cat # MW Storage Soluble Abs EC Em Solvent NotesA-191 175.19 L DMF, DMSO 351 18,000 430 MeOHA-350 427.33 L H2O 353 7,200 520 H2OA-6257 523.39 D,L H2O 424 19,000 505 pH 7A-6289 246.70 D,L DMF, DMSO 425 5,200 531 MeOH 1A-10436 570.48 D,L H2O 493 71,000 517 pH 7A-10437 730.74 D,L H2O 576 86,000 599 pH 7 2A-10438 758.79 D,L H2O 588 97,000 613 pH 7 2A-10439 349.29 L H2O, DMSO 345 13,000 445 pH 7A-10550 445.41 F,D H2O, DMSO <300 noneA-20500 ~950 D,L H2O 624 110,000 643 pH 7A-20501 ~1150 D,L H2O 554 150,000 567 pH 7A-20502 ~1200 D,L H2O 649 250,000 666 pH 7

The aromatic diamine 1,2-diamino-4,5-dimethoxybenzene(DDB, D-1463), which forms heterocyclic compounds withcertain aldehydes and ketones, has been used to selectively detectaromatic aldehydes in the presence of aliphatic aldehydes, includ-ing carbohydrates.121 It has also been employed for fluorometricdetermination of ascorbic acid,122,123 lactic acid,124 α-ketoacids 125–127 and N-acetylneuraminic acids 128 by HPLC. Endoge-nous leucine-enkephalin and other tyrosine-containing peptidescan be detected by HPLC at concentrations as low as 5.6 pico-moles per gram of rat brain tissue using a Reimer–Tiemann reac-tion followed by derivatization with DDB.129 The dimly fluores-cent aromatic diamine 2,3-diaminonaphthalene (D-7918) may be

similarly useful for detection of ketones and α-keto acids throughformation of fluorescent heterocycles.

Alternatively, aldehydes and ketones can be transformed intoprimary aliphatic amines by reductive amination with ammonia,ethylenediamine or other nonfluorescent diamines.130 This chem-istry is particularly useful because the products can then be cou-pled with any of the amine-reactive reagents described in Chapter1 such as the succinimidyl esters of TAMRA 131 (C-1171, C-6121,C-6122; Section 1.6). Derivatization by succinimidyl esters hasbeen extensively utilized for tagging oligosaccharides that are tobe separated by capillary zone electrophoresis with laser-inducedfluorescence detection.131–134

References

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111

Cat # MW Storage Soluble Abs EC Em Solvent NotesA-22840 341.39 L H2O 350 4,200 450 pH 7C-356 493.49 L pH >7, DMF 492 78,000 516 pH 8 3C-687 596.44 L H2O 399 30,000 421 H2O 4, 5D-100 265.33 L EtOH 336 4,400 534 MeOHD-355 275.31 D,L MeCN, DMF 420 46,000 468 MeOHD-1463 241.12 D,L EtOH 298 3,100 359 MeOHD-2371 306.12 F,D,L MeOH, MeCN 503 71,000 510 MeOH 6D-7918 158.20 L DMSO, MeOH 340 5,100 377 MeOH 7F-121 421.43 D,L pH >7, DMF 492 85,000 516 pH 9 3F-6290 254.29 F,D DMF, DMSO 299 5,800 309 MeOH 8L-453 457.24 L H2O 428 12,000 536 H2O 9L-20492 277.27 F,DD H2O <300 noneL-20493 445.42 F,DD DMSO <300 none 10M-20490 209.16 F,L MeCN 487 24,000 none MeOH 11P-101 302.38 D,L MeCN, DMF 341 43,000 376 MeOH 12T-6256 620.74 F,L DMF 582 109,000 602 MeOH

For definitions of the contents of this data table, see “How to Use This Book” on page viii.

Notes1. Spectra of this compound are in methanol containing a trace of KOH.2. Maximum solubility in water is ~8% for A-10437 and A-10438.3. Absorption and fluorescence of fluorescein derivatives are pH dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.4. Cascade Blue dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.5. Maximum solubility in water is ~1% for C-687, ~1% for C-3221 and ~8% for C-3239.6. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.7. Fluorescence of D-7918 is weak. Reaction with α-ketoaldehydes yields fluorescent benzoquinoxaline derivatives (Abs = 365 nm, Em = 540 nm in H20 (pH 8); J Chromatogr B 729,

237 (1999)).8. F-6290 has stronger absorption at 264 nm (EC = 20,000 cm-1M-1). This peak is usually used for excitation in fluorescence-detected HPLC or capillary electrophoresis.9. Maximum solubility in water is ~8% for L-453, ~6% for L-682 and ~1% for L-1177.

10. This product is supplied as a ready-made solution in the solvent indicated under Soluble.11. NBD methylhydrazine reacts with aldehydes and ketones in the presence of strong acid, yielding weakly fluorescent hydrazone products (Anal Chem 71, 1893 (1999)). Abs = 493 nm,

Em = 552 nm in MeOH for reaction product with acetone.12. 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.

Section 3.2

Product List — 3.2 Hydrazines and Aromatic Amines for Modifying Aldehydes and Ketones

Cat # Product Name Unit SizeA-10439 Alexa Fluor® 350 hydrazide, sodium salt .............................................................................................................................................................. 5 mgA-10436 Alexa Fluor® 488 hydrazide, sodium salt .............................................................................................................................................................. 1 mgA-20501 Alexa Fluor® 555 hydrazide, tris(triethylammonium salt) ..................................................................................................................................... 1 mgA-10437 Alexa Fluor® 568 hydrazide, sodium salt .............................................................................................................................................................. 1 mgA-10438 Alexa Fluor® 594 hydrazide, sodium salt .............................................................................................................................................................. 1 mgA-20500 Alexa Fluor® 633 hydrazide .................................................................................................................................................................................. 1 mgA-20502 Alexa Fluor® 647 hydrazide, tris(triethylammonium salt) ..................................................................................................................................... 1 mgA-6289 2-aminoacridone, hydrochloride ........................................................................................................................................................................... 25 mgA-191 7-amino-4-methylcoumarin *reference standard* ............................................................................................................................................... 100 mgA-22840 7-aminonaphthalene-1,3-disulfonic acid, potassium salt (ANDS) *FluoroPure™ grade* ..................................................................................... 1 gA-350 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) ....................................................................................................................... 1 gA-10550 N-(aminooxyacetyl)-N′-(D-biotinoyl) hydrazine, trifluoroacetic acid salt (ARP) .................................................................................................... 10 mgA-6257 8-aminopyrene-1,3,6-trisulfonic acid, trisodium salt (APTS) ............................................................................................................................... 10 mgC-356 5-(((2-(carbohydrazino)methyl)thio)acetyl)aminofluorescein ............................................................................................................................... 25 mgC-687 Cascade Blue® hydrazide, trisodium salt .............................................................................................................................................................. 10 mgD-1463 1,2-diamino-4,5-dimethoxybenzene, dihydrochloride (DDB) ................................................................................................................................ 100 mgD-7918 2,3-diaminonaphthalene ....................................................................................................................................................................................... 100 mgD-355 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH) ............................................................................................................................ 25 mgD-2371 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY® FL hydrazide) ................................................ 5 mgD-100 5-dimethylaminonaphthalene-1-sulfonyl hydrazine (dansyl hydrazine) ................................................................................................................ 100 mgF-6290 N-(9-fluorenylmethoxycarbonyl) hydrazine (FMOC hydrazine) ............................................................................................................................. 25 mgF-121 fluorescein-5-thiosemicarbazide ........................................................................................................................................................................... 100 mgL-20492 N-levulinoyl-D-mannosamine (ManLev) ................................................................................................................................................................ 25 mgL-20493 N-levulinoyl-D-mannosamine, tetraacetate (ManLev tetraacetate) *5 mM solution in DMSO* ............................................................................. 500 µLL-453 lucifer yellow CH, lithium salt ............................................................................................................................................................................... 25 mgM-20490 N-methyl-4-hydrazino-7-nitrobenzofurazan (NBD methylhydrazine) .................................................................................................................... 25 mgP-20495 Pro-Q™ Emerald 300 Lipopolysaccharide Gel Stain Kit *10 minigels* ................................................................................................................. 1 kitP-101 1-pyrenebutanoic acid, hydrazide ......................................................................................................................................................................... 100 mgT-6256 Texas Red® hydrazide *>90% single isomer* ...................................................................................................................................................... 5 mg