Not only are low molecular weight amines abundantly distributed in nature, but numerous drugs, synthetic probes and other molecules of interest also contain amino groups. The sensitive detection, identification and quantitation of amines are important applications of many of the reactive fluorophores in this section. Some of these reagents have also been used to indirectly detect carbohydrates, carboxylic acids, thiols and cyanide.
The preferred reagents for detecting and quantitating amines in solution or on amine-containing polymers are those that are nonfluorescent but form fluorescent conjugates stoichiometrically with amines. It is difficult to compare the sensitivity for amine detection of the different reagents because it depends heavily on the equipment and detection technology used. Many of the assays, however, are rapid, reliable and adaptable to a variety of different sample types and instrumentation.
Fluorescamine (F2332, F20261) is intrinsically nonfluorescent but reacts rapidly with primary aliphatic amines, including those in peptides and proteins, to yield a blue-green–fluorescent derivative (Figure 1.8.1). Modifications to the reaction protocol permit fluorescamine to be used to detect those amino acids containing secondary amines, such as proline. Excess reagent is rapidly converted to a nonfluorescent product by reaction with water, making fluorescamine useful for determining protein concentrations of solutions.
Fluorescamine can also be used to detect proteins in gels and to analyze low molecular weight amines by TLC, HPLC and capillary electrophoresis. An optimized procedure that employs fluorescamine for amino acid analysis in microplates has been published. Chiral separation of fluorescamine-labeled amino acids has been optimized using capillary electrophoresis in the presence of hydroxypropyl-β-cyclodextrin, a method designed for use in extraterrestrial exploration on Mars. Furthermore, a 200-fold increase in sensitivity and improved resolution in these measurements has been obtained by replacing fluorescamine with Pacific Blue succinimidyl ester (P10163, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7).
Analyte Detection with OPA and NDA
The homologous aromatic dialdehydes o-phthaldialdehyde (OPA, P2331MP) and naphthalene-2,3-dicarboxaldehyde (NDA, N1138) are essentially nonfluorescent until reacted with a primary amine in the presence of a thiol such as 2-mercaptoethanol, 3-mercaptopropionic acid or the less obnoxious sulfite, or in the presence of excess cyanide, to yield a fluorescent isoindole (Figure 1.8.2, Figure 1.8.3). Improved detection sensitivity can be obtained by using SAMSA fluorescein (A685, Chemical Crosslinking Reagents—Section 5.2) as the thiol reagent, thereby incorporating fluorescein as the R2 substituent of the isoindole product (Figure 1.8.2). Modified protocols that use an excess of an amine and limiting amounts of other nucleophiles permit the determination of carboxylic acids and thiols, as well as of cyanide in blood, urine and other samples. Without an additional nucleophile, NDA forms fluorescent adducts with both hydrazine and methylated hydrazines (excitation/emission maxima ~403/500 nm).
Figure 1.8.2 Fluorogenic amine-derivatization reaction of o-phthaldialdehyde (OPA, P2331MP).
Figure 1.8.3 Fluorogenic amine-derivatization reaction of naphthalene-2,3-dicarboxaldehyde (NDA, N1138).
Sensitivity of OPA and NDA
Amine adducts of NDA have longer-wavelength spectral characteristics and greater sensitivity than the amine adducts of OPA. The stability and detectability of the amine derivatives of NDA are also superior; the detection of glycine with NDA and cyanide is reported to be 50-fold more sensitive than with OPA and 2-mercaptoethanol. The limit for electrochemical detection of the NDA adduct of asparagine has been determined to be as low as 36 attomoles (36 × 10-18 moles). An optimized procedure that uses NDA for amino acid analysis in microplates has been published.
Applications for OPA and NDA
OPA and NDA are used extensively for both pre- and post-column derivatization of amines (and thiols) separated by HPLC or by capillary electrophoresis. The amines in a single cell have been analyzed by capillary electrophoresis using a sequence of on-capillary lysis, derivatization with NDA and cyanide, and laser-excited detection.
Sensitivity of ATTO-TAG CBQCA and ATTO-TAG FQ
ATTO-TAG CBQCA (A6222, A2333) and ATTO-TAG FQ (A10192, A2334) provide ultrasensitive detection of primary amines, including those in peptides, aminophospholipids and glycoproteins. These reagents combine high sensitivity, visible-wavelength excitation and freedom from background fluorescence, making them useful for research, as well as analytical, forensic and clinical applications. Developed by Novotny and collaborators, the ATTO-TAG reagents are similar to OPA and NDA in that they rapidly react with amines in the presence of thiols or cyanide to form highly fluorescent isoindoles (Figure 1.8.4).
ATTO-TAG CBQCA reagent reacts specifically with amines to form charged conjugates that can be analyzed by electrophoresis techniques. Carbohydrates lacking amines can be detected following reductive amination with ammonia and NaCNBH3. ATTO-TAG CBQCA conjugates are maximally excited at ~456 nm or by the 442 nm spectral line of the He-Cd laser, with peak emission at ~550 nm, whereas ATTO-TAG FQ conjugates are maximally excited at ~480 nm or by the 488 nm spectral line of the argon-ion laser, with peak emission at ~590 nm. Ultrasensitive detection of CBQCA-derivatized amino sugars, amino acids and low molecular weight peptides by capillary electrophoresis has been reported. In capillary electrophoresis, the sensitivity of amine detection of the laser-induced fluorescence is in the subattomole range (<10-18 moles) for ATTO-TAG CBQCA and subfemtomole range (<10-15 moles) for ATTO-TAG FQ. Detection sensitivity of reductively aminated glucose using ATTO-TAG CBQCA is reported to be 75 zeptomoles (75 × 10-21 moles).
ATTO-TAG reagents can, of course, be used in HPLC and other modes of chromatography with either absorption or fluorescence detection. The principal limitation to obtaining ultrasensitive detection using the ATTO-TAG reagents and all other chemical derivatization reagents is that relatively high concentrations of the derivatizing reagent are required to obtain adequate kinetics and quantitative modification of the analyte. A very sensitive assay that uses ATTO-TAG CBQCA for rapid quantitation of protein amines in solution (C6667) is described in Protein Quantitation in Solution—Section 9.2. Similarly, ATTO-TAG CBQCA has proven useful for in situ quantitation of proteins attached to microspheres.
ATTO-TAG Reagents and Kits
Because cyclodextrins have been reported to amplify the signal from ATTO-TAG CBQCA conjugates up to 10-fold, we have included β-cyclodextrin in our ATTO-TAG Amine Derivatization Kits (A2333, A2334). These kits contain:
The ATTO-TAG CBQCA and ATTO-TAG FQ Amine Derivatization Kits supply sufficient reagents for derivatizing approximately 150 and 100 samples, respectively, depending on the amine concentration and sample volume.
NBD chloride (C20260, ) was first introduced in 1968 as a fluorogenic derivatization reagent for amines. NBD fluoride (F486) usually yields the same products as NBD chloride but is much more reactive; for example, the reaction of NBD fluoride with glycine is reported to be 500 times faster than the reaction of NBD chloride with glycine. Reaction of NBD fluoride with alcohols leads to their utility for derivatizing and detecting lipopolysaccharides (LPS). Unlike OPA and fluorescamine, both NBD chloride and NBD fluoride react with secondary amines and are therefore capable of derivatizing proline and hydroxyproline. NBD chloride and NBD fluoride are extensively used as derivatization reagents for chromatographic analysis of amino acids and other low molecular weight amines.
The absorption and fluorescence emission spectra, quantum yields and extinction coefficients of NBD conjugates are all markedly dependent on solvent; in particular, the fluorescence quantum yield in water of NBD adducts of amines can be very low (<0.01), particularly of secondary amines. NBD adducts of aromatic amines are essentially nonfluorescent, a property that we have utilized to prepare our QSY 35 quenchers (see below).
Fluorescence of lysine-modified NBD-labeled actin is sensitive to polymerization. Inactivation of certain ATPases by NBD chloride apparently involves a tyrosine modification followed by intramolecular migration of the label to a lysine residue. NBD is also a functional analog of the dinitrophenyl hapten, and its fluorescence is quenched upon binding to anti-dinitrophenyl antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4).
NBD aminohexanoic acid (NBD-X, N316) and its succinimidyl ester (NBD-X, SE; S1167) are precursors to NBD-labeled phospholipids (Fatty Acid Analogs and Phospholipids—Section 13.2), NBD C6-ceramide (N1154, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4) and other probes.
Many of the sulfonyl chlorides described in Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7, including dansyl chloride (D21), 1-pyrenesulfonyl chloride (P24) and Dapoxyl sulfonyl chloride (D10160), react with amines to yield blue- or blue-green–fluorescent sulfonamides and are particularly useful as chromatographic derivatization reagents. They react with both aliphatic and aromatic amines to yield very stable derivatives. In addition, they are generally good acceptors for fluorescence resonance energy transfer (FRET) from tryptophan, as well as good donors to longer-wavelength dyes such as QSY dyes (Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6) (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Fluorescence of dansyl conjugates in aqueous solutions can be enhanced by adding cycloheptaamylose.
Although dansyl chloride is the most commonly used of these reagents, the stronger absorption of 1-pyrenesulfonamides and large Stokes shift of Dapoxyl sulfonamides () should make these sulfonyl chlorides more sensitive reagents for amine analysis. Note that sulfonyl chlorides are unstable in dimethylsulfoxide (DMSO) and should never be used in that solvent.
Since its development by Weber in 1951, dansyl chloride (D21, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7) has been used extensively to determine the N-terminal amino acid residue of proteins and to prepare fluorescent derivatives of drugs, amino acids, oligonucleotides and proteins for detection by numerous chromatographic methods. Nonfluorescent dansyl chloride reacts with amines to form fluorescent dansyl amides that exhibit large Stokes shifts, along with environment-sensitive fluorescence quantum yields and emission maxima.
Pyrene Sulfonyl Chloride
The absorptivity (and therefore ultimate fluorescence output) of dansyl derivatives is weak compared with that of the more strongly UV light–absorbing fluorophores such as pyrene. Thus, 1-pyrenesulfonyl chloride (P24, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7) should have greater sensitivity for detection of amines. The fluorescence lifetime of pyrenesulfonamides can also be relatively long (up to ~30 nanoseconds), making them useful for fluorescence anisotropy measurements. Fluorescence polarization measurements of DNA probes labeled with 1-pyrenesulfonyl chloride permit homogeneous detection of hybridization.
Dapoxyl Sulfonyl Chloride
Sulfonamides derived from Dapoxyl sulfonyl chloride (D10160, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7) have much higher extinction coefficients than those of dansyl chloride (~22,000 cm-1M-1 versus ~4000 cm-1M-1) and equal or greater quantum yields when dissolved in organic solvents; however, Dapoxyl derivatives have very low fluorescence in water. The huge Stokes shifts (up to ~200 nm) and large extinction coefficients of Dapoxyl derivatives in some solvents () make the reactive Dapoxyl derivatives a good choice for derivatization reagents in chromatographic and electrophoretic analysis.
Isothiocyanates for preparing bioconjugates have been described in several sections of this chapter. However, FITC (F143, F1906, F1907; Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) can also be used for derivatizing low molecular weight amines and, like phenyl isothiocyanate, for microsequencing of peptides as their thiohydantoins. A method for specific derivatization of the N-terminus of peptides by FITC has been described. FITC-labeled amino acids and peptides have been separated by capillary electrophoresis with a detection limit of fewer than 1000 molecules.
Succinimidyl esters have a high selectivity for reaction with aliphatic amines. Most of the succinimidyl ester reagents described elsewhere in this chapter can be used to derivatize low molecular weight amines for subsequent separation by chromatography or capillary electrophoresis. Alexa Fluor, BODIPY, Oregon Green and fluorescein derivatives typically yield the greatest sensitivity, particularly when the conjugate is detected with laser excitation. Use of single isomers of these reactive dyes is essential for all high-resolution analyses. Analysis by capillary electrophoresis shows that carboxyfluorescein succinimidyl ester reacts faster and yields more stable amine conjugates than FITC or DTAF.
The UV light–excitable coumarins described in Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7 have good absorptivity at ~320–420 nm, with purple to bright blue emission at 400–500 nm. Aliphatic polyamines derivatized with 1-pyrenebutanoic acid succinimidyl ester (P130, Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7) have been differentiated from pyrene-labeled monoamines by HPLC using their fluorescent excimer formation.
The Smallest Reactive Fluorophore
N-methylisatoic anhydride (M25) is a useful precursor for preparing esters or amides of the small N-methylanthranilic acid fluorophore. The small size of this fluorophore should reduce the likelihood that the label will interfere with the function of the biomolecule, an important advantage when designing site-selective probes. This amine-acylating reagent is often used to prepare fluorescent derivatives of biologically active peptides and toxins and, in combination with a quencher, to prepare fluorogenic endoprotease substrates.
Chromophoric Succinimidyl Esters: Fluorescence Quenchers
Dabcyl has broad and intense visible absorption (Figure 1.8.5) but no fluorescence, making it useful as an acceptor in FRET applications (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Biomolecules double-labeled with dabcyl and the appropriate fluorophore can be used to monitor proteolytic cleavage, conformational changes and other dynamic spatial movements. Dabcyl succinimidyl ester (dabcyl, SE; D2245; ) is particularly useful in preparing quenched fluorogenic substrates for proteases, including our HIV protease (Figure 1.8.6) and renin substrates (H2930, R2931; Detecting Peptidases and Proteases—Section 10.4), papain, Alzheimer disease–associated proteases and others. Fluorogenic substrates using this quenching group have also been prepared for interleukin-1β–converting enzyme (ICE), a cysteine protease that is proposed to function in the onset of apoptosis. The dabcyl chromophore has been used as the quencher in donor–acceptor labeled oligonucleotides (molecular beacons); unfolding of these probes upon hybridization leads to recovery of the donor dye's fluorescence.
QSY 35 acetic acid succinimidyl ester (Q20133, ) is an essentially nonfluorescent nitrobenzoxadiazole (NBD) derivative. Like the QSY 7, QSY 9 and QSY 21 dyes (Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6), the QSY 35 dye has absorption at longer wavelengths than does the dabcyl dye (Figure 1.8.5), making it a very good acceptor from most blue-fluorescent dyes. A peptide containing the QSY 35 quencher paired with the blue-fluorescent 7-hydroxy-4-methyl-3-acetylcoumarin fluorophore has proven useful in a fluorescence resonance energy transfer (FRET) assay for Bacillus anthracis lethal factor protease. QSY 35 iodoacetamide (Q20348, Thiol-Reactive Probes Excited with Visible Light—Section 2.2) and an FMOC-protected QSY 35 amino acid (Q21931, Peptide Analysis, Sequencing and Synthesis—Section 9.5) are available for automated preparation of FRET-based protease substrates.
Figure 1.8.6 Principle of the fluorogenic response to protease cleavage exhibited by HIV protease substrate 1 (H2930). Quenching of the EDANS fluorophore (F) by distance-dependent resonance energy transfer to the dabcyl quencher (Q) is eliminated upon cleavage of the intervening peptide linker.
N-(t-BOC)-Aminooxyacetic Acid TFP Ester
The tetrafluorophenyl ester (TFP) 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 can then spontaneously react with aldehydes, the reducing ends of saccharides and oligosaccharides and abasic sites in oligonucleotides to form stable adducts (Reagents for Modifying Aldehydes and Ketones—Section 3.3).
|A2333||305.29||F,D,L||MeOH||465||ND||560||MeOH||1, 2, 3, 4|
|A6222||305.29||F,D,L||MeOH||465||ND||560||MeOH||1, 2, 3|
|C20260||199.55||F,D,L||DMF, MeCN||336||9800||none||MeOH||6, 7, 8|
|F486||183.10||F,D,L||MeCN, CHCl3||328||8000||none||MeOH||6, 7|
|N1138||184.19||L||DMF, MeCN||419||9400||493||see Notes||12|