Reagents for Modifying Alcohols—Section 3.2

Page Contents

• Alcohols in Proteins: Serine, Threonine and Tyrosine Residues
• Alcohols in Carbohydrates
• Alcohol Modification in Organic Solvents
• Data Table
• Ordering Information

Although alcohols (including phenols such as tyrosine and the hydroxyl groups in serine, threonine, sterols and carbohydrates) are abundant in biomolecules, their chemical reactivity in aqueous solution is extremely low. Few reagents are selective for alcohols in aqueous solution, especially in the presence of more reactive nucleophiles such as thiols and amines. It is therefore difficult to selectively modify serine, threonine and tyrosine residues in proteins except when they exhibit unusual reactivity, such as by residing at an enzyme's active site.

Serine and Threonine Residues

Nonacylated N-terminal serine and threonine residues in peptides and proteins can be oxidized with periodate to yield aldehydes (Figure 3.2.1) that can be subsequently modified with a variety of hydrazine, hydroxylamine or amine derivatives (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2). In addition, peptides containing serine, threonine or tyrosine residues separated from a histidine residue by a single amino acid can be selectively acylated by the succinimidyl ester of biotin-X (B1582, Biotinylation and Haptenylation Reagents—Section 4.2, Figure 3.2.2). This property may also permit selective modification of these sequences (Ser-x-His, Thr-x-His and Tyr-x-His, where "x" refers to any amino acid) in peptides and proteins with fluorescent succinimidyl esters (Fluorophores and Their Amine-Reactive Derivatives—Chapter 1). O-acylation versus N-acylation can be detected by treatment with hydroxylamine, which cleaves esters but usually not amides.

Figure 3.2.1 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.

Figure 3.2.2 Nucleophilic attack of serine on the carbonyl group (C=O) of biotin-X, SSE results in the stable O-acylated derivative. In addition to histidine-x-serine, this stable intermediate can be formed in the presence of linear sequences of histidine-x-tyrosine and histidine-x-threonine, where "x" refers to any amino acid.

Tyrosine Residues

Modification of tyrosine residues is sometimes a side reaction when proteins are reacted with sulfonyl chlorides, iodoacetamides or other reactive dyes described in Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 and Thiol-Reactive Probes—Chapter 2. For example, NBD chloride (Reagents for Analysis of Low Molecular Weight Amines—Section 1.8) reacts with an active-site tyrosine in Escherichia coli F₁-ATPase, causing strong inhibition.

Tyrosine residues in some proteins can be selectively modified by initial nitration of the ortho position of its phenol using tetranitromethane, and then reduction of the o-nitrotyrosine with sodium dithionite (Na₂S₂O₄) to form an o-aminotyrosine (Figure 3.2.3). Although much less reactive than aliphatic amines, the aromatic amine of o-aminotyrosine can react with most amine-reactive reagents (Fluorophores and Their Amine-Reactive Derivatives—Chapter 1) between pH 5 and pH 7. To obtain selective derivatization in complex samples, it is of course critical to block all aliphatic primary amines, typically by acetylation with acetic anhydride, prior to reduction of nitrotyrosine. Nitration of tyrosine residues by nitric oxide occurs naturally in cells through peroxynitrite radical intermediates (Probes for Nitric Oxide Research—Section 18.3, Reactive oxygen species—Table 18.1), yielding derivatives that can be reduced to o-aminotyrosine and subsequently detected using amine-reactive reagents. Tyrosine residues of peptides and proteins can be selectively coupled to aniline derivatives in the presence of formaldehyde. Chemoselective derivatization of tyrosine has also been accomplished using a fluorescent diazodicarboxamide reagent prepared from carboxy-X-rhodamine (ROX) succinimidyl ester.

Another method for modifying tyrosine groups in peptides is to convert the phenol group in tyrosine residues to a salicylaldehyde derivative, and then to react the salicylaldehyde with 1,2-diamino-4,5-dimethoxybenzene (D1463, Reagents for Modifying Aldehydes and Ketones—Section 3.3) to form a fluorescent benzimidazole.

The tyramide signal amplification (TSA) technology (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2), which was developed by NEN (now a part of PerkinElmer Corporation) and licensed for in-cell and in-tissue applications, permits significant amplification of the detectability of targets by a horseradish peroxidase–mediated scheme. In the TSA method, the labeled tyramide becomes covalently linked to tyrosine residues in or near the target. We have introduced an extensive selection of TSA Kits that utilize an Alexa Fluor tyramide, Oregon Green 488 tyramide or biotin-XX tyramide as the amplification reagents (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2).

Figure 3.2.3 Reaction scheme for the conversion of tyrosine to o-aminotyrosine. Tyrosine undergoes nitration by reaction with tetranitromethane, followed by reduction with sodium dithionite, to yield an o-aminotyrosine.

As with derivatization of alcohols in proteins, it is difficult to selectively modify most carbohydrates in aqueous solution because of their low reactivity and the competing hydrolysis of the reactive reagents. However, several reagents are available for derivatizing reducing sugars (which contain a low equilibrium concentration of the reactive aldehyde function), as well as for modifying aldehydes and ketones obtained by periodate oxidation of various carbohydrates. To pursue this labeling approach, see Reagents for Modifying Aldehydes and Ketones—Section 3.3 for a description of aldehyde- and ketone-reactive reagents.

Dichlorotriazines

Dichlorotriazines readily modify amines in proteins, and they are among the few reactive groups that are reported to react directly with polysaccharides and other alcohols in aqueous solution, provided that the pH is >9 and that other nucleophiles are absent. We offer the 5-isomer of fluorescein dichlorotriazine (5-DTAF, D16), with absorption/emission maxima of ~492/516 nm, as well as Texas Red C₂-dichlorotriazine (T30200), with absorption/emission maxima of ~588/601 nm. 5-DTAF has been used to label a wide range of hydroxylated biopolymers including collagen, cellulose, cyclodextrins and soluble beta-glucan, as well as functionalized carbon nanotubes.

N-Methylisatoic Anhydride

In the absence of other reactive functional groups, N-methylisatoic anhydride (M25) will convert ribonucleotides and certain other carbohydrates to fluorescent esters with excitation/emission maxima of ~350/446 nm in mildly basic aqueous solution. The compactness and moderate environmental sensitivity of this fluorophore, which is a synthetic precursor to blue-fluorescent N-methylanthraniloyl (MANT) amides and esters, may be advantageous for preparing site-selective probes. Low molecular weight alcohols are better derivatized by this reagent in aprotic organic solvents (Figure 3.2.4).

Figure 3.2.4 Reaction of N-methylisatoic anhydride (M25) with an alcohol to produce a blue-fluorescent (~350/446 nm) N-methylanthraniloyl (MANT) ester.

Dansyl Aminophenylboronic Acid

m-Dansylaminophenylboronic acid reacts with vicinal diols (hydroxyl groups on adjacent carbon atoms) and certain amino alcohols to form cyclic complexes (Figure 3.2.5) that have a fluorescence intensity and peak emission dependent on the environment of the dansyl fluorophore. This interesting reagent binds reversibly to cell-wall carbohydrates, as well as to glycosylated (but not deglycosylated) human serum albumin. Dansylaminophenylboronic acid is also used as an HPLC derivatization reagent for vicinal diols and as a detection reagent for glycolipids analyzed by thin-layer chromatography.

Figure 3.2.5 Reaction of m-dansylaminophenylboronic acid with a vicinal diol to form a reversible fluorescent cyclic complex.

Two functional groups—acyl azides and acyl nitriles—react directly with aliphatic amines to yield the same products as do the corresponding succinimidyl esters. When reacted in organic solvents, however, these reagents can also form derivatives of alcohols and phenols, making them extremely useful for sensitive analysis of alcohols by HPLC or capillary electrophoresis.

Isocyanates Prepared from Acyl Azides

Alcohols are much easier to modify in anhydrous organic solvents than in aqueous solution. Perhaps the most effective reagents are isocyanates, which are much more reactive with alcohols (and amines) than are isothiocyanates but are not sufficiently stable to permit their sale. Fortunately, isocyanates can often be prepared by Curtius rearrangement of acyl azides (Figure 3.2.6). When an acyl azide and alcohol are heated together in an organic solvent such as toluene, dioxane or DMF at 80°C, the acyl azide will rearrange to form an isocyanate that then reacts with the alcohol to form a stable urethane. As little as 50 femtograms of the urethane conjugates prepared from coumarin derivatives 7-methoxycoumarin-3-carbonyl azide and 7-diethylaminocoumarin-3-carbonyl azide can be detected using an HPLC fluorescence detector. Alcohol conjugates (urethanes) prepared from the single-isomer carbonyl azides of fluorescein diacetate and tetramethylrhodamine may provide even higher sensitivity, particularly with instruments that employ the argon-ion laser. Following rearrangement and alcohol conjugation, the acetates of the fluorescein derivative can be removed by hydrolysis at pH 9–10. The diacetate of fluorescein-5-carbonyl azide has been used to synthesize a fluorogenic substrate for the anandamide transmembrane carrier. Tetramethylrhodamine-5-carbonyl azide has been successfully conjugated to the hydrophobic poly(ε-caprolactone) (PCL) block of a diblock copolymer micelle in order to follow its cellular internalization and has also been used to prepare riboflavin conjugates for the same purpose.

Figure 3.2.6 Derivatization of an alcohol using the diacetate of fluorescein-5-carbonyl azide. This process consists of three steps: 1) rearrangement of the acyl azide to an isocyanate, 2) reaction of the isocyanate with an alcohol to form a urethane and 3) deprotection of the nonfluorescent urethane derivative using hydroxylamine.

Acyl Nitriles

9-Anthroylnitrile reacts with alcohols, such as steroids and acylglycerols, in organic solvents to yield carboxylate esters that are useful for HPLC. To optimize solid-phase organic synthesis, 9-anthroylnitrile has been used to quantitate the absolute amount of resin-bound hydroxyl groups directly on solid support. In addition, 9-anthroylnitrile has been reported to be useful for the selective labeling of certain serine and threonine residues in myosin. The lipophilicity of 9-anthroylnitrile may make it useful for modifying hydroxyl groups of proteins and hydroxylated fatty acids that are buried within cell membranes.

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