Alcohols in Proteins: Serine, Threonine and Tyrosine Residues

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 ref (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 or sulfosuccinimidyl ester of biotin-X ref (B1582, B6353; 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.ref

Sodium periodate oxidation of an N-terminal serine residue   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.

Nucleophilic attack of serine on the carbonyl groupFigure 3.2.2 Nucleophilic attack of serine on the carbonyl group (C=O) of biotin-X, SSE (B6353) 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 (C20260, Reagents for Analysis of Low Molecular Weight Amines—Section 1.8, structure) reacts with an active-site tyrosine in Escherichia coli F1-ATPase, causing strong inhibition.ref

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.ref 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.ref Tyrosine residues of peptides and proteins can be selectively coupled to aniline derivatives in the presence of formaldehyde.ref Chemoselective derivatization of tyrosine has also been accomplished using a fluorescent diazodicarboxamide reagent prepared from carboxy-X-rhodamine (ROX) succinimidyl ester ref (C1309, Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6).

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.ref

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.

Alcohols in Carbohydrates

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, structure), with absorption/emission maxima of ~588/601 nm. 5-DTAF has been used to label a wide range of hydroxylated biopolymers including collagen,ref cellulose,ref cyclodextrins ref and soluble beta-glucan,ref as well as functionalized carbon nanotubes.ref

N-Methylisatoic Anhydride

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

Reaction of N-methylisatoic anhydride  
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 (D2281, structure) reacts with vicinal diols (hydroxyl groups on adjacent carbon atoms) and certain amino alcohols ref to form cyclic complexes ref (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,ref as well as to glycosylated (but not deglycosylated) human serum albumin.ref Dansylaminophenylboronic acid is also used as an HPLC derivatization reagent for vicinal diols ref and as a detection reagent for glycolipids analyzed by thin-layer chromatography.ref

Reaction of m-dansylaminophenylboronic  
Figure 3.2.5 Reaction of m-dansylaminophenylboronic acid (D2281) with a vicinal diol to form a reversible fluorescent cyclic complex.

Alcohol Modification in Organic Solvents

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 (M1445) and 7-diethylaminocoumarin-3-carbonyl azide (D1446) can be detected using an HPLC fluorescence detector.ref Alcohol conjugates (urethanes) prepared from the single-isomer carbonyl azides of fluorescein diacetate (F6218) and tetramethylrhodamine (T6219) 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.ref 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 ref and has also been used to prepare riboflavin conjugates for the same purpose.ref

Derivatization of an alcohol using the diacetate of fluorescein-5-carbonyl azide
Figure 3.2.6 Derivatization of an alcohol using the diacetate of fluorescein-5-carbonyl azide (F6218). 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 (A1440) reacts with alcohols, such as steroids and acylglycerols, in organic solvents to yield carboxylate esters that are useful for HPLC.ref 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.ref In addition, 9-anthroylnitrile has been reported to be useful for the selective labeling of certain serine and threonine residues in myosin.ref 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.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A1440icon231.25F,D,LDMF, MeCN3617500470MeOH1
D16icon495.28F,D,LpH >6, DMF49283,000516pH 92, 3
D1446icon286.29F,D,LDMF, MeCN43657,000478MeOH 
D2281icon icon370.23D,LDMF, DMSO3374600517MeOH4
F6218icon485.41FF,DDMF, MeCN<300 none  
M25icon177.16DDMF, DMSO3163500386MeOH5
M1445icon245.19FF,D,LDMF, MeCN36025,000415MeOH 
T6219icon455.47FF,D,LDMF, MeCN54590,000578MeOH 
T30200icon796.74F,D,LDMF, DMSO58387,000604MeOH 
  1. The absorption spectrum of A1440 has subsidiary peaks at 380 nm and 344 nm. Emission spectrum is unstructured. Ester derivatives formed by reaction with alcohols have essentially similar spectra.
  2. Unstable in water. Use immediately.
  3. Absorption and fluorescence of fluorescein derivatives are pH dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.
  4. Fluorescence of D2281 when bound to proteins is typically blue shifted (Em ~490 nm).
  5. The amide reaction product of M25 with butylamine has Abs = 353 nm (EC = 5900 cm-1M-1), Em = 426 nm in MeOH. Ester reaction products with alcohols have Abs = 350 nm (EC = 5700 cm-1M-1), Em = 446 nm in water (pH 8).

Ordering Information

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