Chapter 21Reagents for Other Groups

21.1 The Alcoholic OH-Group

The reactivity of alcohols in aqueous solutions is low, most reagents prefer to reactwith more nucleophilic groups like amine or thiol.

Tosyl- or diphenylphosphoryl-modified Ser can undergo “-elimination under for-mation of dehydroalanine, this can be hydrolysed at the C’�N bond under alkalineconditions (0:1 M NaOH) [386]. A similar reaction occurs with O-glycosidic bondson Thr and Ser when treated with alkali, this may be used to identify the sites ofO-glycosylation. Phospho-Ser or -Thr also show this reaction (see Fig. 21.2). Ingeneral, however, the reaction conditions are probably to rough to be useful.

After tosylation the alcohol may be converted into a thiol by incubation withthioacetate at pH 5:5, followed by hydrolysis.

Sulphonyl fluorides and organic phosphoesters can label Ser and Thr OH-groupsin the active centre of enzymes, where they are more reactive. Protease inactivationby PMSF is an example (see Fig. 21.1).

Alternatively, periodate oxidation of alcohols leads to aldehydes which may bemodified with hydrazines or aromatic amines. This reaction is of particular signif-icance for the modification of sugar residues in glycoproteins, it will also workwith Ser but not Thr. Because the sugar residues of glycoproteins are usually notinvolved in protein secondary and tertiary structure, their modification is unlikelyto alter binding and enzymatic properties of proteins; one of the application is thecross-linking of antibodies with horseradish peroxidase via their sugar chains forELISA [432].

Galactose oxidase can introduce aldehyde groups into Gal-residues in glycopro-teins, these in turn may be introduced using Gal-transferase.

N-methylisatoic acid anhydride is used to label molecules like sugars or nu-cleotides, the small size of this probe and its environmentally sensitive fluorescenceallow ligand binding to proteins to be monitored. The Mant-group is probably thesmallest fluorescent label available (see Fig. 6.2 on p. 41).

Phenylboronic acid derivatives form complexes with vincinal alcohols, like sug-ars. They also react with Ser in proteins if there is an adjacent His (catalytic triad!).

E. Buxbaum, Biophysical Chemistry of Proteins: An Introductionto Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 21,© Springer Science+Business Media, LLC 2011

209

210 21 Reagents for Other Groups

Fig. 21.1 PMSF is used tomodify Ser S

O

Sulphonyl fluoride

O

R F SR

O

O

O X+ X-OH

HF+

C Protein

O

CHNH

Protein

CH2

O Pi

OH−

C Protein

O

CNH

Protein

CH2

C Protein

O

CHNH

Protein

CH2

S CH2

CH2

NH2

SH CH2

CH2

NH2Pi

b-elimination Michael-addition

Fig. 21.2 Labelling of phosphoserine residues in proteins. The resulting thioethylamine may beused to identify phosphoserine in mass spectroscopy, or may be used to introduce amine-reactivereagents. Before labelling, phosphoproteins or peptides may be enriched by IMAC on a Fe3C orGa3C column, which have high affinity for phosphoproteins

21.2 The Phenolic OH-Group

The reactivity of Tyr depends on the ionisation state of the OH-group and on themicro-environment inside the protein. The simplest modification is the introduc-tion of iodine (in particular radio-iodine 125I and 131I). Iodination is often achievedfrom KI in combination with an oxidant like Chloramine T or H2O2. The iodideis oxidised to iodine (2I� C 2HC C H2O2 ! I2 C 2H2O). The iodine reacts spon-taneously with Tyr, forming iodotyrosine and HI, the reaction may involve IC aselectrophilic intermediate. Pierce offers Iodo-Gen, a hydrophobic oxidant that isused to coat the test tube in which the iodination is performed (see Fig. 21.3).Since the oxidant is not in solution, damage to proteins is limited. Free radio-iodineand protein are separated by gel chromatography on disposable desalting columns.It is convenient to leave the unbound radio-iodine on the column and dispose ofit as solid waste. Note that radio-iodine is volatile and must be handled only inwell-functioning fume cupboards. As student I once experienced an instructor whoroutinely did iodinations for immunoassays on the open bench, his attitudes changedwhen students, just for fun, held a GEIGER-counter to his throat and picked up a sig-nificant signal.

Sulphonyl chlorides, iodoacetamides and NBD-Cl sometimes react with Tyr.Tyr can be nitrated with tetranitromethane (or peroxynitrite) in the ortho-

position. The o-nitrotyrosine has an absorbance of 4 100 M�1cm�1 at 428 nm inalkaline solution, the pKa of the OH-group is shifted from 7 to 10 by the stronginductive effect of the nitro-group. This can inactivate enzymes which use Tyrin acid-base catalysis. Absorbance is environmentally sensitive and decreases in amore hydrophobic surrounding. This can be used to measure protein conformationalchanges after ligand binding. Nitrotyrosine is an effective quencher of fluorescenceand has been used as acceptor in FRET-studies.

21.2 The Phenolic OH-Group 211

OH

R

OH

R

NO2

XN OH

R

NN

XN

N

N

F

FF

O

R

N N

NF F

N

N

C CH3

O

O

R

CCH3

O

PO O

O

F

CH

CH

OH

R

NH2

PO O

O

CH

CH

CH3

CH3

CH3

CH3

CH3

CH3H3C

H3C

O

R

NN

N N

O

O

Cl

ClCl

Cl

Tyrosine:

Tetranitromethane

Diazonium salt

+

cyanuric fluoride

+

+N-acetylimidazole

−Imidazole

di-isopropyl-fluorophosphate

+

−HF

−HF

−H+

Iodo-Gen

C(NO2)4

−HC(NO2)3

N+

Na2S2O4

Na2S2O4

Fig. 21.3 Reagents to modify tyrosine. For details see text

The nitro-group can be reduced to an amino-group with sodium dithioniteand then labelled with amine-reactive probes. Tetranitromethane reacts also withSH-groups, His, Met, and Trp. In addition, at acidic pH, TNM can cross-link thetyrosine residues of a protein (zero-length cross-linking).

Aromatic diazonium salts react with Tyr to yield azo-dyes. If required, these canbe reduced with dithionite to o-aminotyrosine and then amine modified. Lys and Hismay also be labelled.

Acetylimidazole labels at neutral pH with a change in absorbance of ��278 D1:210 M�1cm�1, the reaction is reversible under alkaline conditions in the presenceof a nucleophile like Tris or hydroxylamine.

Cyanuric fluoride can label Tyr groups under alkaline conditions, when the OH-group is ionised. Diisopropyl fluorophosphate has also been used, but is highlytoxic.

212 21 Reagents for Other Groups

21.3 Carboxylic Acids

Carbodiimides are used for labelling or cross-linking carboxy-groups (seeFig. 21.4). The most common are dicyclohexylcarbodiimide (DCCD) and themore water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). Alsovery reactive toward carboxyl-groups are the WOODWARD-reagents (isoxazolium

Fig. 21.4 Reagents to modify carboxylic acids. The acid is first activated, then coupled to annucleophilic reagent (-YH: alcohol, thiol, amine etc.)

21.4 Histidine 213

fluorobromides). Sulpho-NHS esters are sometimes stable enough to be shipped asintermediates. Like amines, carboxylic acid groups are stronger nucleophiles whendeprotonated, that is, above their pKa.

21.4 Histidine

Diethylpyrocarbonate (ethoxyformic anhydride) labels His specifically at pH5:5–7:5 by transfer of a carboethoxy-group to N3 (see Fig. 21.5), resulting in achange of absorbance at 240 nm, �� � 3 200 M�1cm�1. The reaction is reversibleby incubation with hydroxylamine or other nucleophiles (Tris!) at neutral or al-kaline pH. Higher DEPC/His ratios result in the modification of both N3 and N1,this is not reversible. Instead, incubation with hydroxylamine leads to the openingof the His-ring. In side-reactions DEPC may also react with Cys, Tyr and pri-mary amino groups, Lys –-amino groups may form isopeptides with neighbouringcarboxy-groups.

Before diethylpyrocarbonate became available His was modified by photo-oxidation in the presence of methylene blue, rose bengal, or eosine. Electrons inthese dyes are excited by light to higher orbitals, from where they may enter thetriplet state. The energy is then transferred to nearby oxygen molecules (for whichthe triplet state is the ground state) which become activated and can oxidise othermolecules. Apart from His, photo-oxidation may also affect Trp, Met, and to alesser extend Tyr, Ser and Thr, especially at higher pH.

In microenvironments where the nucleophilicity of His is increased labellingwith iodoacetate, iodoacetamide, or p-bromo-phenacyl bromide (��271 D17 000 M�1cm�1), is possible. This can be used to label reactive His in the cat-alytic centre of enzymes.

21.4.1 Tryptophan

Trp can be oxidised on the indole ring with hydrogen peroxide in 1 M Na2CO3

pH 8.4 (see Fig. 21.6). This results in a reduction of absorbance, ��280D3 490 M�1cm�1.

N-bromosuccimide and 2-hydroxy-5-nitrobenzyl bromide require harsh reactionconditions (10 % acetic or formic acid, 8–10 M urea) and are therefore of limiteduse. The nitro-group in the latter reagent gives the product strong absorbance inalkaline solution (�410 D 18 000 M�1cm�1). To avoid reaction with Cys, the proteinshould be reduced and carboxymethylated.

Formic acid saturated with HCl can formylate the ring nitrogen of Trp, result-ingresulting in an increase in absorbance at 298 nm. The reaction is reversible in apH-stat at pH 9.

KOSHLAND’s reagent (2-hydroxy-5-nitrobenzylbromide) can be used toquantitate Trp-groups since its reaction product has a strong absorbance

214 21 Reagents for Other Groups

Fig. 21.5 Histidine modifying reagents

(��410 D 18 000 M�1cm�1 at pH 12), however, the reaction conditions are harsh(180 mM acetic acid, 10 M urea, 37 °C). If the protein survives these conditions, thereagent has the advantage that its absorbance spectrum depends on environmentalconditions (acidity, hydrophobicity).

21.4 Histidine 215

NH

CH2

H2

NH2

CH2Br

O2N

C CH

CO

OH

NH

CH2

H2

H2

H2

C CH

CO

OH

O

OH

NH

C C CH

CO

OH

OH

N

C C CH

CO

OH

CH

O

+

Tryptophane:

2-hydroxy-5-nitrobenzyl bromide

+ HCOOH

+ H2O2

− H2O

− HBr

− H2O

NH2

NH2

NH2H2

H2

O2N

CH2

Fig. 21.6 Reagents to modify Trp

21.4.2 Arginine

Phenylglyoxal [400], 4-hydroxy-3-nitro-phenylglyoxal (chromogenic, �316 D10 900 M�1cm�1), 2,3-butanedione [440] and 1,2-cyclohexanedione [315] alsolabel Arg (see Fig. 21.7), but also amino- and SH-groups and His, but with lesserefficiency. Borate (50 mM pH 8) accelerates the reaction about 20-fold. The reactionproducts are generally not stable to acid hydrolysis (6 M HCl at 110 °C for 24 h),but can be protected by mercaptoacetic acid.

Ninhydrin [401] also labels Cys and Lys.

21.4.3 Methionine

Methionine is fairly hydrophobic and often buried in the core of the protein, makingmodification more difficult than it should be from the chemists point of view.

Of greatest importance is the specific splitting of peptides and proteins at Met-residues by cyanogen bromide [133] (see Fig. 21.8). This compound is highly toxicand should be handled accordingly. Since Met is a relatively rare residue in proteins(abundance 1:7 %), the resulting fragments are much larger than those obtained withtrypsin or chymotrypsin. A 100-fold excess of the reagent is commonly used underacidic conditions (70 % trifluoroacetic acid) for 4 h in the dark, followed by lyophili-sation (with KOH in the trap!).

216 21 Reagents for Other Groups

−

Fig. 21.7 Labelling of Arg with diones and ninhydrin

The sulphur in methionine may be oxidised step-wise with Chloramine T(accompanied by a change in absorbance at 245 nm), sodium periodate, hy-drogen peroxide, or the hydrophobic t-butylhydroperoxide. Cys interferes withthat reaction, it can be reversed with mercaptoethanol, DTT, and especially N-methylmercaptoacetamide (1 M, 37 °C, 21 h).

21.4 Histidine 217

Fig. 21.8 Modification of proteins at Met-residues. Of the reactions shown the cleavage of pro-teins with cyanogens, bromide is the most important

Halogen-compounds like iodoacetate react much slower with Met than with Cys,and often only at low pH after disruption of protein structure with caotropes. Theresult is a sulphonium-compound.