Iodoacetic acid, alkylation with

Figure 2. Tryptic map of human relaxin B-chain. The peptide was reduced with dithiotreitol and alkylated with iodoacetic acid before digestion with trypsin. The chromatography was performed on a Vydac Cis column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient.

Nitrogens 1 and 3 in the imidazole ring of histidyl residues in proteins may be alkylated with iodoacetic acid (generally in a much slower reaction than alkylation of cysteinyl residues) to give three carboxy-methyl derivatives 1-carboxymethylhistidine, 3-carboxymethyl-histidine and 1,3-dicarboxymethylhistidine ( 3.4). In general, the 3-carboxymethyl derivative is formed most rapidly. These derivatives are stable to acid hydrolysis under the usual conditions (but excess reagent must be removed) and may be analyzed on the long column of most analyzers as described below. 

Auto-oxidation of cysteine residues during cleavage of the disulfide bridge-containing proteins is a potential concern. This concern can be addressed by first reducing those proteins at alkaline pH ( 8.0) with either 2-mercaptoethanol or dithiothreitol (Equation (1)) and then alkylating with iodoacetic acid to S-carboxymethyl derivatives (Equation (2)) The reduction-alkylation process also disrupts the 3D structure of proteins to allow more sites accessible for cleavage. 

Diagnostic losses of certain neutral species are commonly observed from b- and j-ions. For example, Ser, Thr, Asp, and Glu side chains exhibit prominent loss of water (—18 Da), and the Asn-, Gin-, Lys-, and Arg-containing ions similarly show abundant loss of ammonia (—17 Da). The loss of 48 Da (HSCH3) is observed from Met-containing sequence ions, but if Met is in oxidized form, the mass of the expelled neutral ion becomes 64 Da. The Cys-containing ions show a loss of 34 Da (H2S), which shifts to 92 Da (HSCH2COOH) if the precursor protein is alkylated with iodoacetic acid prior to digestion. The peptides that contain a basic residue at the C-terminus are likely to expel the C-terminal residue to produce the (h , + OH) ion. 

Fig. 7.8. The -SH groups are protected by alkylation with iodoacetic acid to prevent re-oxidation.

Example 8.1 A protein with two sulfide bonds and three cysteine residues is first reduced with 2-mercaptoethanol and then alkylated with iodoacetic acid. By how much will the mass of the protein increase ... 

Example 9.1 The molecular mass of a protein is 10,275 Da. Upon reduction with dithiothreitol and alkylation with iodoacetic acid, its mass increased to 10,865 Da. In a separate experiment, the protein was treated only with iodoacetic acid. The molecular mass of the protein was found to be 10,391 Da. Calculate the number of disulfide bonds in this protein. 

Ribonuclease A contains several disulfide bonds but no free sulfhydryl group. The molecular mass of the protein measured before and after reduction and alkylation (with iodoacetic acid) was found to be 13,682 and 14,155 Da, respectively. How many disulfide bonds does this protein contain ... 

In the case of amino acid analysis, the quantification of cysteine can be difficult because it is oxidized to cystine during acid hydrolysis. To circumvent this problem, cysteine can be oxidized to cysteic acid with performic acid prior to analysis. Alternatively, cysteine can be converted to the pyridyl ethyl derivative and subsequently detected by postcolumn reaction with ninhydrin. Still another method involves the production of carboxymethyl cysteine following alkylation with iodoacetic acid. All of these cysteine derivatives can be separated by either reversed-phase precolumn or ion-exchange postcoT umn methods. 

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

Alkylation at pH 8.5 shows reduced rates of reaction at the histidine residues but significant substitution at lysine, particularly Lys 41 118). The histidine reactions show the same general stereospecificity as found at pH 5.5. The inactive Lys 41 derivatives (25, 26, and 27 of Table VI) show alkylation patterns of His 12 and 119 at pH 5.5 which are similar to those of RNase-A although with some differences in detail. When Lys 1 and 7 are acetylated in RNase-S the alkylation pattern with iodoacetic acid is not affected. When PIR is used the alkylation of His 119 is nearly abolished but that at His 12 is accelerated 163). The probable interaction of Asp 121 with His 119 may be important in the alkylation reactions observed in the native enzyme and the various lysine derivatives. In PIR this interaction has, of course, been removed. 

The structures of ebumaminol (155) and larutensine (154) have been confirmed by a synthesis reported by Lounasmaa from the previously available indoloquinolizidine ester 154 (Scheme 8). Successive reduction, acetylation and Fuji oxidation of 156 yielded the enamine 157 which was alkylated with iodoacetic ester followed by NaBH4 reduction to give a mixture of four products. Treatment of two of these, the epimeric esters 158, with ethanolic sodium ethoxide resulted in cyclization to 18-hydroxyebumamonine (159) accompanied by its C(20) epimer. Reduction of 18-hydroxyebumamonine furnished ( )-ebumaminol and 16-epiebumami-nol (160) which on overnight treatment with acid gave ( )-larutensine [114]. 

Cleavage of disulfide bonds occurs before hydrolysis of the protein into peptides. Disulfide bonds may be cleaved oxidatively, or they may be reduced and alkylated. Treatment of the native protein with performic acid, a powerful oxidizing agent, breaks disulfide bonds and converts cystine residues to cysteic acid (Figure 3-11). Reduction of the disulfide linkage by thiols, such as d-mercaptoethanol, yields reactive sulfhydryl groups. These groups may be stabilized by alkylation with iodoacetate or ethyleneimine to yield the carboxymethyl or aminoethyl derivative, respectively. 

Enzymes that contain free sulfhydryl groups at the active site (e.g., glyceraldehyde-3-phosphate dehydrogenase see Chapter 13) react with an alkylating reagent, iodoacetic acid, resulting in inactivation of the enzyme. 

Compound 227 was obtained by alkylation of a substituted adenine with iodoacetic acid. Alkylation of the NH group of compound 211a or of its 7,8-dihydro-derivative 219 with alkyl bromide gave the 3-alkyl- 212 or 3-alkyl-7,8-dihydro-derivatives 221, respectively (97JMC3248) (see Scheme 64). The 8-oxo-compound 226 underwent a hydrolytic cleavage to 227 (69JOC3492). 

Since exposure to air reconverts the sulfhydryl groups to disulfides, it is necessary to stabilize the reduced forms by alkylation with benzyl chloride or with iodoacetic acid... 

To determine the number of disulfide bonds, the protein is reduced and alkylated as described in Section 8.5.1 by reacting it with either dithiothreitol or 2-mercaptoethanol, followed by reaction with iodoacetic acid. The molecular masses of the native protein (Mnat) and reduced and alkylated protein (Mr+a) are determined with MALDl-MS or ESl-MS. S-Carboxymethylation increases the mass of each cysteine residue by 59 Da. From the change in the molecular mass, the number of cysteine residues (Ncys) and hence the number of disulfide bonds (Ns-s) can be estimated using... 

Ribosomal protein L12 was oxidized with 0.3 M H202 at 30°C for 1 h. After dialysis, the protein was incubated in the presence of 0.8 M 2-mercaptoethanol for 48 min at 37 °C and dialyzed. The amount of methionine residues was quantitated by exhaustive alkylation of the protein with [14C]iodoacetic acid. 

Another approach uses reactive alkyl halogen compounds containing a terminal carboxylate group on the other end to form spacer arms off the dextran polymer from each available hydroxyl. In this manner, Brunswick et al. (1988) used chloroacetic acid to modify the hydroxyl groups to form the carboxymethyl derivative. The carboxylates then were aminated with ethylene diamine to create an amine-terminal derivative (Inman, 1985). Finally, the amines were modified with iodoacetate to form a sulfhydryl-reactive polymer (Figure 25.14). 

FIGURE 4 Effect of sample preparation on the fragmentation of an rMAb observed in (A) SDS-PAGE and (B) CE-SDS with LIF detection. SDS-PAGE lanes (Lane I) molecular weight standards bovine serum albumin at (Lane 2) 8 ng and (Lane 3) 2 ng (Lane 4) rMAb control after alkylation with (Lane 5) iodoacetic acid and (Lane 6) iodoacetamide. (See color plate 4.)... 

Antigen unmasking on sections of paraffin-embedded tissues can be accomplished by reduction of disulfide bonds by treatment with 2-mercaptoethanol, followed by alkylation with sodium iodoacetate to prevent the bonds from reforming. This method has been used for unmasking a Kunitz protease inhibitory domain epitope of Alzheimer s amyloid precursor protein in human brain (Campbell et al., 1999). Sections are reduced with a mixture of 0.14 M 2-mercaptoethanol in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 3 hr in the dark at room temperature. After being washed for 3 min in distilled water, the sections are treated with a mixture of 250 mg/ml iodoacetic acid in 0.1 M NaOH, diluted 1 10 in 0.5 M Tris-HCl (pH 8.0) and 1 mM EDTA for 20 min in the dark. 

Friedel-Crafts acylation. Posner el al. have developed a remarkably efficient route to the methyl ether of the steroid 11-oxoequilenin (5) from 2-methyl-2-cyclopentenone (1). jS-Addition of the organocoppermagnesium reagent 2 to 1 followed by a-alkylation with ethyl iodoacetate proceeds stereospecifically to give the secosteroid 3 in 94% yield. The final step requires an intramolecular Friedel-Crafts acylation, a reaction that has proved troublesome in previous syntheses of steroids via 9,11-secosteroids. And indeed attempts to cyclize the free acid corresponding to 3 with HF proceeded in yields of 10%. However, cyclization of the ketal acid 4 gives stereochemically pure 5 in 75% yield based on recovered secosteroid. The overall yield from 2-bromo-6-methoxynaphthalene is 52%. 

In an approach employing oxidative radical alkylation, the pyrrole 61 was converted to the corresponding pyrrol-2-acetic acid derivative 62 by treatment with the xanthate 63 in the presence of dilauroyl peroxide (DLP). This procedure was also useful for the alkylation of other heterocyclic systems (e,g, indole) producing ethyl indole-2-acetate <03CC2316>. Alternatively, pyrrole-2-acetic acids have been obtained by treatment of pyrrole with various substituted iodoacetic acids and Na2S203/n-Bu4NI with propylene oxide as the Hl-trap in... 

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