Practical Protein Chemistry - A. Darbre 1989

Latest methods for solid-phase and liquid-phase amino acid sequence determination
Solid-Phase Analysis. Recent Approaches
Other Sequential Degradation Methods

The solid-phase method allows The Use of approaches distinct from the Edman Degradation for Amino Acid Sequence analysis. Many Reagents suitable for alternative Chemical Reactions cannot be employed in liquid-phase Methods because simple extraction often fails to completely remove excess reagents and side-reaction products.

The basics of several alternative approaches are outlined below, along with their potential and limitations.

16.2.5.1. Determination of the N-Terminal Sequence by Thioacylation and Acid Cleavage. Several sequential cleavage methods are based on the attachment of a thioacyl group R = C = S to the N-terminal amino group of a peptide, followed by the cyclization of the N-terminal residue under acidic conditions and its cleavage as a thiazolinone [4, 75, 84]. This approach differs from the Edman method in both the reagent used at the Condensation stage and the reaction mechanism. Furthermore, at the cleavage stage of The amino acid derivative, a 2-alkyl- or 2-arylthiazolinone is formed instead of a 2-anilinothiazolinone. The formation of these derivatives offers several additional advantages. The weaker electron-withdrawing Nature of the alkyl or aryl groups makes the thiocarbonyl sulfur atom more nucleophilic. Consequently, cyclization (and cleavage) proceeds under milder conditions. Cleavage using TFA is completed at room Temperature within 20 min [4, 84]. By increasing the reaction temperature, weaker acids or diluted TFA can be employed. Thus, it may be possible to mitigate many of the problems encountered during the acidic cleavage stage of the Edman reaction: peptide chain cleavage, Ser and Thr dehydration, succinimide formation from Asn residues, Trp oxidation, and the cyclization of N-terminal Gln to pyroglutamic acid. It should be noted that these thiazolinones are more stable than the corresponding intermediates of Edman reactions and cannot isomerize into thiohydantoins. This important circumstance should significantly increase yields in alternative conversion reactions that yield products intended for identification.

Class="center">Table 16.1. Thioacylating reagents (general formula RC(:S)—X) used for sequential peptide cleavage

For the successful application of this approach to amino acid sequence analysis, it is necessary to identify thioacylating agents and process conditions that match the efficiency of PITC at the peptide condensation stage. General reagents of the form RC(:S)—X have been used, where R is an alkyl or aryl (aromatic component) group, and X is an activating leaving group, such as —SR', —OR', or —N<R'. Thioacyl chlorides (X = Cl) exhibit high reactivity, but they are insufficiently stable. The thioacylating ability increases with the electron-withdrawing strength of the acyl substituent R. However, this trend is accompanied by a decrease in The rate of the cyclization reaction; therefore, a compromise must be struck when selecting a suitable reagent for condensation with the peptide to ensure the efficient progression of both cycle stages. A large number of compounds have been tested, and the most promising reagents for sequential amino acid cleavage are listed in Table 16.1. The presence of charges on the leaving groups (compounds 1, 6, and 7) greatly enhances the solubility of the reagents in aqueous-organic media, although anhydrous solvent systems and uncharged reagents can also be used in the analysis without complication.

Simple alkyl carbodithioates (compounds 8–12) possess very low reactivity, rendering them practically useless except for reactions carried out in the presence of general base catalysts (e.g., triethylammonium acetate) in strongly polar Solvents [83]. Catalysis in the thioacylation reaction can be intramolecular, as in the aminolysis of the Choline ester of m-nitrobenzoic acid [7] (Table 16.1, compound 6). This reagent is exceptionally resistant to Hydrolysis. Cyanomethyl dithioesters (e.g., compound 2) are sufficiently reactive [84], but the eliminated leaving group polymerizes, leading to the formation of precipitates that are difficult to remove. A readily synthesizable reagent (compound 5) has been recently described [19].

An automated solid-phase analysis protocol based on the thioacylation reaction is known [29, 70, 75]. During the structural determination of the Insulin B-chain using thioacetylthioglycolic acid (compound 7), 26 amino acid residues were successfully identified, and sequences of up to 40 Amino Acids were determined for several Proteins. Stepwise yields exceeded >95%. The identification of cleaved residues was performed either after converting ATZ into thioacylamino acids or following back-hydrolysis into the original amino acids. A rapid conversion method of 2-phenylthiazolinone into thiobenzoylpeptide methylamide using an excess of methylamine is known. This mild transformation may play a significant role in future cleavage Procedures involving thioacylating reagents. 2-Phenylthiazolinones have been identified mass spectrometrically [5]. These compounds have also been converted into thiobenzoylamino acid anilides and identified by TLC [6], although this aminolysis variant proceeds more slowly than the analogous reaction with methylamine.

16.2.5.2. C-Terminal Cleavage. A number of methods for sequential chemical cleavage from the carboxyl end of a peptide molecule have been proposed (Chapter 18). Initial attempts in this direction were undertaken even before the Edman reaction was described, but they failed to compete with it. In a promising and thoroughly studied method [98], the C-terminal carboxyl group of a peptide reacts with ammonium thiocyanate in acetic anhydride: the resulting cyclic peptidyl thiohydantoin is treated with an acid (or acetohydroxamate); the products of the latter reaction are the thiohydantoin of the C-terminal residue and a peptide shortened by one C-terminal amino acid. In the original methodology, each cleavage cycle involved lengthy and tedious Separation and drying stages. Only 2–6 residues could be successfully cleaved. However, with The Development of the solid-phase method, new expectations are associated with this approach [26, 27, 58]. The cited works provide a General Overview of C-terminal analysis methods.

The solid-phase approach significantly stimulated work on thiocyanate cleavage, although it has still not been possible to cleave more than 6 amino acids. Particular difficulties arise in the analysis of Asn, Asp, Glu, and Pro residues themselves, as well as the residues following them. Pro is cleaved during the very first operation of its respective cleavage cycle (Treatment with ammonium thiocyanate), causing the subsequent amino acid to appear alongside it in the products of the same cycle [58]. The Cleavage of the other aforementioned amino acids proceeds in low yields. The identification of thiohydantoins has been carried out using various methods without exploiting the advantages of highly sensitive photometry. It is difficult to assess the feasibility of determining long sequence stretches without conducting additional thoughtful and systematic work. We hope that such research will be continued, as C-terminal cleavages can serve as a very useful Complement to N-terminal sequence analysis. Furthermore, the quantitative Attachment of Peptides via their α-amino group to an insoluble support is generally much easier than condensation via the C-terminal carboxyl.

Alternative approaches to determining the C-terminal sequence in combination with the solid-phase method have been proposed. The peptide was attached via a carbodiimide to an S-alkylthiouronium salt, followed by the cleavage of the C-terminal residue as an iminohydantoin using an aqueous base solution at pH 10–11.5. Up to five C-terminal amino acids of short peptides were determined in this manner. The advantage of this method over the thiocyanate approach is that it is carried out under milder conditions, whereas its drawback is the inability to cleave C-terminal Pro; additionally, if Asp or Glu is present in the same position, they tend to form cyclic anhydrides during carbodiimide-mediated condensation [99].

Recently, new possibilities for performing C-terminal analysis on insoluble Supports have been reported. It has been demonstrated that the ester bond can be cyclized into a C-terminal alkoxazole using strong dehydrating agents [83]. Subsequent treatment with an alcohol in an acidic medium yields an amino acid ester. Another sequence analysis scheme, distinct from this approach and utilizing several new synthetic reagents, exists [68]. Both of these approaches currently exist only as laboratory developments; however, they are of interest to the creative chemist as they open new pathways toward a major goal.