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... 652 Lastly, glyoxyloyles can easily be transformed into corresponding amines via transamination reaction in the presence of copper(II) or nickel(II) salts. 604,610,653 The reaction mechanism as well as need for both essential components of the system: the acceptor of the glyoxyloyl (usually aspartic acid or glycine) and the cation of a heavy metal are explained in Fig. 75. Despite being of moderate interest for bioconjugation by itself, this approach has initiated the development of a more general methodology for selective N-terminus modification -transaminative conjugation (Section 2.1.3). ...
Bioconjugation methodologies have proven to play a central enabling role in the recent development of biotherapeutics and chemical biology approaches. Recent endeavours in these fields shed light on unprecedented chemical challenges to attain bioselectivity, biocompatibility, and biostability required by modern applications. In this review the current developments in various techniques of selective bond forming reactions of proteins and peptides were highlighted. The utility of each endogenous amino acid-selective conjugation methodology in the fields of biology and protein science has been surveyed with emphasis on the most relevant among reported transformations; selectivity and practical use have been discussed.
This chapter discusses acylation with dicarboxylic acid anhydrides. Succinic and maleic anhydride, or analogs of these two compounds, is utilized in a variety of protein modification studies. During the course of the reaction most of the anhydride is hydrolyzed. The dicarboxylic acid side product may be separated from the modified protein by dialysis, by passage through a column of anion exchange resin (for example, Amberlite IRA-400 in the chloride form), or by gel exclusion chromatography (for example, with Sephadex G-25). The stability of the half amide adduct depends on the anhydride used in the reaction. Five purposes for which the dicarboxylic anhydrides have been utilized are discussed in the chapter, which include protein dissociation, protein hybridization, mapping of lysine peptides, peptide sequencing, lysine side-chain reactivity and function, and introduction of new functional groups. The relatively high specificity of succinic and maleic anhydride for primary amino groups suggests that these reagents may be useful for studying the reactivities of lysine side chains and for elucidating the roles these residues may play in the biological activities of proteins.
Transamination specifically removes the α-amino group and allows any function that this group may possess to be studied, but at the same time, it also specifically introduces a highly reactive carbonyl group. This allows further specific reactions to be carried out; the main one so far studied is the reaction under comparatively mild conditions with a bifunctional nucleophile that removes the ketoacyl residue, thus leaving the polypeptide chain shorter by one residue. Thus, the function of the whole N-terminal residue may be studied, and the process of modification and scission of the terminal residue may be repeated. Conditions for the transamination of proteins that are mild enough so that the protein would not be denatured are developed from the conditions found suitable for the transamination of amino acids. The electrophilic carbon of the keto group of a transaminated protein can bind a reagent that will specifically attack and break the adjacent peptide bond. In this way, the modified residue is selectively removed, so that the protein molecule is left shorter by one residue. This extends the transamination procedure to permit study of the role of the side chain of the first residue as well as the role of its α-amino group, and, of course, enables the whole two-step process to be repeated.
The first efforts to modify the terminal -amino groups of proteins without reaction of the -amino groups of lysine residues made use of their lower pK values. A pH below 7 favors modification of weaker bases, since the stronger bases, although more reactive, are protected to an even greater extent by protonation. Unfortunately, this approach only favors modification of terminal over side-chain amino groups to a limited extent. N-Terminal serine and threonine residues may be selectively acylated on the amino group by an acyl transfer reaction after a peptide has been selectively acylated on its hydroxyl groups. This approach is severely limited by the need for the peptide to be stable to the acidic and anhydrous conditions necessary for selective O-acylation, and to the alkaline conditions necessary for removing the remaining O-acyl groups. Terminal serine and threonine residues may also be selectively oxidized by periodate, since this reaction is a thousand-fold faster than other oxidations of periodate, e.g., of 1,2-diols or disulfides. Further, it forms glyoxyloyl groups, which may be converted into terminal glycine residues by transamination. The last observation provided the basis for the one general modification of N-terminal residues, namely their conversion into 2-oxoacyl groups by reaction of the -amino group with glyoxylate, a reaction catalysed by a bivalent cation, e.g., Cu2+, and a base, e.g., acetate. Participation of the neighboring peptide bond in the reaction ensures specificity of the reaction for the N-terminus. Scission of the N-terminal residue is possible after such a transamination; hence residues may be removed from the N-terminus under nondenaturing conditions. Other exploitations of transamination may be developed.
Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.
MUCH industry has been devoted in recent years to methods for locating the position of components separated on paper chromatograms, and claims have been made for many reagents of greater or less specificity. We wish to report our experiences in locating compounds on paper chromatograms by the successive application of several reagents with different specificities.
Oxidation of steroid alcohols by ruthenium tetroxide gives corresponding ketones in almost quantitative yields. The reaction provides a simple and convenient procedure for converting secondary alcohols to ketones in neutral media. The reconversion of ruthenium dioxide, produced during the oxidation, into the tetroxide with an appropriate oxygen donor such as sodium metaperiodate makes possible the oxidation of a given steroid alcohol to a ketone in the presence of a catalytic amount of ruthenium tetroxide.
1. Osmium tetroxide in dilute ammonia oxidizes various pyrimidine nucleosides at different rates. Thymidine is oxidized about 45 times as fast as deoxycytidine. The phosphate groups may be eliminated from oxidized thymine nucleotides by successive treatments with alkali and then with diphenylamine in aqueous formic acid. The reactions can be applied to the selective degradation of thymidine in oligodeoxynucleotides.
1. Pyrimidine nucleosides such as thymidine, uridine or cytidine are oxidized readily at 0 degrees by osmium tetroxide in ammonium chloride buffer. There is virtually no oxidation in bicarbonate buffer of similar pH. Oxidation of 1-methyluracil yields 5,6-dihydro-4,5,6-trihydroxy-1-methyl-2-pyrimidone. 2. Osmium tetroxide and ammonia react reversibly in aqueous solution to form a yellow 1:1 complex, probably OsO(3)NH. A second molecule of ammonia must be involved in the oxidation of UMP since the rate of this reaction is approximately proportional to the square of the concentration of unprotonated ammonia. 3. 4-Thiouridine reacts with osmium tetroxide much more rapidly than does uridine. The changes of absorption spectra are different in sodium bicarbonate buffer and in ammonium chloride buffer. They occur faster in the latter buffer and, under suitable conditions, cytidine is a major product. 4. Polyuridylic acid is oxidized readily by ammoniacal osmium tetroxide, but its oxidation is inhibited by polyadenylic acid. Pyrimidines of yeast amino acid-transfer RNA are oxidized more slowly than the corresponding mononucleosides, especially the thymine residues. Appreciable oxidation can occur without change of sedimentation coefficient.
