Selective cleavage of peptide bonds

The sequencing strategy follows the order 1) preparation of pure protein, 2) selective cleavage of peptide bond 3) isolation of cleaved peptide fragments, 4) analysis of primary structure on amino acid sequencer, 5) determination of entire primary structure. Although this order has not changed since our primary structure determination work on AspAT carried out in early 1970, many improvements have been made, e.g. preparation of micro scale amount of sample and introduction of high sensitivity analytical instruments. These improvements have shortened analysis time and lowered the sample amount of required. 

Patchornik A, Lawson WB, Witkop B. Selective cleavage of peptide bonds.2. The tryptophyl peptide bond and the cleavage of glucagon. J. Am. Chem. Soc. 1958 80 4747-4748. 

Survey of Principles and Reactions Employed in the Preferential or Selective Cleavage of Peptide Bonds... 

Chemical and enzymatic methods for the selective cleavage of peptide bonds are not completely separate either-or propositions. In addition chemical modification may well be combined with enzymatic degradation in a manner which promises new usages and more selectivity for proteinases and peptidases in sequence studies. 

Selective activation of peptide side chain functional groups is an important approach in site-selective cleavage of peptide bonds. In 1950, Edman reported a stepwise peptide cleavage fi om the N-terminal using phenyl isothiocyanate [107]. Thereafter, selective peptide bond cleavage methods at specific residues... 

D.xi.a. Hydrolysis and Hydration. The electrophilicity of cationic palladium(II) complexes has been taken advantage of in an interesting biomimetic method of selective cleavage of peptide bonds at histidine, methionine, or tryptophane residues. Further discrimination is possible as peptides with two former amino acids are cleaved in aqueous solution but not cleaved in acetone, while those with tryptophane are cleaved only in acetone in the presence of water. The mechanism of cleavage at tryptophane is particularly interesting as it involves the electrophilic ip o-paUadation of indole ring, while the remaining coordination site of Pd in the ip/ro-cyclopalladated complex is used for the electrophilic activation of carbonyl (Scheme 91). 

Exploratory experiments have shown that glycosylidene carbenes also insert into the N-H bond of sulfonamides, whereas carboxamides yield esters, as illustrated by the product obtained from an A-Boc-asparagine benzyl ester (Figure 7c) [32]. Presumably, such esters are formed by hydrolysis of the initially formed imino ether 0-glycosides, suggesting that glycosylidene diazirines may be used for (selective ) cleavage of peptidic bonds. 

Selective cleavage of peptides and proteins is an important procedure in biochemistry and molecular biology. The half-life for the uncatalyzed hydrolysis of amide bonds is 350 500 years at room temperature and pH 4 8. Clearly, efficient methods of cleavage are needed. Despite their great catalytic power and selectivity to sequence, proteinases have some disadvantages. Peptides 420,423,424,426 an(j proteins428 429 can be hydrolytically cleaved near histidine and methionine residues with several palladium(II) aqua complexes, often with catalytic turnover. 

Another commonly used enzyme for selective cleavage of peptides is chymotrypsin, which hydrolyzes peptide bonds on the carboxyl side of aromatic residues (phenylalanine, tyrosine, and tryptophan) ... 

Because cobalt(III) complexes bind to the N-terminal amino acid residue of peptides, only the N-terminal peptide bond is hydrolyzed. It would be advantageous if the metal complex could coordinate to various parts of a peptide chain, thus allowing for cleavage of other areas of the peptide chain. Recently this has become an active area of research, and there have been several publications on the cleavage of the polypeptide backbone of proteins promoted by metal complexes. The advantage that these complexes have over the cobalt(III) complexes is that they can be selectively introduced on to various amino acid side chains, allowing for cleavage of peptide bonds at locations other than the N-terminus. This is of interest because,... 

The oxidation of trypsin and trypsinogen was carried out in aqueous 0.1 M acetate buffer solutions at room temperature. In this particular case and under these conditions no significant cleavage of peptide bonds next to tryptophan residues occurred. Careful analysis of hydrolyzates of NBS-oxidized trypsinogen and trypsin confirmed the selectivity of the oxidative modification of the protein, as Table XXIV shows. There is no significant loss of tyrosine, histidine, serine, threonine, or cystine, although all of these amino acids will react with NBS but considerably less rapidly than tryptophan. 

Several other methods for the selective chemical cleavage of peptide bonds adjacent to particular amino acids have been described, but none has been used widely. 

T. Hashimoto, S. Saito, K. Ohki, N. Sakura, Highly Selective Cleavage of Pyroglutamyl-Peptide Bond in Concentrated Hydrochloric Acid , Chem. Pharm. Bull. 1996, 44, 877-879. 

Proteases, since their discovery, have played an important role in both basic and applied research because their sometimes exquisite selectivity of peptide bond cleavage can be extremely useful in the elucidation of other proteins structures and function. The following three sub-sections give several examples of how proteases are used in specific aspects of research. 

Picornain 2A [EC 3.4.22.29] catalyzes the hydrolysis of peptide bonds including the selective cleavage of a particular peptide bond in the picornavirus polyprotein. Picornain 3C [EC 3.4.22.28] catalyzes the selective hydrolysis of the Gin—Gly bond in the poliovirus polyprotein. 

Thrombin [EC 3.4.21.5], also known as fibrinogenase, catalyzes the hydrolysis of peptide bonds, exhibiting preferential cleavage for the Arg—Gly peptide bond. The enzyme, a member of the peptidase family SI, activates fibrinogen to fibrin and releases fibrinopeptide A and B. Thrombin, formed from prothrombin, is more selective in peptide hydrolysis than trypsin or plasmin. 

Zhu, J. X. and Aswad, D. W. (2007). Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming. Anal. Biochem. 364,1-17. 

The porcine insulin is first subjected to digestion with chymotrypsin-free trypsin at pH 7.5 for in excess of 45 min. This results in the selective cleavage of the peptide bond linking arginine 22... 

Chemical cleavage at selective amino acid residues is an alternative method for scission of peptide bonds in target molecules. Cleavage at the Met-Xaa bond is achieved by CNBr, at the Trp-Xaa bond by the tryptophan-directed reagent 3-bromo-3-methyl-2-[(2-nitro-phenyl)sulfanyl]-3//-indole (BNPS-skatole) and at the Asp-Xaa bond by 2% formic acid.123 24 All these reactions are carried out at acidic pH under standard conditions for each reagent. 

The critical feature of the Edman degradation is that it allows the N-terminal amino acid to be removed without cleaving any of the other peptide bonds. Let s see how this occurs. The mechanism of the reaction is shown in Figure 26.3. First the nucleophilic nitrogen of the N-terminal amino acid attacks the electrophilic carbon of phenyl isothiocyanate. When anhydrous HF is added in the next step, the sulfur of the thiourea acts as an intramolecular nucleophile and attacks the carbonyl carbon of the closest peptide bond. II is the intramolecular nature of this step and the formation of a five-membered ring that result in the selective cleavage of only the N-terminal amino acid. The mechanism for this part of the reaction is very similar to that for acid-catalyzed hydrolysis of an amide (see Section 19.5). However, because no water is present, only the sulfur is available to act as a nucleophile. The sulfur is ideally positioned for intramolecular attack at the carbonyl carbon of the N-terminal amino acid, so only this amide bond is broken. 

B. Selective Cleavage of Tryptophyl Peptide Bonds in Serum Albumins... 284... 

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