Peptide chains, cleavage

This reaction can be used for selective peptide chain cleavage of cysteine-containing polypeptides and proteins under rather mild conditions. Mild acid treatment of dehydroalanine-containing polypeptides and proteins leads to specific peptide bond cleavage with formation of pyruvate and ammonia (57-59)). 

Above Steps (1) and (2) are peptide chain cleavage, Steps (3) and (4) are regeneration of the thiol by hydrolysis. ... 

When the aminolysis of [14a] was conducted in TFA, the product precipitated when approximately 40% of the benzyl ester had reacted. Conversion of the benzyl ester to amide reached 50% within 24 hrs. and did not change significantly thereafter. However, the extent of peptide chain cleavage continued to increase from a value 7.7% to 17.9% over a total reaction time of 144 hrs. When the product precipitated, the access to the benzyl ester was impeded and subsequent activity was focused on the peptide bonds. Addition of 12.5% DHSO to the THF produced a solvent mixture which dissolved by reactants and products in all conversion ranges. Thus, it was possible to study the aminolysis of [14a] under homogeneous conditions in this solvent mixture. 

The discovery of nbozymes (Section 28 11) in the late 1970s and early 1980s by Sidney Altman of Yale University and Thomas Cech of the University of Colorado placed the RNA World idea on a more solid footing Altman and Cech independently discovered that RNA can catalyze the formation and cleavage of phosphodiester bonds—exactly the kinds of bonds that unite individual ribonucleotides in RNA That plus the recent discovery that ribosomal RNA cat alyzes the addition of ammo acids to the growing peptide chain in protein biosynthesis takes care of the most serious deficiencies in the RNA World model by providing precedents for the catalysis of biologi cal processes by RNA... 

Trypsin, chymotrypsin, and elastase all carry out the same reaction—the cleavage of a peptide chain—and although their structures and mechanisms are quite similar, they display very different specificities. Trypsin cleaves peptides... 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Fig. 19 Structure of LA-PRX (above) and degradation of LA-PRX (below), (a) Threaded a-CDs prevent hydrolysis of PLLA in LA-PRX. (b) LA-PRX converts into LA-pPRX by peptide linkage cleavage at bulky end-capping groups through action of papain, (c) Ester bond hydrolysis in the PLLA chain begins by an exposure of PLLA to water by release of a-CDs from LA-pPRX. Reprinted from [292] with permission...

Figure 20.10 Digestion of IgG class antibodies with pepsin results in heavy chain cleavage below the disulfide groups in the hinge region. The bivalent fragments that are formed are called F(ab )2. The remaining Fc region normally is severely degraded into smaller peptide fragments.

PARACEST agents with a response to enzymatic activity have also been reported. Pagel et al. used a Tm1 DOTA monoamide complex containing a peptide chain which is hydrolyzed by the Caspase-3 enzyme (204,205). Following enzymatic cleavage, the PARACEST effect originating from the amide proton disappears due to the hydrolysis of the amide bond. 

As mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. Carboxypeptidases and ther-molysins are, respectively, exopeptidases, which remove amino acids from the carboxyl terminus of proteins, and endopeptidases, which cleave peptide bonds in the interior of a polypeptide chain. However, they both have almost identical active sites (Figure 12.4) with two His and one Glu ligands to the Zn2+. It appears that the Glu residue can be bound in a mono- or bi-dentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

There are dozens of linkers available for synthesis, and nearly all of them, once acylated by a protected amino acid, provide a benzyl ester or a benzyl amide that has been sensitized to cleavage by acid by the presence of electron-donating moieties such as alkoxy, phenyl, or substituted phenyl. There are cases in which a peptide chain is bound to a support through two different linkers in series. This allows for versatility in synthesis. The distinction between designation of a moiety affixed to a support as a handle or linker is sometimes arbitrary. 

FIGURE 6.15 Imide formation from a dipeptide sequence containing an aspartyl residue with side-chain functional group in various states followed by generation of two peptide chains resulting from cleavage at the bonds indicated by the dashed arrows. The reaction is catalyzed by base52 or acid. [Merrifield, 1967]. The table shows the effect of the nature of the substituent on the extent of this side reaction. Dmpn = 2,4-dimethylpent-3-yl. 

Lenard, J., and G. P. Hess An approach to the specific cleavage of peptide bonds. II. Specific cleavage of peptide chains based on the hydrogen fluoride induced nitrogen to oxygen. shift. Journ. Biol. Chem. 239, 3275 -3281 (1964). 

Afterward, the peptide chain was elongated following the standard Fmoc-based protocol. Before cleavage of the peptide the incorporated Mmt-protected cysteine was deprotected using 1% TFA. Under these very mild conditions the farnesyl moiety was not harmed. Palmitoylation could he achieved using an excess of palmitoyl chloride. Cleavage with copper acetate and methanol as a nucleophile gave the farnesylated and palmitoylated N-Ras sequence with the C-terminal methyl ester 78. ... 

Larsen et al. reported the enzymatic cleavage of a desB30 insuhn B-chain from a presequence (Lys(Boc))6. This spacer shifts the conformation of the growing peptide chain from a y9-structure to a random coil conformation and reduces pep-tide-chain aggregation, which otherwise causes serious synthetic problems. Nova-syn KA [18] was employed as a solid support, but unfortunately, no information about the enzyme used was reported [19]. 

There are no complementary tRNAs for the stop codons. Instead, two releasing factors bind to the ribosome. One of these factors (RF-1) catalyzes hydrolytic cleavage of the ester bond between the tRNA and the C-ter-minus of the peptide chain, thereby releasing the protein. 

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