Cleavage methods

In another method for sequencing, the DNA is labeled at internal base positions by enzymes which repHcate DNA strands in the presence of specific chain terrninators, thus producing an array of labeled fragments which reflect the sequence of the DNA. The rest of the procedure is the same as described for the chemical cleavage method (22). 

Solvolysis in MeOH/H20 at 21°. This method was developed for a series of penicillin derivatives where conventional cleavage methods resulted in partial /3-lactam cleavage. ... 

It is important to recognize that the following analytical methods essentially determine EO-PO ratio ( H NMR, IR, cleavage methods) or even simply alkylene oxide content (compleximetric methods) of the analyte, and as such are not specific quantitative or qualitative methods for poloxamers, since EO-PO copolymers of a different structure (for instance, random copolymers, or PO-EO-PO block copolymers) may respond to the methods in a way indistinguishable from poloxamers. The principal technique that permits definitive identification of a sample as a poloxamer is C NMR, which allows structural details, such as the distribution of EO and PO units along the polymer chain, to be elucidated [10]. 

Recent work has shown that there are many assumptions required in order to use the silane-cleavage method. However, an advantage of this approach is that it can be nsed to determine acidities at regiospecific positions in very weak acids, including those for which the conjugate base anion is unstable with respect to electron detachment. 

Maxam, A. M. Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980, 65, 499-560. 

DS King, CG Fields, GB Fields. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Ini J Pept Prot Res 36, 255, 1990. 

Fig. 5. Schematic representation of the domain structure of albumin, the position of the disulfide loops, and the fragments of the molecule that have been isolated. Included in the figure are the cleavage methods used to obtain the fragments plus the regions to which the restricted antibody populations used in these experiments are directed. Reprinted, with permission, from Teale and Benjamin (1976). Copyright by the American Society of Biological Chemists, Inc.

Adjacent half-cystine residues are present in many peptides and proteins. In most of the cases they form two disulfide bonds with other cysteines in the molecule, unless the peptide bond between them is cis (see Section 6.1.5.1 ).t40 41 Specific enzymes that cleave the Cys-Xaa bond have not yet been discovered, although there are a few reports of cleavage of the Cys-Cys bond by enzymes such as elastase and pepsinJ42-43 For peptides with Cys-Cys bonds the cleavage method in Section 6.1.6.2.4 is recommended. 

Alternatively, cleavage can be done chemically, but here specificity of cleavage at a single defined position is complicated, since most chemical cleavage methods react with single amino acids (Table 5.13). For instance, chemical cleavage at the C-terminal side of methionine residue can be effected using CNBr, but the use of this method is limited, since most proteins are likely to contain internal methionine residues. 

Table 5.13 Specific cleavage methods for proteins. The peptide bond susceptible to the cleavage is marked, (adapted from Nilsson et at, 1988)...

Removal of the auxiliary is straightforward and consists of treatment of the alkylated A -acyl-sultam 5 with an excess of lithium hydroxide/30% hydrogen peroxide in tetrahydrofuran. Other mild cleavage methods are available20. 

For the regeneration of sensitive, optically active aldehydes and ketones from their SAMP- or RAMP-hydrazones, two mild cleavage methods are used routinely. 

Starting Hydrazoneb Reaction Conditions Alkylated Hydrazone Cleavage Method Config. of Stereogenic Center Formed eef (%) Yield (%) Over- all Md (c, solvent)11 Ref... 

After use of this cleavage method, the chiral auxiliary may be partially recycled by neutralization and extraction of the aqueous layer. In this way a mixture of methylated hydrazine, the SAMP-hydrazone of formaldehyde and SAMP (1 7 2) is obtained, which is subjected to air oxidation and hydrolysis8. 

Since the above cleavage methods proceed without racemization, the enantiomeric excess of the alkylated carbonyl compounds obtained can be safely determined by measuring the diastereomer-ic ratio of the alkylated hydrazones (see Section I.I.I.4.2.3.). The diastereomeric excess of SAMP-hydrazones derived from aldehydes was further determined by gas chromatography41. 

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