Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 408

like amines to form an amide bond accompanied by formation of a byproduct called
dicyclohexylurea.
In this method, all the required reagents are used in stoichiometrically equimolar
concentrations in aprotic solvents like chloroform, dimethylformamide, and so forth,
at 0°C temperature and is allowed to attain room temperature overnight. To avoid
formation of N -acylurea, temperature is preferably kept low. In a modified technique,
N -hydroxysuccinimide is usually added to DCC-mediated coupling reactions to
reduce the widespread racemization that is seen with DCC alone.
8.6.1.3 Mixed Anhydride (Classical Method)
This method was developed back in 1951. It basically involves formation of an
asymmetric or mixed anhydride from amino acid and an alkyl chlorocarbonate. This
is subsequently followed by reaction with an amine component to create the peptide
bond. The reaction must be carried out under stringent conditions as it involves for-
mation of numerous byproducts and intermediates if the process is not controlled.
One of the salient characteristics of this method is the production of volatile and
readily extractable byproducts like isobutanol, carbon dioxide, and others. However,
limitations mainly consist of numerous side reactions and generation of various
byproducts leading to impure product.
8.6.1.4 Azide Method
The azide-coupling procedure involves the reaction of an azide with an amino deriv-
ative. The method has an edge over existing methods like coupling and the mixed
anhydride method in terms of the absence of extensive racemization in this method.
Numerous procedures are available for the preparation of peptide azides, follow-
ing the preparation of a peptide hydrazide. The azide is formed by the reaction of a
nitrite in the presence of an acid. The azide coupling is performed immediately fol-
lowing azide formation by buffering the solution at pH of 7-8 and lowering the tem-
perature to 0°C. Normally, the reaction is carried out in polar solvents like dimethyl
formamide. A reaction sometimes may take days to complete due to mild reactivity
of azides.
The chief advantages of azide reaction are low racemization rates. The method is
useful for synthesis and purification of large peptides. However, the method suffers
from limitations like being cumbersome and tedious, and from the plethora of side
reactions involved.
8.6.2 Solid Phase Peptide Synthesis
Solid phase peptide synthesis involves the attachment of side-chain-protected amino
acid to an insoluble polymeric solid support. The amino protecting group is removed
in the next step and salt of amine is converted to free amine by the addition of a
free weak organic base. The solvents and other residual reagents are removed, and
the entire procedure is repeated several times until the final peptide sequence is